Double-protein microcapsule and application thereof

Abstract

The invention discloses a double-protein microcapsule and application thereof. The microcapsule is prepared by combining a double-protein compound formed by lactoferrin and alpha-lactalbumin, beta-lactoglobulin or casein with sodium alginate and encapsulating probiotics through an endogenous emulsification method. The core advantages are that the double-protein compound enhances gastric acid tolerance through space barrier effect, and the probiotic effect cooperates with the controlled release characteristic of sodium alginate, so that the intestinal activity retention and targeted release efficiency of probiotics is greatly improved; the microcapsule is endowed with 28-day excellent storage stability by a physical protection-nutrition synergy dual mechanism. According to the invention, double-protein specific interaction is combined with a polysaccharide encapsulation technology, reproducible optimization parameters and theoretical basis are provided, the problem of'activity-release-storage 'synergy in probiotic delivery is effectively solved, and reference is provided for precise design of synbiotic microcapsules.

Description

Technical Field

[0001] This invention relates to the field of drug delivery, and more specifically to a dual-protein microcapsule and its application. Background Technology

[0002] Probiotics, especially Bifidobacteria, play a vital role in maintaining gut health, regulating the gut microbiota, and enhancing the immune system. However, the delivery of probiotics in the gastrointestinal tract still faces significant challenges, primarily due to the harsh environment of the stomach and small intestine (such as low pH, bile acids, and digestive enzymes) leading to a loss of probiotic activity and affecting their efficacy. Microencapsulation technology, as an effective method to improve the stability and activity of probiotics in the gastrointestinal tract, has become a research hotspot. Among these methods, endogenous emulsification effectively mixes the oil and aqueous phases through emulsification to form a stable emulsion, which can effectively encapsulate probiotics within microcapsules, thereby enhancing their protective effect in the gastrointestinal tract.

[0003] Lactoferrin (LF) is a multifunctional iron-binding globulin widely found in the milk of mammals, especially in colostrum where it is present in high concentrations. LF possesses important biological functions, including antibacterial, antiviral, antioxidant, and immunomodulatory effects, making it crucial in the immune defense of infants and newborns. However, using lactoferrin alone as a carrier or functional component of probiotics presents several technical limitations, restricting its effectiveness and feasibility in practical applications.

[0004] First, lactoferrin has poor stability, especially in the gastrointestinal environment. Influenced by gastric acid and digestive enzymes, its structure and function are easily altered, leading to a significant reduction in its activity. Second, lactoferrin has low bioavailability; it is often not effectively absorbed in the gastrointestinal tract, hindering its full functional expression and reducing its effectiveness in vivo. Furthermore, the production and purification processes of lactoferrin are complex and costly, limiting its large-scale application and commercialization.

[0005] Therefore, given the shortcomings of using lactoferrin alone, there is an urgent need for a new technological approach to improve the stability, activity, and bioavailability of lactoferrin in the gastrointestinal environment, while reducing production costs, thus enabling its wider application. This invention addresses these challenges in existing technologies by proposing an innovative dual-protein complex and microcapsule carrier system that effectively solves the aforementioned problems and significantly enhances the delivery efficiency and clinical application potential of lactoferrin. Summary of the Invention

[0006] Technical problem to be solved: In view of the above-mentioned technical problems, the purpose of this invention is to provide a dual-protein microcapsule and its application.

[0007] Technical solution: A dual-protein microcapsule, wherein the dual-protein microcapsule is prepared from a dual-protein complex and sodium alginate.

[0008] Furthermore, the dual-protein complex is a lactoferrin (LF) / α-lactalbumin (ALA) complex, a lactoferrin (LF)-β-lactoglobulin (BLG) complex, or a lactoferrin (LF)-casein (CN) complex.

[0009] Furthermore, the preparation method of the lactoferrin-α-lactalbumin (ALA-LF) complex is as follows: weigh lactoferrin and α-lactalbumin powders separately, dissolve them in distilled water to prepare 1 mg / mL lactoferrin solution and α-lactalbumin solution, and stir at room temperature for 2-2.5 h to fully hydrate the protein; mix the α-lactalbumin solution and lactoferrin solution at a molar ratio of 1.5-2.0, and incubate in a water bath at 45-55℃ for 30 min to obtain the final product.

[0010] Furthermore, the preparation method of the lactoferrin-β-lactoglobulin (BLG-LF) complex is as follows: weigh lactoferrin and β-lactoglobulin powders separately, dissolve them in distilled water to prepare 1 mg / mL β-lactoglobulin solution and lactoferrin solution, and stir at room temperature for 2-2.5 h to fully hydrate the protein; mix the β-lactoglobulin solution and lactoferrin solution at a molar ratio of 2.0, and incubate in a water bath at 45-55℃ for 30 min to obtain the final product.

[0011] Furthermore, the preparation method of the lactoferrin-casein (CN-LF) complex is as follows: Weigh lactoferrin powder and dissolve it in distilled water to prepare a 1 mg / mL lactoferrin solution, and stir at room temperature for 2-2.5 h to fully hydrate the protein; Weigh 0.1 g of casein powder into a mortar, moisten it with a small amount of distilled water, slowly add 4 mL of 0.2 mol / L NaOH, grind thoroughly, boil in a water bath for 15 min, cool, and bring the volume to 100 mL to obtain a casein solution; Mix the casein solution and the lactoferrin solution at a molar ratio of 0.4-0.6, and incubate in a water bath at 20-30℃ for 30 min to obtain the final product.

[0012] The above-mentioned dual-protein microcapsules are used in the delivery of probiotics.

