Preparation method and application of protein nanocage loaded with tuna peptide

Abstract

The present invention provides a preparation method and application of a protein nanocage loaded with tuna peptides, specifically including the construction and modification of ferritin nanocages, and the extraction, screening, and embedding of tuna active peptides. The tuna active peptides are encapsulated in ferritin nanocages modified with ferulic acid and delivered to the intestines in a targeted manner. They interact with anorexigenic hormone receptors, regulate the production of anorexigenic hormones such as cholecystokinin, glucagon-like peptide 1, and PYY, control appetite after a normal diet, and enhance the body's sense of fullness, thereby reducing food intake and weight loss, thereby playing a role in regulating obesity. In the present invention, ferritin nanocages are modified with ferulic acid. The carboxyl and phenolic hydroxyl groups in the ferulic acid interact with the amino end of the outer surface of the ferritin nanocage, giving the ferritin nanocages the function of being resistant to gastric juice environment. They can exist stably in the stomach of the body without being decomposed, thereby being decomposed in the intestines and exerting their precise targeted effects, laying the foundation for anti-obesity research.

Description

Technical Field

[0001] The present invention belongs to the technical field of functionalized application of seafood, and particularly relates to a preparation method and application of a protein nanocage loaded with tuna peptide. Background Art

[0002] Tuna is a deep-sea fish rich in nutrients and boasts numerous health benefits. It contains not only essential peptides such as glutathione, carnosine, and anserine, but also trace elements such as zinc and selenium. These components work synergistically to maintain a healthy balance in the human body. However, tuna processing produces by-products, comprising approximately 50-70% of the total weight. These by-products include fish scraps, offal, minced meat, heads, skin, and bones. This results in significant waste of tuna resources and negative impacts on the ecological environment. However, tuna processing by-products, which contain over 80% protein, are ideal raw materials for the production of tuna peptides.

[0003] Tuna peptides, with their unique structure and diverse bioactivities, have become a hot topic in scientific research. They can significantly lower blood pressure, promote blood circulation, and safeguard cardiovascular health. As powerful natural antioxidants, they can scavenge free radicals in the body, enhance immunity, and slow the aging process. Of particular note, specific components in tuna peptides have a significant effect on improving sleep quality, regulating the circadian rhythm, promoting deep sleep, and alleviating the troubles of insomnia for modern people. Furthermore, the DHA component in tuna peptides is particularly important for brain health, promoting nerve cell development, and enhancing memory and learning abilities, making them an ideal supplement for those who engage in mental work. However, their effectiveness in combating obesity has been less well-documented.

[0004] Tuna peptides are a general term for peptides composed of natural amino acids in varying compositions and arrangements, ranging from dipeptides to complex linear and cyclic structures. Oligopeptides, such as dipeptides and tripeptides, are digested and absorbed more efficiently by the human body than polypeptides and amino acids of the same composition. Oligopeptides have lower osmotic pressure than amino acids and are highly permeable in the small intestine, enabling efficient absorption and utilization. However, most bioactive peptides are oligopeptides, which are easily degraded by enzymes and the acidic pH in the mouth and gastric juice, thus losing their biological activity.

[0005] Existing bioactive peptide delivery systems are roughly divided into emulsions, liposomes, and microgels. Most of them will be decomposed by the complex enzyme system and extremely acidic environment in the stomach of the body, exposing the active peptides and causing them to be decomposed and inactivated, so the delivery system exists stably in gastric juice; ferritin nanocages in the existing technology have good stability, but ferritin nanocages will dissociate at pH 2 or pH 11. Modified ferritin nanocages can adapt to the gastric juice environment to ensure that the structure is not destroyed. Therefore, it is urgent to invent a preparation method and application of marine-derived bioactive peptides based on protein cage stabilization to ensure that the bioactive peptides can stably play their relevant roles in the body and achieve the purpose of targeted release, so as to provide a reference for further research on marine-derived bioactive peptides and be conducive to market development and utilization. This is of great significance to improving the level of deep processing and high-value utilization of bioactive peptides at home and abroad and making full use of abundant marine protein resources. Summary of the Invention

