Preparation method of ariovum peptide microcapsule and ariovum peptide microcapsule

Abstract

The application discloses a preparation method of a fish-roe-derived peptide microcapsule and the fish-roe-derived peptide microcapsule, and belongs to the technical field of food biotechnology and microcapsule technology. The double-layer microcapsule prepared by the application has high embedding rate, low water content, good thermal stability, and the hygroscopicity of the microcapsule is reduced by about 38% compared with that of free peptides, the microcapsule has excellent gastric acid tolerance and intestinal slow-release characteristics, and can effectively shield the fishy smell of fish. The preparation process of the application is simple and mild, the prepared microcapsule product has good stability and excellent slow-release performance, and the application provides a reference for industrial application of the fish-roe-derived peptide microcapsule, and can be widely applied in the fields of functional food and health-care products.

Description

Technical Field

[0001] This invention belongs to the field of food biotechnology and microcapsule technology, specifically relating to a method for preparing shad egg-derived peptide microcapsules and the shad egg-derived peptide microcapsules thereof. Background Technology

[0002] Shad roe, a byproduct of aquatic product processing, is rich in high-quality protein, with crude protein and crude fat content of 5.33% and 1.14%, respectively. The total amino acid content and essential amino acid content of dried roe samples are 61.41% and 27.05%, respectively. The abundance of essential amino acids makes it a high-quality raw material for preparing bioactive peptides. However, bioactive peptides are difficult to store long-term under normal storage conditions due to their molecular structure, and they are easily degraded by gastric acid and digestive enzymes in the complex environment of the gastrointestinal tract, resulting in significantly reduced bioavailability and posing a serious challenge to their practical application.

[0003] Microencapsulation technology, as a highly efficient stabilization method, can encapsulate solid, liquid, or gaseous core materials within tiny, closed capsules. The physical barrier effect of the wall material isolates the capsules from adverse external factors, while also masking undesirable flavors and controlling release. Sodium alginate, a natural polysaccharide, is frequently used as a microcapsule wall material due to its excellent biocompatibility, gel properties, and pH responsiveness; it can crosslink with calcium ions to form a stable gel network. Chitosan, another natural cationic polysaccharide, can form a polyelectrolyte composite membrane with sodium alginate through electrostatic interactions, further enhancing the structural density and environmental responsiveness of the microcapsules, providing a feasible pathway for the stabilization of bioactive peptides.

[0004] Currently, there are no reports on the preparation of shad roe peptide bilayer microcapsules using sodium alginate (SA) and chitosan (CS) as wall materials via a sharp-pore coagulation bath method. Therefore, developing a shad roe peptide microcapsule product with high encapsulation efficiency, good stability, sustained-release function, and the ability to mask unpleasant odors is of great significance for improving the high-value utilization of shad roe by-products and expanding their application in functional foods. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to solve the technical problems of poor stability, easy degradation, low bioavailability and strong fishy smell of shad egg-derived peptide (ASEP) in storage and gastrointestinal environment, and to provide a shad egg-derived peptide microcapsule with high encapsulation rate, excellent physicochemical properties, sustained release function and antioxidant activity and its preparation method.

[0006] Technical solution: To solve the above technical problems, the present invention provides a method for preparing shad roe peptide microcapsules. The method for preparing shad roe peptide microcapsules uses shad roe peptide as core material and sodium alginate as wall material, and prepares single-layer microcapsules using a sharp-pore coagulation bath method, and further constructs double-layer microcapsules using chitosan as secondary wall material.

[0007] The preparation method of the shad egg-derived peptide microcapsules specifically includes the following steps:

[0008] (1) Sharp-pore-coagulation bath method: Add shad egg peptide freeze-dried powder to sodium alginate solution, then add Tween 80 and stir to obtain core wall mixture; then use a syringe to drop the core wall mixture into the coagulation bath above the prepared CaCl2 coagulation bath, and after solidification, filter and wash to obtain sodium alginate monolayer microcapsules.

[0009] (2) The sodium alginate monolayer microcapsules obtained in step (1) are immersed in chitosan solution and gently stirred to form a second membrane. After filtration and washing, sodium alginate-chitosan bilayer shad egg peptide microcapsules are obtained. After vacuum freeze-drying, peptide-loaded dry microspheres are obtained.

[0010] In step (1), the mass ratio of shad egg peptide to sodium alginate is 1:3.

[0011] The sodium alginate solution in step (1) has a mass fraction of 1.0~3.0% (g / g).

[0012] In step (1), the mass fraction of Tween 80 is 0.5-2.5% (g / g), and the mass fraction of CaCl2 is 1.0-5.0% (g / mL).

[0013] In step (1), the curing time is 10~50 min.

[0014] The concentration of the chitosan solution in step (2) is 1% (g / g).

[0015] In step (2), the chitosan solution is obtained by dissolving a 1% chitosan solution in a 1% acetic acid solution.

[0016] The present invention also includes shad egg-derived peptide microcapsules prepared by the aforementioned preparation method.

[0017] The free shad egg-derived peptide spectrum of the microcapsules exhibits typical protein and polypeptide characteristic absorption bands. The amide I band mainly originates from the C=O stretching vibration, with the peak position concentrated at 1640 cm⁻¹. -1 Nearby; the amide II band is generated by the coupling of NH bending vibration and CN stretching vibration, with a peak position of 1540 cm⁻¹. -1The peak position of amide III is 1270 cm⁻¹. -1 .