[0013] Furthermore, the application method is as follows: S1. Take Bifidobacterium animalis, activate and culture it in MRS broth, 4000 ×g Centrifuge for 10 min to collect the precipitate, wash twice with physiological saline, and resuspend in protein solution to obtain probiotic suspension; S2. Mix the probiotic suspension with the dual protein complex and sodium alginate complex solution, add CaCO3, stir evenly to obtain a suspension; S3. Add the suspension to soybean oil, mechanically stir for 13-18 min, add glacial acetic acid, continue stirring for 30 min, add CaCl2 solution, and stir for 10 min; S4. Remove the oil phase to obtain gel-formed microcapsules, wash with physiological saline until no oil phase residue remains, and collect by centrifugation to obtain double protein microcapsules encapsulating probiotics.

[0014] Furthermore, the number of effective live bacteria in the probiotic suspension in S1 is 10. 8 -10 10 CFU / mL.

[0015] Furthermore, in S2, the volume ratio of the probiotic suspension to the compound solution of the dual protein complex and sodium alginate is 1:4; the concentration of the dual protein complex in the compound solution of the dual protein complex and sodium alginate is 1.0-1.5 mg / mL; the concentration of sodium alginate is 1.5-2.5%; the concentration of CaCO3 solution is 1.5%, and the amount added is 75% of the probiotic suspension.

[0016] Furthermore, in S3, the volume ratio of the suspension to soybean oil is 5:12; the soybean oil contains 0.5% Tween 80; the amount of glacial acetic acid added is 0.2-0.25% of the volume of the W / O emulsion; the concentration of CaCl2 solution is 2%, and the amount added is 115-120% of the volume of the W / O emulsion.

[0017] Beneficial effects:

[0018] 1. This invention constructs a dual-protein complex system of lactoferrin (LF) with α-lactalbumin (ALA), β-lactoglobulin (BLG), and casein (CN), respectively. By utilizing the electrostatic interactions, hydrogen bonds, and hydrophobic interactions between proteins to form a stable complex, and combining it with the ionic cross-linked gel network of sodium alginate, the invention encapsulates Bifidobacterium animalis F1-7 using an endogenous emulsification method, thereby enhancing the structural stability of the microcapsule and significantly improving the encapsulation rate of probiotics (>90%).

[0019] 2. This invention resists gastric acid erosion through the spatial barrier effect formed by the dual protein complex, and the controlled release characteristics of sodium alginate in gastric juice (protonated to form a gel to delay gastric acid penetration) and in intestinal juice (deprotonated to dissolve and release bacteria) achieve the effect of enhancing the acid resistance and enteric solubility of microcapsules, and improving the retention of probiotic intestinal activity and the efficiency of targeted release.

[0020] 3. This invention provides nutritional support to probiotics through the prebiotic effect of the dual protein complex, and combines the physical barrier of sodium alginate to resist adverse factors such as oxygen and humidity, forming a dual mechanism of "physical protection-nutritional synergy", which achieves the effect of giving microcapsules excellent storage stability, so that probiotics can still maintain a high number of live bacteria after being stored at 4°C for 28 days.

[0021] 4. This invention optimizes the preparation conditions of the dual-protein complex (50℃, ALA / LF=1.5; 50℃, BLG / LF=2; 25℃, CN / LF=0.5) to promote the complementary secondary structures of proteins (α-helix / β-sheet / random coil) to form stable aggregates. Combined with polysaccharide encapsulation technology, it achieves synergistic optimization of probiotic "activity-release-storage" and solves the problem that a single encapsulation system is difficult to balance stability and functional activity.

[0022] 5. This invention regulates the combination of the dual protein complex and sodium alginate to enable microcapsules to form specific structures (a rigid network of ALA-LF and a porous structure of CN-LF). By utilizing the secondary structure of proteins to regulate ice crystal formation and interfacial stability, it achieves the effect of meeting different functional requirements such as long-term storage or efficient intestinal targeted release.

[0023] 6. This invention forms a hydrophobic-hydrophilic balanced tight interface between the α-helix of LF and the secondary structure of ALA / BLG in the dual protein complex, and enhances the stability of the complex with disulfide bonds, making it perform well in terms of resistance to gastric acid corrosion, with a viable bacterial count of about 7.5 log CFU / g or even higher.

[0024] 7. This invention uses the random coils of CN to fill the gaps in the rigid structure of LF to form a porous network. Combined with the degradation of protein complexes by trypsin, it achieves the effect of accelerating the disintegration of microcapsules in intestinal fluid, promoting the rapid release of probiotics and maintaining an activity of about 8 log CFU / g.

[0025] 8. This invention utilizes a dual-protein complex with Ca 2+ The synergistic effect of sodium alginate promotes the formation of an "egg-box" structured gel network, enhancing the mechanical strength and thermal stability of microcapsules. This achieves the effect of improving the microcapsules' resistance to environmental stress and ensuring the stable activity of probiotics during processing and storage. Attached Figure Description

[0026] Figure 1 Flowchart for the preparation of dual-protein microcapsules encapsulating probiotics; Figure 2The effects of different temperatures and ratios on the zeta potential of the dual-protein complex are shown in Figure A, where A represents the effect of different temperatures on the zeta potential of the dual-protein complex; B represents the effect of different ratios on the zeta potential of the dual-protein complex; different letters indicate significant differences at the p < 0.05 level. Figure 3 The effects of different temperatures and ratios on the particle size and PDI of the dual-protein complex are shown. In this paper, A represents the effect of different temperatures on the average particle size of the dual-protein complex, and B represents the effect of different ratios on the average particle size of the dual-protein complex. Different letters indicate significant differences at the p < 0.05 level. Figure 4 The images show the fluorescence spectra of the dual-protein complex at different temperatures and ratios. In A, the effect of different temperatures on the fluorescence intensity of the dual-protein complex is shown; in B, the effect of different ratios on the fluorescence intensity of the dual-protein complex is shown. Figure 5 The effects of different temperatures and proportions on the circular dichroism chromatograms of the dual-protein complex are shown; where A represents the effect of different temperatures on the circular dichroism chromatograms of the dual-protein complex; and B represents the effect of different proportions on the circular dichroism chromatograms of the dual-protein complex. Figure 6 The particle size distributions of different microcapsules prepared in Example 4 and Comparative Examples 1-4 are shown, where (a) is LF+F1-7, ALA+F1-7, A50-1.5+F1-7; (b) is BLG+F1-7, B50-2+F1-7; (c) is CN+F1-7, C25-0.5+F1-7; (d) is A25-0.5+F1-7; (e) is B75-1.5+F1-7; and (f) is C75-1+F1-7. Figure 7 The images shown are DSC thermal images and XRD spectra of different microcapsules prepared in Example 4 and Comparative Examples 1-4, where A is a DSC thermal image and B is an XRD spectrum. Figure 8 The images are SEM images of the microcapsules, where A is A50-1.5, A1 is 50x magnification, and A2 is 200x magnification; B is B50-2, B1 is 50x magnification, and B2 is 200x magnification; C is C25-0.5, C1 is 50x magnification, and C2 is 200x magnification. Figure 9 For naked and double protein encapsulation Bif. Survival and release curves of F1-7 in simulated oral-gastric-intestinal fluid; Figure 10 For naked and double protein encapsulation Bif. Storage stability of F1-7 at 4°C for 28 days. Detailed Implementation