[0006] Technical Problem to Be Solved: To address the aforementioned technical problems, the present invention aims to provide a method for preparing and applying tuna peptide-loaded protein nanocages. Specifically, the method involves constructing and modifying ferritin nanocages, and extracting, screening, and encapsulating tuna active peptides. The tuna active peptides are encapsulated in ferulic acid-modified ferritin nanocages and delivered to the intestines, where they interact with anorexigenic hormone receptors, regulating the production of anorexigenic hormones such as cholecystokinin, glucagon-like peptide 1, and PYY. This controls appetite after a normal diet and enhances satiety, reducing food intake and weight loss, thereby playing a role in regulating obesity. In this invention, ferritin nanocages are modified with ferulic acid. The carboxyl and phenolic hydroxyl groups in the ferulic acid interact with the amino termini on the outer surface of the ferritin nanocages, endowing the ferritin nanocages with gastric resistance. These nanocages can remain stable in the stomach without decomposition, allowing them to decompose and precisely target their effects in the intestines, laying the foundation for anti-obesity research.

[0007] Technical solution: A method for preparing protein nanocages loaded with tuna peptides, comprising the following steps:

[0008] Step 1. Weigh human heavy chain ferritin, add water to dissolve evenly, and prepare a human heavy chain ferritin aqueous solution with a concentration of 250-1000 μg / mL;

[0009] Step 2. Add 0.1-1 M HCl solution to the human heavy chain ferritin aqueous solution, adjust the pH to 2.0-2.8, maintain for 15-20 minutes, then add 0.1-1 M NaOH solution, adjust the pH to 7.0-8.0, maintain for 1-2 hours, and dialyze three times to obtain a ferritin nanocage solution;

[0010] Step 3. Add 0.2-1.0% ferulic acid to the ferritin nanocage solution, adjust the pH to 2.0-2.8 with 0.1-1 M HCl solution, incubate at 37°C for 2-6 hours, and dialyze three times to obtain a modified ferritin nanocage solution;

[0011] Step 4. Take the modified ferritin nanocage solution and add HCl solution to adjust the pH to 2.0-2.8, slowly add tuna peptide, keep at room temperature for 15-20 minutes, then adjust the pH to 7.0-8.0 with NaOH solution, keep for 1-2 hours, and dialyze 3 times to obtain protein nanocages loaded with tuna peptide.

[0012] Furthermore, in step 4, the volume ratio of the modified ferritin nanocage solution to the tuna peptide is (100-400):1.

[0013] Furthermore, the extraction and screening method of tuna peptides comprises the following steps:

[0014] S1. Take the tuna minced meat scraps, add water according to the material-liquid ratio of 1: (2-4) and evenly blend to obtain a tuna minced meat homogenate;

[0015] S2. The pH of the tuna minced meat homogenate was adjusted to 9-11, and alkaline protease was added to the tuna minced meat homogenate for enzymatic hydrolysis, and then the enzyme was inactivated to obtain a tuna minced meat hydrolysate;

[0016] S3. Centrifuge the tuna minced meat enzymatic hydrolysate at 8000-10000 rpm and 4°C for 15-20 min to obtain the supernatant.

[0017] S4. The supernatant was subjected to ultrafiltration separation, evaporation concentration, and freeze-drying to obtain tuna peptides of different molecular weights;

[0018] S5. Conduct cell experiments with tuna peptides of varying molecular weight to evaluate anorexigenic hormone secretion levels and screen for highly active tuna peptides. Furthermore, the enzymatic hydrolysis conditions in step S2 are as follows: alkaline protease activity of 200,000 U / g, alkaline protease addition of 2,500-4,500 U / g, hydrolysis temperature of 45-55°C, and hydrolysis time of 4-6 hours.

[0019] Furthermore, the conditions for inactivating the enzyme in step S2 are: an enzyme inactivation temperature of 90-95° C. and an enzyme inactivation time of 10-20 min.

[0020] Furthermore, the ultrafiltration separation conditions in step S4 are as follows: the supernatant is ultrafiltered step by step through 3 kDa and 5 kDa ultrafiltration membranes to retain supernatant ultrafiltration fractions with molecular weights <3 kDa, 3-5 kDa and >5 kDa.

[0021] Application of the tuna peptide-loaded protein nanocage prepared by any of the above preparation methods in foods, health products and medicines for improving obesity.

[0022] Beneficial effects:

[0023] 1. The tuna peptide of the present invention can interact with the secretory receptors of cholecystokinin (CCK), glucagon-like peptide 1 (GLP-1), and PYY in the intestinal tract of the body, promote the secretion of cholecystokinin (CCK), glucagon-like peptide 1 (GLP-1), and PYY in the intestinal tract of the body, regulate the behavioral activities of the body's digestive tract and delay gastric emptying, thereby making the body feel full, suppressing appetite, and reducing food intake other than normal diet, thereby achieving the purpose of regulating obesity.