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention uses shad roe peptide as the core material and sodium alginate and chitosan as the wall material. Based on the single-layer microcapsules prepared by the sharp-pore-coagulation bath method, a double-layer structure of shad roe peptide microcapsules is prepared by using chitosan as the secondary wall material, which greatly improves the stability and sustained-release performance of shad roe peptide. The preparation process of the present invention is simple, the encapsulation effect is good, the antioxidant activity retention rate is high, and the fishy smell of fish eggs can be improved. Attached Figure Description

[0019] Figure 1 The graph shows the effect of sodium alginate mass fraction on microcapsule encapsulation efficiency.

[0020] Figure 2 The graph shows the effect of CaCl2 mass fraction on the microcapsule encapsulation efficiency.

[0021] Figure 3 The graph shows the effect of curing time on the microcapsule encapsulation efficiency.

[0022] Figure 4 The graph shows the effect of Tween 80 mass fraction on the microcapsule encapsulation efficiency.

[0023] Figure 5 The images show the morphology of single-layer and double-layer microcapsules before and after drying.

[0024] Figure 6 SEM images of single-layer and double-layer microcapsules;

[0025] Figure 7 A comparison diagram of the hygroscopic properties of bilayer microcapsules and shad egg-derived peptides;

[0026] Figure 8 A comparison of the thermal stability of bilayer microcapsules and shad egg-derived peptides;

[0027] Figure 9 Fourier transform infrared spectra of shad egg-derived peptides, monolayer and bilayer microcapsules;

[0028] Figure 10 A comparative diagram showing the in vitro sustained-release properties of shad egg-derived peptides, monolayer and bilayer microcapsules;

[0029] Figure 11 This is an analysis diagram of the electronic nose containing bilayer microcapsules and shad egg-derived peptides. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0031] The freeze-dried shad roe peptide powder of the present invention was prepared according to the method of Example 18 in Chinese patent application with publication number CN120366415A, entitled "A Green Preparation Method of Shad Roe Peptide and Shad Roe Peptide and Its Application".

[0032] Preparation of shad roe peptide solutions: 2.0, 4.0, 6.0, 8.0, and 10.0 mg / mL of lyophilized shad roe peptide powder were dissolved in deionized water to prepare shad roe peptide solutions of different concentrations.

[0033] The evaluation metrics and structural characterization methods used in the following examples are as follows:

[0034] (1) Determination of embedding rate

[0035] A: Determination of peptide content in microcapsules

[0036] ① Biuret reagent: Dissolve 1.5g CuSO4 and 6g C4H4KNaO6·4H2O in 500 mL of distilled water, add 300 mL of NaOH (10%, g / mL) solution and mix, then bring the volume to 1 L. Prepare and use immediately.

[0037] ② Preparation of standard curve: Prepare a series of shad roe peptide solutions with concentration gradients (0.0, 2.0, 4.0, 6.0, 8.0, 10.0 mg / mL). Take 1 mL of each solution, mix it with 4 mL of biuret reagent, shake well, and let it stand for 30 min. Measure the absorbance at 540 nm. Repeat each experiment three times. Plot a standard curve with the concentration of shad roe peptide solution on the x-axis and the net absorbance on the y-axis.

[0038] ③ Sample determination: Take 0.1g of ground shad egg-derived peptide microcapsule powder, add 20 mL of 0.1mol / L sodium citrate solution, and magnetically stir for 30 min to completely release the peptides in the microcapsules. Then centrifuge (4℃, 9000 r / min, 10 min), take 2 mL of supernatant, add 2 mL of (10%, g / mL) trichloroacetic acid solution, let stand for 10 min, and centrifuge again (4℃, 9000 r / min, 10 min). Take 1 mL of supernatant, mix with 4 mL of biuret reagent, let stand for 30 min, and measure its absorbance at 540 nm using an ELISA reader. Each group of experiments is repeated three times, and the average value is substituted into the standard curve equation to obtain the sample concentration.

[0039] B: Determination of microcapsule encapsulation efficiency

[0040] The peptide concentration of the sample was determined according to method A in (1) for determining the encapsulation efficiency, with the blank microcapsule group without core material (SA-CS) as a control. The encapsulation efficiency was calculated as follows:

[0041]

[0042] In the formula: M0(g) is the actual mass of the encapsulated shad egg peptide; M1(g) is the initial mass of the added shad egg peptide.

[0043] (2) SEM analysis

[0044] Dry sodium alginate-chitosan bilayer shad egg peptide microcapsules (ASEP-SA-CS) were evenly spread on the sample stage. Blank microcapsules (SA-CS) without core material were used as a control. Excess powder was blown off and gold was sprayed on. The accelerating voltage was 10 kV. The microstructure of the microcapsules was observed at different magnifications using a scanning electron microscope (SEM).

[0045] (3) Determination of hygroscopicity

[0046] Accurately weigh 1.0 g of sample and place it into a petri dish, using raw shad egg peptide (AESP) and blank microcapsules without core material (SA-CS) as controls. Place in a desiccator containing saturated potassium nitrate (KNO3) solution (75% relative humidity) and equilibrate at room temperature for 7 days. The moisture absorption rate is calculated using the following formula:

[0047]

[0048] In the formula: M(g) is the mass of the sample after absorbing moisture; 1 is the mass of the sample before absorbing moisture (1g).