[0027] This invention proposes a dual-protein microcapsule and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following will provide a more detailed description of the invention with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0028] Lactoferrin (LF, 95%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. α-lactalbumin (ALA, 85%) was purchased from Beijing Heng Hui Biotechnology Co., Ltd. β-lactoglobulin (BLG, 95%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Casein (CN), pepsin (1:3000), trypsin (1:250), and α-amylase (1:8) were purchased from Beijing Solarbio Biotechnology Co., Ltd. MRS broth was purchased from Qingdao Hongbo Biotechnology Co., Ltd. Agar was purchased from Biofroxx AG, Germany. Soybean oil was purchased from a local supermarket in Wuhan. Other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. The experimental water was double-distilled deionized water, prepared using a Milli-Q™ ultrapure water system (Millipore, USA).

[0029] Example 1 The preparation method of the ALA-LF complex is as follows: S1. Weigh out LF and ALA powders respectively and dissolve them in distilled water to prepare a 1 mg / mL solution. Stir at room temperature for 2 h to fully hydrate the protein and obtain LF solution and ALA solution respectively. S2. ALA solution and LF solution were mixed at molar ratios of 0.33, 0.5, 1.0, 1.5 and 2.0, respectively, and then subjected to water baths at 25 ℃, 50 ℃, 62.5 ℃, 75 ℃ and 100 ℃ for 30 min, respectively, to obtain ALA-LF complex solutions.

[0030] Example 2 The preparation method of the BLG-LF complex is as follows: S1. Weigh out LF and BLG powders respectively and dissolve them in distilled water to prepare a 1 mg / mL solution. Stir at room temperature for 2 h to fully hydrate the protein and obtain LF solution and BLG solution respectively. S2. BLG solution and LF solution were mixed at molar ratios of 0.33, 0.5, 1.0, 1.5 and 2.0, respectively, and then subjected to water baths at 25 ℃, 50 ℃, 62.5 ℃, 75 ℃ and 100 ℃ for 30 min, respectively, to obtain BLG-LF complex solution.

[0031] Example 3 The preparation method of the CN-LF complex is as follows: S1. Weigh LF powder and dissolve it in distilled water to prepare a 1 mg / mL solution. Stir at room temperature for 2 h to fully hydrate the protein and obtain the LF solution. S2. Weigh 0.1 g of casein powder into a mortar, moisten it with a small amount of distilled water, slowly add 4 mL of 0.2 mol / L NaOH, grind thoroughly, transfer to a 100 mL volumetric flask, boil in a water bath for 15 min, cool and make up to volume to obtain CN solution; S3. The CN solution and LF solution were mixed at molar ratios of 0.33, 0.5, 1.0, 1.5 and 2.0, respectively, and then subjected to water baths at 25 ℃, 50 ℃, 62.5 ℃, 75 ℃ and 100 ℃ for 30 min, respectively, to obtain CN-LF complex solutions.

[0032] Performance testing: (1) Particle size distribution and zeta potential analysis The particle size distribution and zeta potential of the dual-protein complexes prepared at different ratios and temperatures were measured using a Zetasizer Nano ZS90 (Malvern Instruments, UK). The mixture was gently stirred before measurement to ensure uniform dispersion. (2) Fluorescence spectrum Fluorescence measurements were performed using a fluorescence spectrometer (F-4600, Hitachi, Japan). The sample concentration was adjusted to 0.5 mg / mL, and fluorescence spectra were recorded at an excitation wavelength of 295 nm, with emission spectra recorded from 300 to 600 nm. (3) Circular dichroism The secondary structure characteristics of the samples were determined using a circular dichroism chromatograph (J-1500, Jasco, Japan). Measurements were performed in the far-ultraviolet range (190-260 nm) at 25 °C under nitrogen atmosphere.

[0033] Zeta potential reflects the electrostatic potential near the particle surface in a colloidal system and is an important indicator of particle stability and interparticle interactions. The zeta potentials of three biprotein complexes—ALA-LF, BLG-LF, and CN-LF—were measured at different temperatures and proportions. The results are as follows: Figure 2 As shown.

[0034] Acidic and basic proteins may aggregate through electrostatic interactions. When LF was mixed separately with solutions of three other proteins, the potential of the complex showed a decreasing trend, indicating that an internal reaction occurred. For two substances bound by electrostatic interactions, the closer the complex potential is to 0, the more stable the complex is, meaning the two substances bind better. Therefore, ALA and LF bind better at 50°C and when ALA / LF = 1.5 or 2; BLG and LF bind better at 50°C and when BLG / LF = 2; and CN and LF bind better at 25°C and when CN / LF = 0.5.

[0035] By measuring the particle size and PDI of ALA-LF, BLG-LF, and CN-LF composites at different temperatures and ratios, their structural characteristics can be understood. The results are as follows: Figure 3 As shown.