[0024] 2. The present invention utilizes protein nanocages for targeted delivery of tuna peptides, precisely transporting them to the intestinal tract to exert their effects. Furthermore, the protein nanocages are modified with ferulic acid. The carboxyl and hydroxyl groups in the ferulic acid can undergo cross-linking reactions with the amino and thiol groups on the ferritin nanocages, forming a more stable interaction. This further enhances the structural stability of the ferritin nanocages and promotes close integration between the interior and exterior of the ferritin nanocages, forming a denser and more stable nanostructure that can exist stably in the harsh gastric fluid environment, ensuring that the activity of the tuna peptide is not destroyed by the complex enzyme system and extreme pH in the gastric fluid. The peptide is then delivered to the intestines of the body, where it binds to the corresponding receptors and exerts its effects, achieving the purpose of targeted release.

[0025] 3. The present invention not only provides a reference for further research on tuna peptides, but also facilitates the market development and utilization of tuna peptides, improves the level of deep processing and high-value utilization of tuna peptides, makes full use of abundant marine protein resources, and demonstrates great potential in the fields of nutrition and health. It provides a new direction for the innovation and development of the bio-peptide functionalization industry and demonstrates great application potential and value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The changes of J1, J2, and J3 on the secretion of CCK, PYY, and GLP-1 by STC-1 cells in Example 1;

[0027] Figure 2 The inhibitory effects of J1, J2, and J3 on triglyceride in mouse 3T3-L1 cells in Example 1;

[0028] Figure 3 The changes of the simulated gastric digestion components of Example 10 and Comparative Examples 1-4 on the secretion of CCK, PYY and GLP-1 by STC-1 cells;

[0029] Figure 4The changes of the simulated intestinal digestion components of Example 10 and Comparative Examples 1-4 on the secretion of CCK, PYY and GLP-1 by STC-1 cells;

[0030] Figure 5 The figure shows the inhibitory effect of the simulated intestinal digestion components of Example 10 and Comparative Examples 1-4 on triglyceride in mouse 3T3-L1 cells. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with examples, which are provided to explain the present invention and are not limited to the following examples:

[0032] Materials used in the present invention: human heavy chain ferritin (molecular weight of approximately 22 kDa), purchased from MedChemExpress, USA; STC-1 cells, purchased from Shanghai Fuheng Biotechnology Co., Ltd.; mouse 3T3-L1 cells, purchased from Guangzhou Saiye Biotechnology Co., Ltd.; CCK kit, purchased from Wuhan Cloud Clone Technology Co., Ltd.; GLP-1 detection kit, purchased from Wuhan Huamei Bioengineering Co., Ltd.; PYY detection kit, purchased from RayBiotech, USA; triglyceride detection kit, purchased from Shanghai Beibo Biotechnology Co., Ltd.

[0033] Reagents used in the present invention: Induction differentiation solution A solution (200 μL, 50 μL, and 1 mL of insulin solution, dexamethasone solution, and 3-isobutyl-1-methylxanthine solution, respectively, were added to 98.75 mL of DMEM high-glucose culture medium containing 10% (v / v) fetal bovine serum and 1% (v / v) double antibody); Induction differentiation solution B solution (200 μL of insulin solution was dissolved in 98.8 mL of DMEM high-glucose culture medium containing 10% (v / v) fetal bovine serum and 1% (v / v) double antibody)

[0034] Example 1

[0035] A method for extracting and screening tuna peptides comprises the following steps:

[0036] S1. Take 100g of tuna minced meat scraps, add 400g of water and stir evenly to obtain a tuna minced meat homogenate;

[0037] S2. The pH of the tuna minced meat homogenate was adjusted to 9, 4500 U / g alkaline protease was added to the tuna minced meat homogenate, and enzymatic hydrolysis was performed at 55°C for 5 h, followed by enzyme inactivation at 95°C for 15 min to obtain a tuna minced meat enzymatic hydrolysate.

[0038] S3. Centrifuge the tuna minced meat enzymatic hydrolysate at 10,000 rpm and 4°C for 20 min to obtain the supernatant.