[0049] (4) Determination of thermal stability

[0050] 1g of shad egg-derived peptides ASEP, ASEP-SA, and ASEP-SA-CS microcapsules were uniformly dispersed in petri dishes and placed in a 100℃ constant temperature oven in the dark for 10 h. A certain amount of sample was taken out every 2 h, and the ASEP content in the microcapsules was determined according to method 4.3.5. The retention rate of ASEP was analyzed. Each group was set up in 3 replicates to compare the effect of temperature on the stability of shad egg-derived peptide microcapsules.

[0051]

[0052] In the formula: m0(g) is the content of shad ovum peptide in the microcapsules before storage; m(g) is the content of shad ovum peptide in the microcapsules after storage.

[0053] (5) Determination of Fourier Transform Infrared Spectroscopy (FTIR)

[0054] ASEP, blank microcapsules without AESP, ASEP-SA-CS microcapsule powder, and potassium bromide (KBr) were dried and prepared for use. Each sample was ground uniformly with a certain amount of KBr, compressed into pellets, and used for analysis. The spectra of the wall material, core material, and microcapsules were determined using Fourier transform infrared spectroscopy (FTIR). The sample scans were performed 32 times, with a wavenumber range of 400–4000 cm⁻¹. -1 The resolution is 4 cm. -1 .

[0055] (6) Determination of the in vitro sustained-release performance of microcapsules

[0056] Preparation of artificial gastric juice: Weigh 2.0 g of sodium chloride and place it in a beaker. Add a certain amount of ultrapure water and stir until dissolved. Adjust the pH to 1.2 with hydrochloric acid and then add 3.2 g of pepsin. Stir for 10 min and transfer to a 1000 mL volumetric flask. Make up to volume with ultrapure water and shake well.

[0057] Preparation of artificial intestinal fluid: Weigh 6.8 g of potassium dihydrogen phosphate, place it in a beaker, add a certain amount of ultrapure water, stir until dissolved, adjust the pH to 6.8 with 0.2 mol / L NaOH solution, add 10.0 g of trypsin, stir for 10 min, transfer to a 1000 mL volumetric flask, dilute to volume with ultrapure water, and shake well.

[0058] Determination of the cumulative release of microcapsules in simulated gastrointestinal fluid: Weigh 0.1 g of dried microcapsules into an Erlenmeyer flask, add 100 mL of artificial simulated gastric fluid, and then place it in a dark 37°C, 160 rpm constant temperature water bath and shake well. Enzymatic hydrolysis for 2 h, take 1 mL every 1 h, and add an equal amount of simulated solution after each sampling. Inactivate enzyme in an 80°C water bath for 5 min to terminate the reaction, cool in an ice water bath, centrifuge at 8000 × g for 15 min, filter with a 0.45 μm filter membrane, take the filtrate and determine the peptide content according to method (1)A, calculate the release rate, and use blank microcapsules as a control. After the simulated gastric digestion process is completed, adjust the pH of the solution to 6.8, add 23 mL of artificial simulated intestinal fluid, and react for another 3 h under the same conditions. Similarly, take samples every 1 h to determine the peptide content and calculate the release rate. The formula for calculating the release rate is as follows:

[0059]

[0060] In the formula: m4(g) is the total amount of peptides released in simulated gastrointestinal fluid; m5(g) is the total amount of peptides contained in the microcapsules in simulated gastrointestinal fluid.

[0061] (7) Measurement of electronic nose

[0062] The odor of shad egg-derived peptides and microcapsules was determined using a PEN3 electronic nose, which comprises 10 sensors. 1 g of sample was dissolved in 5 mL of water, and 2 mL of the sample was transferred to a sample vial. The vial was incubated in a 50 °C water bath for 10 min before measurement using the electronic nose. Injection parameters: sensor cleaning time 180 s, detection time 120 s, flow rate 300 mL / min. The measured data were combined with radar plots for principal component analysis (PCA).

[0063] Example 1: Preparation of shad egg-derived peptide microcapsules

[0064] (1) Preparation of monolayer microcapsules

[0065] A certain mass of sodium alginate was weighed and dissolved in deionized water, and stirred in a 50℃ water bath for 3 h to ensure complete dissolution and the absence of bubbles. Shad roe peptide lyophilized powder was added to a 2% sodium alginate solution at a core-to-wall ratio of 1:3, followed by 0.5% Tween 80 to obtain a core-to-wall mixture. The mixture was stirred for 30 min to promote microcapsule membrane formation. A 4% CaCl2 solution was prepared and cooled to room temperature to obtain a coagulated solution. The core-to-wall mixture was dropped into the coagulated solution 10 cm above the surface using a 5 mL syringe. After solidification for 30 minutes, the solution was filtered and washed to obtain sodium alginate monolayer microcapsules (ASEP-SA).

[0066] (2) Preparation of bilayer microcapsules

[0067] A 1% chitosan solution was prepared using a 1% acetic acid solution. The sodium alginate monolayer microcapsules (ASEP-SA) obtained in step (1) were immersed in the chitosan solution and gently stirred to form a second membrane. After filtration and washing, sodium alginate-chitosan bilayer shad egg peptide microcapsules (ASEP-SA-CS) were obtained. After pre-freezing at -80℃, vacuum freeze-drying was performed to obtain peptide-loaded dry microspheres.

[0068] Example 2: Single-factor experiment

[0069] Based on the preparation method of Example 1, namely, sodium alginate mass fraction of 1.5%, CaCl2 mass fraction of 2%, curing time of 20 min, and Tween 80 mass fraction of 1%, the effects of each factor on the encapsulation rate of shad egg-derived peptide microcapsules were studied through single-factor experiments.