[0036] The ALA-LF composite exhibits the largest particle size at 50℃ or an ALA / LF ratio of 1.5, forming micron-sized particles and demonstrating strong self-aggregation ability. In contrast, the composites under other conditions are nanoparticles. The BLG-LF composite shows the largest particle size at 50℃ and a BLG / LF ratio of 2, forming micron-sized particles, while under other conditions, it is mostly nanoparticles. For the CN-LF composite, the largest particle size is achieved at 62.5℃ or a CN / LF ratio of 0.5.

[0037] The polydispersity index (PDI) is a key parameter for assessing the uniformity of particle distribution. A lower PDI value indicates a more uniform particle distribution, while a higher value indicates greater particle size variation. A lower PDI value suggests a more concentrated particle size distribution in the complex, resulting in better dispersibility and stability. Under the above conditions, the three dual-protein complexes generally exhibited low PDI values, indicating that the formed aggregates were relatively uniform and had good dispersibility. Choosing a larger particle size is intended to improve the self-aggregation ability of the complex, enhancing its stability and survival rate in the gastrointestinal tract. Larger particles may better protect the encapsulated Bifidobacteria, improving their resistance to pressure in the acidic gastric environment, thereby increasing the gastrointestinal survival rate of the microcapsules.

[0038] Fluorescence spectroscopy is a highly sensitive technique for studying protein interactions and conformational changes. Its fluorescence characteristics are mainly related to tryptophan and tyrosine residues, providing insights into protein conformation, molecular environment, and binding behavior. When proteins bind, a decrease in fluorescence intensity usually indicates that the fluorophore is embedded due to steric hindrance or conformational changes, thus weakening the solvent quenching effect; while a redshift of characteristic peaks indicates that hydrophobic groups are exposed to a polar environment, at which point the peptide chain extends. By analyzing the spectral changes at different temperatures and ratios, the binding state and structural stability of protein complexes can be further revealed. Figure 4 These are fluorescence spectra of the dual-protein complex at different temperatures and ratios. For the ALA-LF complex, compared to the LF complex, the fluorescence intensity decreased at all temperatures and ratios. This may be due to the conformational change of the two proteins after interaction, resulting in a more tightly packed fluorophore group (such as tryptophan) and reduced contact with fluorescence quenching substances in the solvent, thus decreasing fluorescence intensity. A significant red shift of the peaks was observed at temperatures of 62.5℃, 75℃, and 100℃. At these temperatures, the hydrophobicity of the environment decreased, and the peptide chain extension increased. This may be because excessively high temperatures cause the protein structure to extend, exposing the internal active groups. The fluorescence intensity of the ALA-LF complex decreased significantly at 25℃ and 50℃, or at ALA / LF = 1.5 or 2, suggesting that the binding effect of the two substances was better at these temperatures.

[0039] For the BLG-LF complex, compared to the monomer, the fluorescence intensity of the complex at 25℃ and 50℃ was significantly reduced. At temperatures of 62.5℃, 75℃, and 100℃, the peak of the complex showed a red shift. The BLG-LF complex at different ratios all exhibited reduced fluorescence intensity, with a significant decrease in fluorescence intensity at BLG / LF=2, indicating better binding at this point.

[0040] For the CN-LF complex, compared with LF, the fluorescence intensity of the complex at 25℃ and 50℃ was significantly reduced. When the temperature of the complex was 62.5℃, 75℃, and 100℃, a significant red shift of the peak was observed. The CN-LF complex at different ratios all showed a decrease in fluorescence intensity, and the fluorescence intensity of the curves at CN / LF=1.5 or 2 was significantly reduced, suggesting a stronger binding affinity at these ratios.

[0041] Circular dichroism (CD) is a common method for investigating the secondary structure of proteins by measuring the difference in absorption of left- and right-circularly polarized light. A decrease in ellipticity usually indicates a change in protein structure, possibly due to structural rearrangement caused by electrostatic interactions or conformational changes during binding. An increase in α-helices and β-sheets generally indicates a more ordered and stable protein structure, while a decrease in random coils further supports the high structural stability of the complex under these conditions. Figure 5 These are circular dichroism chromatograms of the complex at different temperatures and ratios. Compared to ALA, the ellipticity of the complex peak is significantly reduced, indicating that electrostatic interactions may have led to a transformation in the secondary structure of the complex. The reduction in ellipticity is more pronounced at temperatures of 25°C and 50°C for the ALA-LF complex, suggesting better binding at these temperatures. Compared to ALA, the ellipticity of the complex peak is significantly reduced at all ratios. This may indicate that electrostatic interactions have caused a transformation in the secondary structure of the complex. The secondary structure of the complex may be more stable at ALA / LF ratios of 0.33 or 0.5. Compared to BLG, the ellipticity of the complex peak is significantly reduced, which may indicate that electrostatic interactions have caused a transformation in the secondary structure of the complex. The reduction in ellipticity is more pronounced at temperatures of 25°C and 50°C for the BLG-LF complex, suggesting better binding at these temperatures. Compared to BLG, the ellipticity of the complex peak is significantly reduced at all ratios, which may indicate that electrostatic interactions have caused a transformation in the secondary structure of the complex. When BLG / LF = 0.33 or 0.5, the secondary structure of the complex may be more stable. Compared to CN, the ellipticity of the complex peak is significantly reduced, which may indicate that electrostatic interactions have altered the secondary structure of the complex. The reduction in ellipticity of the CN-LF complex peak is more significant at temperatures of 25°C and 50°C, suggesting that the two components may bind better at these temperatures. Compared to CN, the ellipticity of the complex peak is reduced at all ratios, which may indicate that electrostatic interactions have altered the secondary structure of the complex. When CN / LF = 0.5 or 1, the ellipticity of the complex peak is significantly reduced, suggesting that the binding effect may be better at these ratios.