[0039] S4. The supernatant was separated by ultrafiltration through 5kDa and 3kDa ultrafiltration membranes, and the supernatant ultrafiltration fractions with molecular weights <3kDa, 3-5kDa, and >5kDa were retained. The supernatant ultrafiltration fractions were then evaporated, concentrated, and freeze-dried to obtain tuna peptides with molecular weights <3kDa, 3-5kDa, and >5kDa;

[0040] S5. Tuna peptides with molecular weights <3kDa, 3-5kDa, and >5kDa were used for in vitro cell experiments to evaluate the secretion of anorexigenic hormones and screen for highly active tuna peptides.

[0041] Among them, tuna peptides with a molecular weight of <3kDa were marked as J1, tuna peptides with a molecular weight of 3-5kDa were marked as J2, and tuna peptides with a molecular weight of >5kDa were marked as J3.

[0042] Performance Testing

[0043] (1) STC-1 cell in vitro experiments

[0044] STC-1 cell culture: STC-1 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, and 1% NEAA, and stored at 37°C in a 5% CO2 humidified atmosphere. When the confluence reached 80-90%, the cells were routinely trypsinized and subcultured, using 10-20 generations of STC-1 cells.

[0045] Evaluation of intestinal hormone secretion: STC-1 cells were seeded in 24-well plates and cultured at 37°C for 24 h. The cells were washed twice with HBSS buffer (pH 7.4) to remove the culture medium and incubated in a culture incubator with buffer containing J1, J2, and J3, respectively, for 2 h. The cell supernatants were collected for analysis using CCK, GLP-1, and PYY detection kits.

[0046] Depend on Figure 1 It can be seen that J1, J2, and J3 can all act on STC-1 cells to promote their secretion of intestinal anorexigenic hormones CCK, GLP-1, and PYY, but J1 has the highest secretion promotion level because J1's molecular weight is <3kDa, which is easier to be absorbed and utilized by the body's cells. At the same time, J1 contains more small molecule peptides with high biological activity, which can promote STC-1 cells to secrete more intestinal anorexigenic hormones.

[0047] (2) Evaluation of anti-obesity effects in vitro

[0048] Cell culture: Mouse 3T3-L1 cells were cultured in high-glucose DMEM medium containing 10% (v / v) newborn calf serum. When the cells reached the logarithmic phase, they were seeded in 6-well plates and the medium was changed every two days. When the cells grew to contact inhibition, fresh medium containing 10% (v / v) newborn calf serum was replaced and cultured for another 48 hours. The cells were then divided into 5 groups (including blank group, model group, J1 group, J2 group, and J3 group).

[0049] Target component culture: J1, J2, and J3 groups were cultured with Induction Differentiation Solution A containing the J1, J2, and J3 components, respectively, for 60 hours. They were then cultured with Induction Differentiation Solution B containing the corresponding J1, J2, and J3 components, respectively, for 60 hours. Finally, the cells were cultured with DMEM medium containing 10% (v / v) fetal bovine serum and the same peptide concentrations for 72 hours. The blank group received DMEM medium containing 10% (v / v) newborn calf serum instead of the Induction Differentiation Solution, and the model group received culture medium instead of the peptide solution.

[0050] Triglyceride detection: Use a triglyceride kit to measure the triglyceride content in cells.

[0051] Depend on Figure 2 It can be seen that J1, J2, and J3 can all act on mouse 3T3-L1 cells to inhibit the production of triglycerides, and J1 has the highest inhibitory effect, indicating that the molecular active peptides produced after enzymatic hydrolysis of tuna have the effect of regulating obesity. Combined with the in vitro test of STC-1 cells, it is shown that tuna peptides promote the secretion of anorexigenic hormones in the intestine, such as cholecystokinin (CCK), glucagon-like peptide 1 (GLP-1), and PYY, which make the body feel full, suppress appetite, and reduce food intake other than normal diet, thereby achieving the purpose of regulating obesity.

[0052] Example 2

[0053] A method for preparing a modified ferritin nanocage loaded with highly active tuna peptide comprises the following steps:

[0054] Step 1. Weigh 250 μg of human heavy chain ferritin and dissolve it evenly in 1 mL of water to prepare a human heavy chain ferritin aqueous solution with a concentration of 250 μg / mL;

[0055] Step 2. Slowly add 1 M HCl solution to the human heavy chain ferritin aqueous solution to adjust the pH to 2.0, maintain for 15 minutes, then add 0.1 M NaOH solution to adjust the pH to 7.4, maintain for 2 hours, and dialyze three times to obtain the ferritin nanocage solution;

[0056] Step 3. Add 0.6 mg of ferulic acid to the ferritin nanocage solution, adjust the pH to 2.5 with 1 M HCl solution, incubate at 37°C for 4 h, then adjust the pH to 7.4 with 1 M NaOH solution, and dialyze three times against pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution.