[0070] With a fixed CaCl2 mass fraction of 2%, a curing time of 20 min, and a Tween 80 mass fraction of 1%, the effects of sodium alginate concentrations (1.0%, 1.5%, 2.0%, 2.5%, and 3.0%) were investigated. Experimental results are shown below. Figure 1The encapsulation efficiency of ASEP-SA microcapsules initially increased and then decreased with increasing sodium alginate concentration, reaching a maximum of (81.84±0.91)% at a sodium alginate concentration of 2.0%. This may be because sodium alginate, as a natural polysaccharide, has encapsulation performance significantly affected by solution concentration. When the sodium alginate mass fraction is below 2.0%, more molecular chains fully extend and form a uniform network structure with increasing concentration, resulting in a denser and more stable microcapsule structure as the wall material, thus gradually increasing the encapsulation efficiency. When the sodium alginate mass fraction exceeds 2.0%, the encapsulation efficiency gradually decreases, possibly due to excessively high sodium alginate concentration leading to excessive molecular chain entanglement and excessively high solution viscosity, resulting in gelation and reduced encapsulation efficiency. On the other hand, sodium alginate concentration also affects the mechanical strength, molding, and appearance of the microspheres. Too low a concentration causes the dried microspheres to collapse; while too high a concentration makes granulation difficult, easily causing clumping and uneven formation. In summary, a sodium alginate mass fraction of 2.0% was selected as the optimal process parameter for further optimization.

[0071] With a fixed sodium alginate mass fraction of 2.0%, a curing time of 20 min, and a Tween 80 mass fraction of 1%, the effect of CaCl2 mass fractions (1.0%, 2.0%, 3.0%, 4.0%, and 5.0%) was investigated. Figure 2 As shown, with the increase of CaCl2 mass fraction, the encapsulation efficiency of ASEP-SA microcapsules exhibits a trend of first increasing and then significantly decreasing, reaching a maximum value of (85.65±1.05)% at 4%. When the CaCl2 mass fraction is below 4%, the encapsulation efficiency gradually increases with increasing CaCl2 mass fraction. This is because calcium chloride acts as a cross-linking agent in the preparation of ASEP-SA microcapsules. With increasing concentration, the cross-linking effect of calcium ions gradually strengthens, which is beneficial to enhancing the density and continuity of the sodium alginate gel network, reducing the leakage of shad roe peptides, and thus improving the encapsulation efficiency. However, when the CaCl2 mass fraction exceeds 4%, the excessively high ionic strength disrupts the molecular dissolution balance of the wall material, and the excessively rapid cross-linking rate causes excessive aggregation and entanglement of molecular chains, resulting in hardening of the microcapsule surface but uneven internal structure and overall instability. This may even further damage the sodium alginate microcapsule structure, leading to leakage of shad roe peptides and a decrease in encapsulation efficiency. Therefore, the optimal CaCl2 mass fraction is 4%.

[0072] With sodium alginate at a constant mass fraction of 2.0%, calcium chloride at 4.0%, and Tween 80 at 1%, the effect of curing time (10, 20, 30, 40, 50 min) was investigated. Figure 3As shown, the encapsulation efficiency of ASEP-SA microcapsules initially increased and then stabilized within a curing time range of 10–50 min, reaching its maximum value of (85.89 ± 1.11)% at a curing time of 30 min. This result indicates that appropriately extending the curing time is beneficial for the full cross-linking of sodium alginate and calcium ions to form a stable gel structure. However, the encapsulation efficiency tends to stabilize after the curing time exceeds 30 min, indicating that the cross-linking reaction is basically complete, and further extending the curing time does not significantly improve the encapsulation effect. Considering actual production, a curing time of 30 min is chosen to balance encapsulation efficiency and process cost. Therefore, 30 min is selected as the optimal curing time for subsequent optimization.

[0073] With sodium alginate mass fraction fixed at 2.0%, calcium chloride mass fraction at 4.0%, and curing time at 30 min, the effect of Tween 80 mass fractions (0.5%, 1.0%, 1.5%, 2.0%, 2.5%) was investigated. Figure 4 As shown, the encapsulation efficiency of ASEP-SA microcapsules initially increased and then decreased with increasing Tween 80 mass fraction, reaching a maximum of (86.04±1.83)% when the Tween 80 mass fraction was 1%. Low concentrations of Tween 80 improved the emulsifying properties of sodium alginate, promoting uniform dispersion of peptides in the system and thus increasing the encapsulation efficiency. The encapsulation efficiency gradually decreased after the concentration exceeded 1.0%, possibly due to excessive surfactant interfering with gel network formation and inhibiting structural stability, thereby reducing the encapsulation efficiency. Therefore, a Tween 80 mass fraction of 1.0% was selected as the optimal concentration for subsequent optimization.

[0074] Example 3: Orthogonal Experimental Design and Results

[0075] Following the method of Example 1, based on the results of single-factor experiments and considering actual production needs, the encapsulation rate was used as the index. The mass fraction of sodium alginate (A), the mass fraction of CaCl2 (B), the curing time (C), and the mass fraction of Tween 80 (D) were selected as the main influencing factors. Considering the interaction effect, an L9 (34) orthogonal experiment with 4 factors and 3 levels was conducted, as shown in Table 1, to determine the optimal process conditions for preparing microcapsules by encapsulating ASEP with sodium alginate.

[0076] Table 1. Factor Levels of Orthogonal Experiment

[0077]

[0078] The orthogonal experimental design and experimental results are shown in Table 2.