[0042] Example 4 Bifidobacterium animalis F1-7 ( Bifidobacterium animalis , Bif F1-7 (accession number: CCTCC M 2020833) is a laboratory-preserved strain.

[0043] S1. Activation and culture of Bifidobacterium animalis strain F1-7 in MRS broth, 4000 ×gCentrifuge for 10 min to collect the precipitate, wash twice with physiological saline, and resuspend in protein solution to obtain an effective viable count of 10-1. 10 CFU / mL Bifidobacterium animalis F1-7 bacterial suspension; S2. Take 3 mL of each of the following complexes prepared in Examples 1-3 (ALA-LF complex (temperature 50℃, ALA / LF=1.5), BLG-LF complex (temperature 50℃, BLG / LF=2), CN-LF complex (temperature 25℃, CN / LF=0.5), ALA-LF complex (temperature 25℃, ALA / LF=0.5), BLG-LF complex (temperature 75℃, BLG / LF=1.5), and CN-LF complex (temperature 75℃, CN / LF=1) and mix them with 1 mL of 2% sodium alginate to obtain a dual-protein complex and a sodium alginate complex solution. S3. Mix 1 mL of bacterial suspension with 4 mL of dual protein complex and sodium alginate complex solution, add 750 µL of 1.5% CaCO3 suspension, stir well to obtain a suspension; S4. Add this suspension to soybean oil (containing 0.5% Tween 80) at a water-to-oil ratio of 5:12, mechanically stir to form a W / O emulsion, add 40 µL of glacial acetic acid after 15 min, continue stirring for 30 min, add 20 mL of 2% CaCl2 solution, and stir for 10 min. S5. Remove the oil phase to obtain gel-formed microcapsules, wash with physiological saline until no oil phase residue remains, centrifuge to collect the double protein microcapsules encapsulating probiotics, and store at 4°C.

[0044] The microcapsules obtained from the ALA-LF complex (at a temperature of 50℃, ALA / LF=1.5) are abbreviated as A50-1.5+F1-7; The microcapsules obtained from the BLG-LF complex (at a temperature of 50℃, BLG / LF=2) are abbreviated as B50-2+F1-7; The microcapsules obtained from the CN-LF complex (at a temperature of 25℃, CN / LF=0.5) are abbreviated as C25-0.5+F1-7; The microcapsules obtained from the ALA-LF complex (at a temperature of 25℃, ALA / LF=0.5) are abbreviated as A25-0.5+F1-7; The microcapsules obtained from the BLG-LF complex (at a temperature of 75℃, BLG / LF=1.5) are abbreviated as B75-1.5+F1-7; The microcapsules obtained from the CN-LF complex (at a temperature of 75℃, CN / LF=1) are abbreviated as C75-1.0+F1-7.

[0045] Comparative Example 1 The preparation method of LF+F1-7 microcapsules is as follows: Bifidobacterium animalis F1-7 ( Bifidobacterium animalis , Bif F1-7 (accession number: CCTCC M 2020833) is a laboratory-preserved strain.

[0046] S1. Activation and culture of Bifidobacterium animalis strain F1-7 in MRS broth, 4000 ×g Centrifuge for 10 min to collect the precipitate, wash twice with physiological saline, and resuspend in protein solution to obtain an effective viable count of 10-1. 10 CFU / mL Bifidobacterium animalis F1-7 bacterial suspension; S2. Take 3 mL of 1 mg / mL LF solution and mix it with 1 mL of 2% sodium alginate to obtain a composite solution of LF and sodium alginate. S3. Mix 1 mL of bacterial suspension with 4 mL of LF and sodium alginate composite solution, add 750 µL of 1.5% CaCO3 suspension, stir well to obtain a suspension; S4. Add this suspension to soybean oil (containing 0.5% Tween 80) at a water-to-oil ratio of 5:12, mechanically stir to form a W / O emulsion, add 40 µL of glacial acetic acid after 15 min, continue stirring for 30 min, add 20 mL of 2% CaCl2 solution, and stir for 10 min. S5. Remove the oil phase to obtain gel-formed microcapsules. Wash with physiological saline until no oil phase residue remains. Centrifuge to collect the lactoferrin microcapsules encapsulating probiotics and store them at 4°C.

[0047] Comparative Example 2 The preparation method of ALA+F1-7 microcapsules is as follows: Bifidobacterium animalis F1-7 ( Bifidobacterium animalis , Bif F1-7 (accession number: CCTCC M 2020833) is a laboratory-preserved strain.

[0048] S1. Activation and culture of Bifidobacterium animalis strain F1-7 in MRS broth, 4000 ×g Centrifuge for 10 min to collect the precipitate, wash twice with physiological saline, and resuspend in protein solution to obtain an effective viable count of 10-1. 10 CFU / mL Bifidobacterium animalis F1-7 bacterial suspension; S2. Take 3 mL of 1 mg / mL ALA solution and mix it with 1 mL of 2% sodium alginate to obtain a composite solution of ALA and sodium alginate. S3. Mix 1 mL of bacterial suspension with 4 mL of ALA and sodium alginate composite solution, add 750 µL of 1.5% CaCO3 suspension, stir well to obtain a suspension; S4. Add this suspension to soybean oil (containing 0.5% Tween 80) at a water-to-oil ratio of 5:12, mechanically stir to form a W / O emulsion, add 40 µL of glacial acetic acid after 15 min, continue stirring for 30 min, add 20 mL of 2% CaCl2 solution, and stir for 10 min. S5. Remove the oil phase to obtain gel-formed microcapsules, wash with physiological saline until no oil phase residue remains, centrifuge to collect the α-lactalbumin microcapsules encapsulating probiotics, and store at 4°C.

[0049] Comparative Example 3 The preparation method of BLG+F1-7 microcapsules is as follows: Bifidobacterium animalis F1-7 ( Bifidobacterium animalis , Bif F1-7 (accession number: CCTCC M 2020833) is a laboratory-preserved strain.