[0057] Step 4. Take 4 mL of modified ferritin nanocage solution and add 1 M HCl solution to adjust the pH to 2.0. Slowly add 40 μL of 20 mg / mL J1 solution (molecular weight <3 kDa) dropwise while stirring. Keep at room temperature for 20 minutes, then adjust the pH to 7.4 with 1 M NaOH solution and keep it for 2 hours. Dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptide.

[0058] Example 3

[0059] A method for preparing a modified ferritin nanocage loaded with highly active tuna peptide comprises the following steps:

[0060] Step 1. Weigh 500 μg of human heavy chain ferritin and dissolve it evenly in 1 mL of water to prepare a human heavy chain ferritin aqueous solution with a concentration of 500 μg / mL;

[0061] Step 2. Slowly add 1 M HCl solution to the human heavy chain ferritin aqueous solution to adjust the pH to 2.5, maintain for 20 minutes, then add 0.1 M NaOH solution to adjust the pH to 7.0, maintain for 2 hours, and dialyze three times to obtain the ferritin nanocage solution;

[0062] Step 3. Add 0.6 mg of ferulic acid to the ferritin nanocage solution, adjust the pH to 2.0 with 1 M HCl solution, incubate at 37°C for 4 h, then adjust the pH to 7.4 with 1 M NaOH solution, and dialyze three times against pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution.

[0063] Step 4. Take 4 mL of modified ferritin nanocage solution and add 1 M HCl solution to adjust the pH to 2.0. Slowly add 40 μL of 20 mg / mL J1 solution (molecular weight <3 kDa) dropwise while stirring. Keep at room temperature for 20 minutes, then adjust the pH to 7.4 with 1 M NaOH solution and keep it for 2 hours. Dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptide.

[0064] Example 4

[0065] A method for preparing a modified ferritin nanocage loaded with highly active tuna peptide comprises the following steps:

[0066] Step 1. Weigh 750 μg of human heavy chain ferritin and dissolve it evenly in 1 mL of water to prepare a human heavy chain ferritin aqueous solution with a concentration of 750 μg / mL;

[0067] Step 2. Slowly add 1 M HCl solution to the human heavy chain ferritin aqueous solution to adjust the pH to 2.0, maintain for 20 minutes, then add 0.1 M NaOH solution to adjust the pH to 7.0, maintain for 2 hours, and dialyze three times to obtain the ferritin nanocage solution;

[0068] Step 3. Add 0.6 mg of ferulic acid to the ferritin nanocage solution, adjust the pH to 2.0 with 1 M HCl solution, incubate at 37°C for 4 h, then adjust the pH to 7.4 with 1 M NaOH solution, and dialyze three times against pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution.

[0069] Step 4. Take 4 mL of modified ferritin nanocage solution and add 1 M HCl solution to adjust the pH to 2.0. Slowly add 40 μL of 20 mg / mL J1 solution (molecular weight <3 kDa) dropwise while stirring. Keep at room temperature for 20 minutes, then adjust the pH to 7.4 with 1 M NaOH solution and keep it for 2 hours. Dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptide.

[0070] Example 5

[0071] A method for preparing a modified ferritin nanocage loaded with highly active tuna peptide comprises the following steps:

[0072] Step 1. Weigh 1000 μg of human heavy chain ferritin and dissolve it evenly in 1 mL of water to prepare a human heavy chain ferritin aqueous solution with a concentration of 1000 μg / mL;

[0073] Step 2. Slowly add 1 M HCl solution to the human heavy chain ferritin aqueous solution to adjust the pH to 2.4, maintain for 15 minutes, then add 0.1 M NaOH solution to adjust the pH to 7.4, maintain for 2 hours, and dialyze three times to obtain the ferritin nanocage solution;

[0074] Step 3. Add 0.6 mg of ferulic acid to the ferritin nanocage solution, adjust the pH to 2.0 with 1 M HCl solution, incubate at 37°C for 4 h, then adjust the pH to 7.4 with 1 M NaOH solution, and dialyze three times against pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution.