[0079] Table 2. Orthogonal experimental design and results

[0080]

[0081] The range analysis in Table 2 shows that the influence of each factor on the encapsulation rate of shad roe peptide microcapsules, from largest to smallest, is: curing time (C) > sodium alginate mass fraction (A) > Tween 80 mass fraction (D) > CaCl2 mass fraction (B). Based on the K-value analysis, the optimal process combination for preparing shad roe peptide microcapsules is A2B2C2D1. Then, an orthogonal experiment was conducted to verify the optimal process conditions. The results showed that the encapsulation rate of ASEP in the microcapsules prepared under these conditions was (88.69±0.43)%, higher than the better combination in the orthogonal experiment, indicating the accuracy of the orthogonal experiment prediction. Therefore, the optimal preparation process parameters for monolayer sodium alginate shad roe peptide microcapsules were finally determined to be: sodium alginate mass fraction 2.0%, calcium chloride mass fraction 4%, curing time 30 min, and Tween 80 mass fraction 0.5%. The encapsulation effect of this invention is relatively good, laying a technological foundation for practical production.

[0082] Example 4 Morphological characterization of the microcapsules prepared in Example 1

[0083] 1. Real photo of microcapsules

[0084] Figure 5 (A) and Figure 5 (C) are actual photos of the ASEP-SA monolayer microcapsules prepared in Example 1 before and after drying; Figure 5 (B) and Figure 5 (D) are actual photos of the ASEP-SA-CS bilayer microcapsules prepared in Example 1 before and after drying. It can be seen that the surfaces of the two types of microcapsules are relatively smooth, uniform in size, without cracks, and spherical or elliptical in shape before drying, with no obvious difference. The ASEP-SA-CS bilayer microcapsules are better shaped after drying, with full, round, white particles and no obvious collapse. However, the ASEP-SA monolayer microcapsules show some collapse after drying, which may be due to the insufficient mechanical strength of the monolayer sodium alginate wall material and the resulting sparse gel network, which leads to the collapse of the microspheres.

[0085] 2. SEM Analysis Results

[0086] like Figure 6 The scanning electron microscope image of the shad egg-derived peptide microcapsules shows that... Figure 6 In (A), the surface of the ASEP-SA monolayer microcapsule is relatively smooth but has a few fine wrinkles, but the structure is intact and there are no obvious cracks or damage. Figure 6In (B), the ASEP-SA-CS bilayer microcapsules formed after secondary chitosan encapsulation still maintain a good spherical morphology, but their surface structure has changed significantly. Compared with monolayer microcapsules, the surface of the bilayer microcapsules becomes denser and rougher, exhibiting a typical layered wrinkle and lamellar texture structure, which is a typical characteristic of the polyelectrolyte composite film formed by the electrostatic interaction between chitosan and sodium alginate. This dense surface structure can increase the thickness and density of the wall material, further enhancing the physical barrier function of the microcapsules.

[0087] Example 5: Performance experiment of the microcapsules prepared in Example 1

[0088] 1. Hygroscopicity of microcapsules and shad egg-derived peptides

[0089] The hygroscopicity of a powder refers to its ability to absorb moisture from its environment, and it is a core indicator for evaluating its stability. For example... Figure 7 As shown, under high humidity (RH: 75%), the hygroscopicity of exposed shad roe peptide powder was significantly higher than that of microencapsulated shad roe peptide. The hygroscopic rate was extremely rapid in the initial exposure phase, then slowed down, reaching equilibrium around day seven with a final equilibrium hygroscopic rate of (63.37±1.20)%. In contrast, after encapsulation with sodium alginate-chitosan microencapsulation, the hygroscopic process became extremely slow, with a cumulative hygroscopic rate of only (24.70±1.12)% after reaching equilibrium on day seven, a reduction of approximately 38% compared to exposed ASEP. This result clearly demonstrates that SA-CS as a wall material can effectively block water molecule penetration. This is because peptides are polymers composed of amino acids linked by amide bonds, and their structure is rich in various strongly polar groups, exhibiting a strong interaction with water molecules. Their hygroscopic process follows typical porous media adsorption rules. As for ASEP-SA-CS, the SA-CS double-layer wall material forms a dense diffusion barrier. This process is diffusion-controlled. Water molecules must first be adsorbed on the surface of the microcapsule and then slowly penetrate the wall material network to contact the internal peptides. This can significantly reduce the damage of external humidity to the core material, increase the stability of the active peptides of shad eggs, and also provide a guarantee for its subsequent processing, storage in humid environments and commercial applications, laying a key technological foundation.

[0090] 2. Thermal stability of microcapsules and shad egg-derived peptides

[0091] Heat treatment is a common method in food processing, but it can cause peptide chains to loosen, leading to amino acid desulfurization and isomerization. The thermal stability of microcapsules directly determines the retention efficiency of active ingredients during high-temperature processing and storage, and is a core indicator for evaluating their industrial application potential. The thermal stability of each sample is as follows: Figure 8As shown, in the accelerated thermal stability evaluation under extreme conditions of 100 ℃, the retention rate of free shad ovum peptide (ASEP) decreased the fastest with increasing heat treatment time, followed by sodium alginate monolayer microcapsules (ASEP-SA), while sodium alginate-chitosan bilayer microcapsules (ASEP-SA-CS) showed the slowest decrease. After 10 h of heat treatment, the retention rates of shad ovum peptide were (39.18±1.27)%, (75.67±0.95)%, and (81.44±0.77)%, respectively. This indicates that microencapsulation can significantly improve the thermal stability of peptides, and the protective effect of the bilayer encapsulation system is better than that of the monolayer. The reason for this may be that the calcium alginate gel network can encapsulate peptide molecules within a three-dimensional network, limiting their direct contact with the heat medium. At the same time, the gel skeleton has a certain buffering effect on heat conduction, thereby delaying the thermal degradation of peptides. The introduction of the chitosan layer further increases the wall thickness and density, forming a polyelectrolyte composite membrane on the calcium alginate surface. This membrane fills the pores on the gel surface, reduces the molecular diffusion coefficient, and inhibits both the rate of external heat transfer to the interior and the rate of peptide molecule leakage to the exterior. Therefore, bilayer microcapsules provide a stronger diffusion barrier than monolayer microcapsules.