[0050] S1. Activation and culture of Bifidobacterium animalis strain F1-7 in MRS broth, 4000 ×g Centrifuge for 10 min to collect the precipitate, wash twice with physiological saline, and resuspend in protein solution to obtain an effective viable count of 10-1. 10 CFU / mL Bifidobacterium animalis F1-7 bacterial suspension; S2. Take 3 mL of 1 mg / mL BLG solution and mix it with 1 mL of 2% sodium alginate to obtain a BLG and sodium alginate composite solution; S3. Mix 1 mL of bacterial suspension with 4 mL of BLG and sodium alginate composite solution, add 750 µL of 1.5% CaCO3 suspension, stir well to obtain a suspension; S4. Add this suspension to soybean oil (containing 0.5% Tween 80) at a water-to-oil ratio of 5:12, mechanically stir to form a W / O emulsion, add 40 µL of glacial acetic acid after 15 min, continue stirring for 30 min, add 20 mL of 2% CaCl2 solution, and stir for 10 min. S5. Remove the oil phase to obtain gel-formed microcapsules. Wash with physiological saline until no oil phase residue remains. Centrifuge to collect the β-lactoglobulin microcapsules encapsulating probiotics and store at 4°C.

[0051] Comparative Example 4 The preparation method of CN+F1-7 microcapsules is as follows: Bifidobacterium animalis F1-7 ( Bifidobacterium animalis , Bif F1-7 (accession number: CCTCC M 2020833) is a laboratory-preserved strain.

[0052] S1. Activation and culture of Bifidobacterium animalis strain F1-7 in MRS broth, 4000 ×g Centrifuge for 10 min to collect the precipitate, wash twice with physiological saline, and resuspend in protein solution to obtain an effective viable count of 10-1. 10 CFU / mL Bifidobacterium animalis F1-7 bacterial suspension; S2. Take 3 mL of 1 mg / mL CN solution and mix it with 1 mL of 2% sodium alginate to obtain a CN and sodium alginate composite solution; S3. Mix 1 mL of bacterial suspension with 4 mL of CN and sodium alginate composite solution, add 750 µL of 1.5% CaCO3 suspension, stir well to obtain a suspension; S4. Add this suspension to soybean oil (containing 0.5% Tween 80) at a water-to-oil ratio of 5:12, mechanically stir to form a W / O emulsion, add 40 µL of glacial acetic acid after 15 min, continue stirring for 30 min, add 20 mL of 2% CaCl2 solution, and stir for 10 min. S5. Remove the oil phase to obtain gel-formed microcapsules, wash with physiological saline until no oil phase residue remains, centrifuge to collect casein microcapsules encapsulating probiotics, and store at 4°C.

[0053] Performance testing: (4) Characterization of microcapsules (4.1) Size distribution of microcapsules A small amount of uniformly dispersed microcapsule solution was taken and added dropwise to the sample cell. Distilled water was used as the dispersion. The sample was measured using a Mastersizer 2000 laser particle size analyzer (Malvern Instruments, UK). The laser intensity was washed to above 75%. The laser shading was kept at 4%-6% when the sample was added. D [4, 3] was used as the main particle size index.

[0054] (4.2) Differential Scanning Calorimetry (DSC) Measurement Thermodynamic analysis was performed using a DSC 300 system (Netzsch, Germany). 5-15 mg of the lyophilized microcapsule powder was weighed and placed in a DSC crucible at atmospheric pressure. The crucible lid was compacted and perforated, then placed on the autosampler. The sample temperature was set to 30-200℃, the heating rate to 15℃ / min, and the cooling rate to 30℃ / min. The nitrogen gas flow was adjusted.

[0055] (4.3) X-ray diffraction (XRD) measurement The crystal properties of the samples were determined using an X-ray diffractometer (D8 Advance, Bruker, Germany). A certain amount of powder sample was placed in the sample trough of a special support plate and compacted with a glass plate at room temperature. The measurement conditions were as follows: Cu-k α Ray (λ=1.5418, 40 kV and 40 mA), scanning angle 5° ≤ 2θ ≤ 75°, scanning speed 8° / min, step size 0.02°.

[0056] (4.4) Scanning electron microscopy (SEM) observation The morphology of the lyophilized microcapsules was observed using scanning electron microscopy. The microcapsules were fixed to the sample stage using conductive tape, vertically sputtered with gold under vacuum, and then characterized using SEM at an accelerating voltage of 10 kV, with magnifications of 50x and 200x.

[0057] (5) In vitro digestion simulation Based on the standardized in vitro digestion simulation method proposed by M. Minekus et al., simulated saliva (SSF), simulated gastric juice (SGF), and simulated intestinal juice (SIF) were prepared according to the components and proportions in Table 1. All solutions were filtered using a 0.22 μm filter.

[0058] Table 1. Composition of simulated digestive fluid

[0059] Take 1 mL of sample and add 0.7 mL SSF, 0.1 mL α-amylase (1500 U / mL), 5 μL CaCl2 solution (0.3 M), and 195 μL deionized water sequentially. Incubate at 37 °C for 2 min. Then, add 1.5 mL SGF, 0.32 mL pepsin (1500 U / mL), 1 μL CaCl2 solution (0.3 M), and 139 μL deionized water sequentially to the system, and continue incubation under the same conditions for 2 h. After the reaction is complete, discard the reaction solution, and then add 2.2 mL SIF, 1 mL trypsin (800 U / mL), 8 μL CaCl2 solution (0.3 M), and 262 μL deionized water sequentially, and incubate at 37 °C for 2 h. At 0 h, 1 h, 2 h, 4 h, 5 h, and 6 h after the start of the oral-gastro-intestinal reaction, 100 μL of the solution was added to sterile PBS solution, and the viable bacteria count was performed by plate plating. Uncoated Bifidobacteria were used as a control.