[0075] Step 4. Take 4 mL of modified ferritin nanocage solution and add 1 M HCl solution to adjust the pH to 2.0. Slowly add 40 μL of 20 mg / mL J1 solution (molecular weight <3 kDa) dropwise while stirring. Keep at room temperature for 20 minutes, then adjust the pH to 7.4 with 1 M NaOH solution and keep it for 2 hours. Dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptide.

[0076] Example 6

[0077] A method for preparing a modified ferritin nanocage loaded with highly active tuna peptide comprises the following steps:

[0078] Step 1. Weigh 1000 μg of human heavy chain ferritin and dissolve it evenly in 1 mL of water to prepare a human heavy chain ferritin aqueous solution with a concentration of 1000 μg / mL;

[0079] Step 2. Slowly add 1 M HCl solution to the human heavy chain ferritin aqueous solution to adjust the pH to 2.0, maintain for 15 minutes, then add 0.1 M NaOH solution to adjust the pH to 7.4, maintain for 2 hours, and dialyze three times to obtain the ferritin nanocage solution;

[0080] Step 3. Add 0.2 mg of ferulic acid to the ferritin nanocage solution, adjust the pH to 2.4 with 1 M HCl solution, incubate at 37°C for 4 h, then adjust the pH to 7.4 with 1 M NaOH solution, and dialyze three times against pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution.

[0081] Step 4. Take 4 mL of modified ferritin nanocage solution and add 1 M HCl solution to adjust the pH to 2.0. Slowly add 40 μL of 20 mg / mL J1 solution (molecular weight <3 kDa) dropwise while stirring. Keep at room temperature for 20 minutes, then adjust the pH to 7.4 with 1 M NaOH solution and keep it for 2 hours. Dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptide.

[0082] Example 7

[0083] A method for preparing a modified ferritin nanocage loaded with highly active tuna peptide comprises the following steps:

[0084] Step 1. Weigh 1000 μg of human heavy chain ferritin and dissolve it evenly in 1 mL of water to prepare a human heavy chain ferritin aqueous solution with a concentration of 1000 μg / mL;

[0085] Step 2. Slowly add 1 M HCl solution to the human heavy chain ferritin aqueous solution to adjust the pH to 2.0, maintain for 20 minutes, then add 0.1 M NaOH solution to adjust the pH to 7.4, maintain for 2 hours, and dialyze three times to obtain the ferritin nanocage solution;

[0086] Step 3. Add 0.4 mg of ferulic acid to the ferritin nanocage solution, adjust the pH to 2.4 with 1 M HCl solution, incubate at 37°C for 4 h, then adjust the pH to 7.4 with 1 M NaOH solution, and dialyze three times against pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution.

[0087] Step 4. Take 4 mL of modified ferritin nanocage solution and add 1 M HCl solution to adjust the pH to 2.0. Slowly add 40 μL of 20 mg / mL J1 solution (molecular weight <3 kDa) dropwise while stirring. Keep at room temperature for 20 minutes, then adjust the pH to 7.4 with 1 M NaOH solution and keep it for 2 hours. Dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptide.

[0088] Example 8

[0089] A method for preparing a modified ferritin nanocage loaded with highly active tuna peptide comprises the following steps:

[0090] Step 1. Weigh 1000 μg of human heavy chain ferritin and dissolve it evenly in 1 mL of water to prepare a human heavy chain ferritin aqueous solution with a concentration of 1000 μg / mL;

[0091] Step 2. Slowly add 1 M HCl solution to the human heavy chain ferritin aqueous solution to adjust the pH to 2.4, maintain for 20 minutes, then add 0.1 M NaOH solution to adjust the pH to 8.0, maintain for 2 hours, and dialyze three times to obtain the ferritin nanocage solution;

[0092] Step 3. Add 0.8 mg of ferulic acid to the ferritin nanocage solution, adjust the pH to 2.4 with 1 M HCl solution, incubate at 37°C for 4 h, then adjust the pH to 7.4 with 1 M NaOH solution, and dialyze three times against pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution.

[0093] Step 4. Take 4 mL of modified ferritin nanocage solution and add 1 M HCl solution to adjust the pH to 2.0. Slowly add 40 μL of 20 mg / mL J1 solution (molecular weight <3 kDa) dropwise while stirring. Keep at room temperature for 20 minutes, then adjust the pH to 7.4 with 1 M NaOH solution and keep it for 2 hours. Dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptide.