[0092] 3. Fourier transform infrared spectroscopy analysis

[0093] Figure 9 FTIR spectra of shad roe peptide, blank microcapsules, and shad roe peptide bilayer microcapsules prepared in Example 1 are shown. The free shad roe peptide spectrum exhibits typical protein and peptide characteristic absorption bands, with an amide I band (1600~1700 cm⁻¹). -1 The peaks are mainly derived from C=O stretching vibrations, concentrated around 1640 cm⁻¹, indicating an α-helical or random coil conformation; the amide II band (1500~1600 cm⁻¹) is also present. -1 It is generated by the coupling of NH bending vibration and CN stretching vibration, with a peak position of 1540 cm. -1 Amide III band (approximately 1200–1350 cm) -1 The peak position is 1270 cm. -1 This reflects the expansion and contraction of CN and the in-plane bending of NH. Furthermore, due to... Figure 9 It can be seen that, compared with free shad egg-derived peptides, the peptide-loaded bilayer microcapsules exhibit higher activity at 1640 cm⁻¹. -1 The characteristic peak at the amide I band was significantly weakened and shifted, indicating that the peptide was successfully encapsulated within the sodium alginate or chitosan via electrostatic interactions rather than being adsorbed on the surface; compared with the blank microcapsules, the peptide-loaded bilayer microcapsules showed a significant decrease in peak value at 3400 cm⁻¹. -1At this point, the stretching vibrations of OH and NH undergo a blue shift, resulting in a broadened peak and a significantly weakened absorption peak. This is likely due to the cross-linking reaction between the hydroxyl and amino groups in the shad ovum-derived peptides and the functional groups in the wall material sodium alginate or chitosan, forming a new hydrogen bond network. This leads to changes in vibrational energy, thereby weakening the absorption peak. In conclusion, the above results indicate that the shad ovum-derived peptides were successfully encapsulated within the microcapsules, and that an interaction occurred between the two.

[0094] 4. In vitro sustained-release properties of microcapsules and shad egg-derived peptides

[0095] To investigate the effect of microencapsulation on the long-acting release characteristics, sustained-release performance, and stability of shad egg-derived peptides in the gastrointestinal environment, this invention uses an in vitro simulated digestion model for evaluation. Referring to human physiological parameters, the average residence time of food in the stomach is 1–2 h, and in the small intestine it is 2–6 h. Therefore, the incubation time in simulated gastric juice was set to 2 h, and the incubation time in simulated small intestinal juice was set to 4 h. Figure 10The retention rates of shad roe peptide and shad roe peptide microcapsules prepared in Example 1 in simulated gastrointestinal fluid were measured after incubation in simulated gastric fluid for 120 min. Subsequently, the free peptides were rapidly hydrolyzed and inactivated by gastric acid and pepsin, with the release amount decreasing to (40.85±0.46)%, showing no sustained-release behavior. In contrast, the sodium alginate monolayer microcapsules (ASEP-SA) and the sodium alginate-chitosan bilayer shad egg-derived peptide microcapsules (ASEP-SA-CS) prepared in Example 1 remained intact at this stage, with peptide release amounts of only (24.30±0.98)% and (8.40±1.03)%, respectively. The bilayer microcapsules were lower than the monolayer microcapsules, indicating that the bilayer microcapsules were better able to achieve sustained release of shad egg-derived peptides. This difference may be due to the fact that after the microcapsules enter the gastric environment, calcium alginate is protonated to form insoluble alginate, and the network shrinks and becomes dense, providing a certain physical isolation effect against pepsin. Meanwhile, the gel microsphere structure formed by the mixture of chitosan and calcium chloride hardly swells in the acidic environment, thus effectively preventing the release of active substances as core materials and ensuring that most active peptides can smoothly enter the intestinal environment. After the samples were transferred from the gastric environment to the intestinal environment, the peptides in both types of microcapsules exhibited explosive release. After 4 hours of incubation in intestinal fluid, the release rates of ASEP-SA and ASEP-SA-CS peptides reached (74.68±0.62)% and (63.55±1.03)%, respectively. This phenomenon was caused by a rapid increase in intestinal pH from acidic to neutral to weakly alkaline, along with a dramatic change in the types and strengths of ions. The cross-linking degree of the calcium alginate network in the monolayer capsule continuously decreased, the backbone gradually disintegrated, and the encapsulated peptides were rapidly released, forming the steep rise of the cumulative release curve. In contrast, the chitosan membrane of the bilayer microcapsule deprotonated in the early stage of entering the intestinal environment due to the pH exceeding its solubility range. This enhanced the hydrogen bonds between molecular chains, caused the membrane to shrink, and remained insoluble. It could continue to provide an outer physical barrier when the inner layer of calcium alginate began to dissolve, resulting in a flatter release curve and a longer release period. This further demonstrates that microcapsules can improve the stability of shad egg-derived peptides and slowly release their core material in the intestine.