[0060] (6) Storage stability The microcapsule samples were stored at 4°C for 28 days. Viable bacterial counts were calculated every 7 days using the dilution plating method to evaluate their storage stability. Uncoated Bifidobacterium animalis was used as a control. The survival rate of Bifidobacterium during storage was calculated using the following formula:

[0061] In the formula, N This represents the number of viable bacteria (log CFU / g) after a period of storage. N 0 This represents the original number of viable bacteria before storage (log CFU / g).

[0062] (7) Data Analysis Data are expressed as “Mean ± SD”. Graphs were created using Origin 2019 and Graphpad Prism 6.01 software. Statistical analysis was performed using IBM SPSS Statistics 19 and Excel 2016 software. Comparative analyses, such as one-way ANOVA, were also used. p <0.05 indicates that there is a significant difference between the data.

[0063] DSC and XRD analysis of microcapsules DSC reflects the thermal stability of a material by measuring the energy changes during the endothermic / exothermic process of a sample. Figure 7 As shown.

[0064] from Figure 7As shown in Figure A, the peak temperatures (Tm) of LF, ALA, and BLG are relatively high, possibly due to the high content of disulfide bonds, α-helices, and β-sheets within them, which endow them with high thermal stability. However, casein has a relatively low Tm, possibly because its random coil conformation leads to insufficient stability. But the Tm of Bifidobacterium microcapsules encapsulated with ALA-LF is lower, possibly due to the steric hindrance of the α-helices of LF and ALA, resulting in a loose complex structure. However, A50-1.5 has a higher endothermic peak than A25-0.5, possibly indicating higher stability. The peak temperature of microcapsules encapsulated with BLG-LF is higher, possibly because the α-helices of LF and the β-sheets of BLG complement each other through hydrophobic interactions, forming a more stable cross-linked network. The Tm of microcapsules encapsulated with CN-LF is significantly increased, possibly because the random coils of CN fill the rigid structural voids of LF, forming a dense complex and significantly improving its stability. C25-0.5 has a more significant endothermic peak than C75-1, indicating that its thermal stability may be stronger.

[0065] Generally, crystalline compounds exhibit diffraction patterns consisting of clear, narrow, and sharp peaks, while amorphous compounds give diffraction patterns composed of noise signals and diffuse peaks. For example... Figure 7 As shown in Figure B, different types of microcapsules exhibit the same peak shape. The increase in a series of main sharp diffraction peaks (approximately 27.50°, 31.80°, 45.60°, 56.60°, and 66.30°) indicates improved crystal structure and stability of the samples. The appearance of each diffraction peak likely originates primarily from CaCO3, with a small portion possibly from other crystalline substances. All microcapsules use CaCO3 as a cross-linking agent, forming an "egg-box" structure with sodium alginate (Ca²⁺ bound to carboxylate ions). The crystalline phase of CaCO3 (calcite) dominates the XRD signal, resulting in consistent peak shapes for microcapsules encapsulated by different proteins.

[0066] Morphology of microcapsules The lyophilized microcapsules were observed using SEM, such as... Figure 8 As shown.

[0067] Observations revealed that microcapsules A50-1.5 were regular spherical with smooth surfaces and showed no significant collapse after freeze-drying, indicating that the ALA-LF complex may form a rigid network through α-helical crosslinking, effectively resisting ice crystal stress and exhibiting structural stability suitable for long-term storage. Magnification revealed uniform nanopores on the surface, with a relatively dense surface layer, which is related to the structure of ALA, possibly due to its hydrophobic core (containing four disulfide bonds) inhibiting excessive pore expansion. Microcapsules B50-2 exhibited slight ellipsoidal deformation and significantly higher surface roughness than A50-1.5. This may be because the β-sheet structure of BLG partially unfolded during freeze-drying, lowering its glass transition temperature. Magnification revealed some cracks on the surface and a honeycomb-like porous structure. This loose structure and numerous pores may lead to premature decapsulation in gastric juice, allowing it to permeate and reducing gastric juice tolerance; however, in intestinal juice, the structure easily disintegrates, resulting in rapid release. The C25-0.5 microcapsule surface is highly wrinkled, likely due to the uneven shrinkage of casein's random coil conformation during freeze-drying. Wrinkled surfaces typically increase surface area, potentially allowing for greater attachment of microorganisms or enzymes, thus promoting fermentation. Magnification reveals numerous surface pores, likely due to casein's flexible structure allowing for the formation of large ice crystals. This loose structure facilitates substrate entry, making it easier for bacteria to decompose them and generate SCFAs. Furthermore, its surface is covered with many nanoparticles, possibly because certain sites on the CN layer, after phosphorylation modification, more readily attract Ca²⁺, promoting the formation of microcrystals. These particles may act as attachment sites, aiding in probiotic colonization, and this structure may increase SCFA production. During freeze-drying, protein secondary structures (α-helices / β-sheets / random coils) significantly influence the morphology and function of microcapsules by regulating ice crystal formation and interfacial stability. The rigid network of ALA-LF is suitable for long-term storage, while the porous structure of CN-LF is more conducive to targeted release.

[0068] Survival and release under simulated digestion conditions Simulating the oral-gastric-intestinal system in an in vitro environment, exposing the naked... Bif F1-7 and each microcapsule were reacted in an oral environment for 2 min, in gastric juice for 2 h, and in intestinal juice for 4 h. The viable bacteria count was observed on a plate every 1 h to test the microcapsules' resistance to gastric juice corrosion and their enteric solubility.