[0094] Example 9

[0095] A method for preparing a modified ferritin nanocage loaded with highly active tuna peptide comprises the following steps:

[0096] Step 1. Weigh 1000 μg of human heavy chain ferritin and dissolve it evenly in 1 mL of water to prepare a human heavy chain ferritin aqueous solution with a concentration of 1000 μg / mL;

[0097] Step 2. Slowly add 1 M HCl solution to the human heavy chain ferritin aqueous solution to adjust the pH to 2.0, maintain for 15 minutes, then add 0.1 M NaOH solution to adjust the pH to 7.0, maintain for 2 hours, and dialyze three times to obtain the ferritin nanocage solution;

[0098] Step 3. Add 0.8 mg of ferulic acid to the ferritin nanocage solution, adjust the pH to 2.0 with 1 M HCl solution, incubate at 37°C for 4 h, then adjust the pH to 7.4 with 1 M NaOH solution, and dialyze three times against pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution.

[0099] Step 4. Take 4 mL of modified ferritin nanocage solution and add 1 M HCl solution to adjust the pH to 2.0. Slowly add 20 μL of 20 mg / mL J1 solution (molecular weight <3 kDa) dropwise while stirring. Keep at room temperature for 20 minutes, then adjust the pH to 7.4 with 1 M NaOH solution and keep it for 2 hours. Dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptide.

[0100] Example 10

[0101] A method for preparing a modified ferritin nanocage loaded with highly active tuna peptide comprises the following steps:

[0102] Step 1. Weigh 1000 μg of human heavy chain ferritin and dissolve it evenly in 1 mL of water to prepare a human heavy chain ferritin aqueous solution with a concentration of 1000 μg / mL;

[0103] Step 2. Slowly add 1 M HCl solution to the human heavy chain ferritin aqueous solution to adjust the pH to 2.4, maintain for 20 minutes, then add 0.1 M NaOH solution to adjust the pH to 8.0, maintain for 2 hours, and dialyze three times to obtain the ferritin nanocage solution;

[0104] Step 3. Add 0.8 mg of ferulic acid to the ferritin nanocage solution, adjust the pH to 2.4 with 1 M HCl solution, incubate at 37°C for 4 h, then adjust the pH to 7.4 with 1 M NaOH solution, and dialyze three times against pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution.

[0105] Step 4. Take 4 mL of modified ferritin nanocage solution and add 1 M HCl solution to adjust the pH to 2.0. Slowly add 13.30 μL of 20 mg / mL J1 solution (molecular weight <3 kDa) dropwise while stirring, keep at room temperature for 20 minutes, then adjust the pH to 7.4 with 1 M NaOH solution, keep for 2 hours, and dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptide.

[0106] Example 11

[0107] A method for preparing a modified ferritin nanocage loaded with highly active tuna peptide comprises the following steps:

[0108] Step 1. Weigh 1000 μg of human heavy chain ferritin and dissolve it evenly in 1 mL of water to prepare a human heavy chain ferritin aqueous solution with a concentration of 1000 μg / mL;

[0109] Step 2. Slowly add 1 M HCl solution to the human heavy chain ferritin aqueous solution to adjust the pH to 2.4, maintain for 20 minutes, then add 0.1 M NaOH solution to adjust the pH to 8.0, maintain for 2 hours, and dialyze three times to obtain the ferritin nanocage solution;

[0110] Step 3. Add 0.8 mg of ferulic acid to the ferritin nanocage solution, adjust the pH to 2.4 with 1 M HCl solution, incubate at 37°C for 4 h, then adjust the pH to 7.4 with 1 M NaOH solution, and dialyze three times against pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution.

[0111] Step 4. Take 4 mL of modified ferritin nanocage solution and add 1 M HCl solution to adjust the pH to 2.0. Slowly add 10 μL of 20 mg / mL J1 solution (molecular weight <3 kDa) dropwise while stirring. Keep at room temperature for 20 minutes. Then adjust the pH to 7.4 with 1 M NaOH solution and keep it for 2 hours. Dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptide.

[0112] Comparative Example 1

[0113] The difference between this comparative example and Example 9 is that ferulic acid modification was not used.