[0096] 5. Electronic nose analysis

[0097] Figure 11 (A) demonstrates a significant difference in odor fingerprints between shad roe peptides and microcapsules. The peptide group exhibits abundant volatile components and high response values; while the microcapsules effectively shield volatile components, resulting in an overall decrease in response values ​​and a smaller radar map area compared to the peptide group. The most significant difference is observed in the response values ​​of the W5S (sensitive to nitrogen oxides) and W1W (sensitive to sulfides) sensors, further illustrating the superior shielding effect of the bilayer microcapsule wall material on the fishy odor components of the shad roe peptides. Meanwhile, Figure 11Principal component analysis (PCA) plot (B) shows that PC1 and PC2 contribute 82.3% and 11.9% respectively, with a cumulative contribution of 94.2%, covering most of the biological sample information and capable of distinguishing the odor characteristics of different samples. The shad egg peptide, without wall material embedding, is located on the negative axis of PC1, with volatile components directly released into the headspace. Its odor fingerprint shows a systematic difference from the embedded sample, thus being significantly distinguished in the PC1 dimension with the highest contribution rate. The microcapsules are clearly separated from the peptides in the PC2 dimension. The dense structure of the bilayer wall material and the chemisorption of the chitosan layer may selectively retain specific volatile components, making the odor fingerprint unique in the secondary dimension (PC2). In summary, the results of this electronic nose analysis are consistent with the previous physicochemical properties and release behavior studies, jointly demonstrating the barrier enhancement effect of the bilayer microcapsule wall material structure in the embedding system from multiple perspectives.

[0098] As can be seen from Examples 1 to 3, single-layer microcapsules were prepared using shad roe peptide as the core material and sodium alginate as the wall material. Using the encapsulation rate as the response value, the optimal preparation process parameters for single-layer microcapsules were determined through single-factor and orthogonal experiments as follows: sodium alginate mass fraction 2.0%, calcium chloride mass fraction 4%, curing time 30 min, and Tween 80 mass fraction 0.5%. Under these conditions, the highest microcapsule encapsulation rate was (88.69±0.43)%, which is 7% higher than the encapsulation rate of (81.84±0.91)% obtained under the initial preparation process. Morphological characterization in Example 4 shows that the microcapsule surface is dense and rough, exhibiting a polyelectrolyte composite membrane layered structure. FTIR analysis confirms that ASEP is successfully encapsulated inside the microcapsule and has hydrogen bonding or electrostatic interaction with the wall material. Performance testing in Example 5 shows that the bilayer microcapsules exhibit good gastric acid tolerance and intestinal sustained-release characteristics in simulated gastrointestinal digestion. Electronic nose analysis results indicate that the bilayer microcapsules have a significant shielding effect on the fishy odor components of shad roe peptides, with an overall decrease in radar response values. PCA analysis can effectively distinguish the odor characteristics of different samples, further verifying the physical barrier effect of the bilayer microcapsules.

[0099] Comparative example:

[0100] This comparative example aims to investigate the effect of sodium alginate-chitosan bilayer wall material and core material (shad roe peptide) at different mass ratios on the sustained-release performance of the prepared microcapsules. By screening out the optimal range of ratios for sustained-release effect and comparing it with single-layer microcapsules and other non-optimal ratios, the purpose is to highlight that the selection of specific ratios in the technical solution of this invention is not obvious.

[0101] 1. Experimental Materials and Preparation Methods

[0102] Wall materials: Sodium alginate (SA), chitosan (CS).

[0103] Core material: anchovy egg-derived peptide.

[0104] Methods: Microcapsules were prepared according to the method in Example 1. The sustained-release performance under simulated gastrointestinal conditions was evaluated using the cumulative release rate in simulated gastric fluid (2 h) and the cumulative release rate in simulated intestinal fluid (4 h) as evaluation indicators.

[0105] 2. Experimental group design

[0106] To investigate the effect of proportions on performance, the ratio of sodium alginate, chitosan, and shad roe peptides was set as the main variable. The preparation process parameters (such as calcium chloride mass fraction 4%, curing time 30 min, and Tween 80 mass fraction 0.5%) were kept constant, and the following experimental groups (including a comparative example) were set up:

[0107] Table 3 Comparative Experimental Design Table

[0108]

[0109] 3. Comparative test results

[0110] The results of the comparative experiments are shown in Table 4. Compared with Comparative Example 1 (double-layer) and Comparative Example 1 (single-layer), under the same core-to-wall ratio of 1:3, the release rate of the double-layer microcapsules in gastric juice significantly decreased from 24.30% to 8.40%. This indicates that the double-layer wall material does indeed construct a denser protective barrier, solving the technical problem of easy degradation of single-layer microcapsules in gastric juice, leading to the inactivation of active peptides. Compared with Comparative Example 1 (1:3) and Comparative Examples 2 (1:5) and 3 (1:1), when the core material ratio is too low (1:5), although the gastric juice protection is good, the drug loading per unit mass of microcapsules is low, resulting in poor production efficiency. Furthermore, an excessively thick wall material may lead to incomplete release at the target site in the intestine, with a 4-hour release rate of 48.87% in intestinal juice, failing to fully exert the drug efficacy. When the core material ratio is too high (1:1), the wall material cannot completely and tightly encapsulate the core material, resulting in a loose microcapsule structure and a 2-hour release rate as high as 39.88% in gastric juice, thus losing the significance of enteric protection. When the concentration ratio of sodium alginate to chitosan was improper (Comparative Example 4), the membrane structure formed was defective due to insufficient electrostatic interaction, resulting in a significantly inferior sustained-release effect (P < 0.05) compared to Example 1. Example 1 (core-to-wall ratio of 1:3) achieved the optimal balance between gastric juice release rate (8.40%) and intestinal juice release rate (63.55%), protecting the peptides as they passed through the stomach while ensuring their efficient release in the intestines—an effect unpredictable at other ratio ranges.