[0069] Depend on Figure 9 It can be seen that, whether Bif.Both F1-7 and the microcapsules exhibited the same trend: upon entering the stomach, the viable bacterial count decreased, reaching its lowest point at 2 hours, but still remaining above 6.1 log CFU / g; subsequently, upon entering the intestines, the microcapsules slowly released bacteria, resulting in a significant rebound in the viable bacterial count, which then stabilized at approximately 8 log CFU / g, demonstrating the microcapsules' excellent acid resistance and enteric solubility. This may be due to the protonation of the carboxylic acid groups (-COO⁻) of sodium alginate in gastric juice, forming a gel network that slows gastric acid penetration; while in intestinal juice, the carboxylic acid groups of sodium alginate deprotonate (-COO⁻), dissolving the gel network and releasing the encapsulated bacteria, and trypsin further disrupting the protein complex, accelerating bacterial release. Furthermore, the microcapsules encapsulated by ALA-LF and BLG-LF showed excellent resistance to gastric acid corrosion, with viable bacterial counts reaching approximately 7.5 log CFU / g or even higher. This may be because the α-helix of LF and the secondary structure (α-helix / β-sheet) of ALA / BLG form a tight interface through hydrophobic-hydrophilic balance, resisting gastric acid corrosion, and the disulfide bonds also enhance the stability of the complex. The CN-LF-encapsulated microcapsules exhibit a faster release rate in intestinal fluid, presumably because the random coils of CN fill the gaps in the rigid structure of LF, forming a porous network that allows for rapid dissolution in intestinal fluid.

[0070] Storage stability of encapsulated probiotics Exposed Bif. F1-7 and its microcapsules were stored at 4°C for 28 days, and viable bacterial counts were measured every 7 days using plate swabs. Figure 10 It can be seen that the viable cell count did not change significantly in the first 14 days. Until day 21, the viable cell counts decreased to varying degrees, with the smallest decrease observed in the microcapsules encapsulated with BLG-LF, where the viable cell count remained at 9.18 log CFU / g, significantly higher than that of the unencapsulated microcapsules. Bif. F1-7 (7.91 log CFU / g). By day 28, the change in viable cell count was more pronounced, with the viable cell counts of microcapsules encapsulated by BLG-LF and CN-LF being significantly higher than those of unencapsulated microcapsules. Bif. The viable bacterial count of the F1-7, BLG-LF encapsulated microcapsules remained at 6.27 log CFU / g, indicating that the microcapsules have good storage stability and can achieve the purpose of protecting Bifidobacteria.

[0071] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A dual-protein microcapsule, characterized in that, The dual-protein microcapsules are prepared from a dual-protein complex and sodium alginate; the dual-protein complex is a lactoferrin-α-lactalbumin complex, a lactoferrin-β-lactoglobulin complex, or a lactoferrin-casein complex.

2. The dual-protein microcapsule according to claim 1, characterized in that, The preparation method of the lactoferrin-α-lactalbumin complex is as follows: weigh lactoferrin and α-lactalbumin powders separately, dissolve them in distilled water to prepare 1 mg / mL lactoferrin solution and α-lactalbumin solution, and stir at room temperature for 2-2.5 h; mix the α-lactalbumin solution and lactoferrin solution at a molar ratio of 1.5-2.0, and incubate in a water bath at 45-55℃ for 30 min to obtain the final product.

3. The dual-protein microcapsule according to claim 1, characterized in that, The preparation method of the lactoferrin-β-lactoglobulin complex is as follows: weigh lactoferrin and β-lactoglobulin powders separately, dissolve them in distilled water to prepare 1 mg / mL β-lactoglobulin solution and lactoferrin solution, and stir at room temperature for 2-2.5 h; mix the β-lactoglobulin solution and lactoferrin solution at a molar ratio of 2.0, and incubate in a water bath at 45-55℃ for 30 min to obtain the final product.

4. The dual-protein microcapsule according to claim 1, characterized in that, The preparation method of the lactoferrin-casein complex is as follows: Weigh lactoferrin powder and dissolve it in distilled water to prepare a 1 mg / mL lactoferrin solution, and stir at room temperature for 2-2.5 h; weigh casein powder into a mortar, moisten it with a small amount of distilled water, slowly add 0.2 mol / L NaOH, grind it thoroughly, boil it in a water bath for 15 min, cool it and make up the volume to obtain a casein solution; mix the casein solution and the lactoferrin solution at a molar ratio of 0.4-0.6, and bathe them in a water bath at 20-30℃ for 30 min to obtain the final product.

5. The application of the dual-protein microcapsule according to claim 1 in the delivery of probiotics.

6. The application according to claim 5, characterized in that, The application method is as follows: S1. Take Bifidobacterium animalis, activate and culture it in MRS broth, 4000 ×g Centrifuge for 10 min to collect the precipitate, wash twice with physiological saline, and resuspend in protein solution to obtain probiotic suspension; S2. Mix the probiotic suspension with the dual protein complex and sodium alginate complex solution, add CaCO3, stir evenly to obtain a suspension; S3. Add the suspension to soybean oil, mechanically stir for 13-18 min, add glacial acetic acid, continue stirring for 30 min, add CaCl2 solution, and stir for 10 min; S4. Remove the oil phase to obtain gel-formed microcapsules, wash with physiological saline until no oil phase residue remains, and collect by centrifugation to obtain double protein microcapsules encapsulating probiotics.

7. The application according to claim 6, characterized in that, The number of viable bacteria in the probiotic suspension in S1 is 10. 8 -10 10 CFU / mL.

8. The application according to claim 6, characterized in that, The volume ratio of the probiotic suspension to the compound solution of the dual protein complex and sodium alginate in S2 is 1:4; the concentration of the dual protein complex in the compound solution of the dual protein complex and sodium alginate is 1.0-1.5 mg / mL; the concentration of sodium alginate is 1.5-2.5%; the concentration of CaCO3 solution is 1.5%, and the amount added is 75% of the probiotic suspension.

9. The application according to claim 6, characterized in that, The volume ratio of the suspension to soybean oil in S3 is 5:12; the soybean oil contains 0.5% Tween 80; the amount of glacial acetic acid added is 0.2-0.25% of the volume of the W / O emulsion; the concentration of CaCl2 solution is 2%, and the amount added is 115-120% of the volume of the W / O emulsion.

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