[0111] Table 4 Comparison of cumulative release rates of microcapsules with different proportions in vitro

[0112]

[0113] In summary, the experimental data above demonstrate that not all bilayer microcapsules can achieve the effects of this invention. During the research and development process, it was discovered that the ratio of core material to wall material (especially 1:3) plays a decisive role in sustained-release performance. When the ratio is too high, the wall material is insufficient to form complete encapsulation (see Comparative Example 3); when the ratio is too low, although gastric acid protection is strong, intestinal release is hindered, and bioavailability decreases significantly (P<0.05) (see Comparative Example 2). This invention, through extensive experimental screening, has achieved a synergistic effect of low gastric acid release and high intestinal fluid release at a specific ratio. This equilibrium point between gastric acid tolerance and intestinal sustained-release produced at a specific ratio cannot be easily derived by those skilled in the art from conventional experiments.

[0114] In summary, this invention optimizes the preparation process of ASEP microcapsules using the sharp-pore coagulation bath method. Based on the combined results of single-factor and orthogonal experiments, the optimal process conditions for preparing monolayer microcapsules (ASEP-SA) were determined to be 2.0% sodium alginate, 4% calcium chloride, 30 min curing time, and 0.5% Tween 80. Based on these conditions, bilayer microcapsules (ASEP-SA-CS) were prepared using chitosan as the secondary wall material. The results showed that the ASEP-SA-CS bilayer microcapsules exhibited higher hygroscopicity, thermal stability, and in vitro sustained-release properties than the ASEP-SA monolayer microcapsules. SEM analysis revealed that ASEP-SA-CS had a more stable structure than ASEP-SA. FTIR analysis showed that ASEP was successfully encapsulated within the microcapsules and interacted with the wall material. Electronic nose analysis indicated that the ASEP-SA-CS bilayer microcapsules had a superior shielding effect on the fishy odor components of shad egg-derived peptides. The microcapsule preparation process used in this invention is simple, mild, and has a good encapsulation effect. The resulting microcapsule products have good stability and excellent sustained-release performance, providing a reference for the industrial application of ASEP microcapsules.

Claims

1. A method for preparing shad egg-derived peptide microcapsules, characterized in that, The method for preparing the shad egg-derived peptide microcapsules is to use shad egg-derived peptides as the core material and sodium alginate as the wall material to prepare single-layer microcapsules using the sharp-pore-coagulation bath method, and then further construct double-layer microcapsules using chitosan as the secondary wall material.

2. The method for preparing shad egg-derived peptide microcapsules according to claim 1, characterized in that, Includes the following steps: (1) Add shad egg peptide freeze-dried powder to sodium alginate solution, then add Tween 80 and stir to obtain core wall mixture; then use a syringe to drop the core wall mixture into the coagulation bath above the prepared CaCl2 coagulation bath, filter and wash after solidification to obtain sodium alginate monolayer microcapsules. (2) The sodium alginate monolayer microcapsules obtained in step (1) are immersed in chitosan solution and gently stirred to form a second membrane. After filtration and washing, sodium alginate-chitosan bilayer shad egg peptide microcapsules are obtained. After vacuum freeze-drying, peptide-loaded dry microspheres are obtained.

3. The method for preparing shad egg-derived peptide microcapsules according to claim 2, characterized in that, The mass ratio of shad egg peptide to sodium alginate in step (1) is 1:

3.

4. The method for preparing shad egg-derived peptide microcapsules according to claim 2, characterized in that, The sodium alginate solution in step (1) has a mass fraction of 1.0~3.0%.

5. The method for preparing shad egg-derived peptide microcapsules according to claim 2, characterized in that, In step (1), the mass fraction of Tween 80 is 0.5-2.5%, and the mass fraction of CaCl2 is 1.0-5.0%.

6. The method for preparing shad egg-derived peptide microcapsules according to claim 2, characterized in that, The curing time in step (1) is 10~50 min.

7. The method for preparing shad egg-derived peptide microcapsules according to claim 2, characterized in that, The concentration of the chitosan solution in step (2) is 1%.

8. The method for preparing shad egg-derived peptide microcapsules according to claim 2, characterized in that, The chitosan solution mentioned in step (2) is obtained by dissolving a 1% chitosan solution in a 1% acetic acid solution.

9. Shad egg-derived peptide microcapsules prepared by the preparation method according to any one of claims 1 to 8.

10. The shad egg-derived peptide microcapsules according to claim 9, characterized in that, The free shad egg-derived peptide spectrum of the microcapsules exhibits typical protein and polypeptide characteristic absorption bands. The amide I band mainly originates from the C=O stretching vibration, with the peak position concentrated at 1640 cm⁻¹. -1 Nearby; the amide II band is generated by the coupling of NH bending vibration and CN stretching vibration, with a peak position of 1540 cm⁻¹. -1 The peak position of amide III is 1270 cm⁻¹. -1 .

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