[0114] A method for preparing ferritin nanocages loaded with high secretion levels of tuna peptides comprises the following steps:

[0115] Step 1. Weigh 1000 μg of human heavy chain ferritin and dissolve it evenly in 1 mL of water to prepare a human heavy chain ferritin aqueous solution with a concentration of 1000 μg / mL;

[0116] Step 2. Slowly add 1 M HCl solution to the human heavy chain ferritin aqueous solution to adjust the pH to 2.0, maintain for 15 minutes, then add 0.1 M NaOH solution to adjust the pH to 7.0, maintain for 2 hours, and dialyze three times to obtain the ferritin nanocage solution;

[0117] Step 3. Take 4 mL of modified ferritin nanocage solution and add 1 M HCl solution to adjust the pH to 2.0. Slowly add 20 μL of 20 mg / mL J1 solution (molecular weight <3 kDa) dropwise while stirring, keep at room temperature for 20 minutes, then adjust the pH to 7.4 with 1 M NaOH solution, keep for 2 hours, and dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptide.

[0118] Comparative Example 2

[0119] The difference between this comparative example and Example 9 is that genipin is used for modification.

[0120] A method for preparing a modified ferritin nanocage loaded with highly active tuna peptide comprises the following steps:

[0121] Step 1. Weigh 1000 μg of human heavy chain ferritin and dissolve it evenly in 1 mL of water to prepare a human heavy chain ferritin aqueous solution with a concentration of 1000 μg / mL;

[0122] Step 2. Slowly add 1 M HCl solution to the human heavy chain ferritin aqueous solution to adjust the pH to 2.0, maintain for 15 minutes, then add 0.1 M NaOH solution to adjust the pH to 7.0, maintain for 2 hours, and dialyze three times to obtain the ferritin nanocage solution;

[0123] Step 3. Add 0.8 mg of genipin to the ferritin nanocage solution, adjust the pH to 2.0 with 1 M HCl solution, incubate at 37°C for 4 h, then adjust the pH to 7.4 with 1 M NaOH solution, and dialyze three times against pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution.

[0124] Step 4. Take 4 mL of modified ferritin nanocage solution and add 1 M HCl solution to adjust the pH to 2.0. Slowly add 20 μL of 20 mg / mL J1 solution (molecular weight <3 kDa) dropwise while stirring. Keep at room temperature for 20 minutes, then adjust the pH to 7.4 with 1 M NaOH solution and keep it for 2 hours. Dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptide.

[0125] Comparative Example 3

[0126] The difference between this comparative example and Example 9 is that sodium tripolyphosphate was used for modification.

[0127] A method for preparing a modified ferritin nanocage loaded with highly active tuna peptide comprises the following steps:

[0128] Step 1. Weigh 1000 μg of human heavy chain ferritin and dissolve it evenly in 1 mL of water to prepare a human heavy chain ferritin aqueous solution with a concentration of 1000 μg / mL;

[0129] Step 2. Slowly add 1 M HCl solution to the human heavy chain ferritin aqueous solution to adjust the pH to 2.0, maintain for 15 minutes, then add 0.1 M NaOH solution to adjust the pH to 7.0, maintain for 2 hours, and dialyze three times to obtain the ferritin nanocage solution;

[0130] Step 3. Add 0.8 mg of sodium tripolyphosphate to the ferritin nanocage solution, adjust the pH to 2.0 with 1 M HCl solution, incubate at 37°C for 4 h, then adjust the pH to 7.4 with 1 M NaOH solution, and dialyze three times against pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution.

[0131] Step 4. Take 4 mL of modified ferritin nanocage solution and add 1 M HCl solution to adjust the pH to 2.0. Slowly add 20 μL of 20 mg / mL J1 solution (molecular weight <3 kDa) dropwise while stirring. Keep at room temperature for 20 minutes, then adjust the pH to 7.4 with 1 M NaOH solution and keep it for 2 hours. Dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptide.

[0132] Comparative Example 4

[0133] The difference between this comparative example and Example 9 is that the delivery system is replaced by liposomes instead of ferritin nanocages.

[0134] A method for preparing liposomes loaded with highly active tuna peptides comprises the following steps:

[0135] Step 1. Weigh 20 mg of soy lecithin and 4 mg of cholesterol and dissolve them in 100 mL of anhydrous ethanol to prepare a liposome base liquid. Step 2. Add 1 mL of 20 mg / mL J1 solution (molecular weight <3 kDa) to the liposome base liquid, slowly add it to PBS buffer with a syringe, rotary evaporate, and then pass through a liposome extruder to prepare liposomes loaded with high secretion levels of tuna peptide.

[0136] Performance Testing

[0137] (1) Average particle size

[0138] Examples 2-11 and Comparative Examples 1-4 were diluted 10 times, and the particle sizes were measured at 25° C. using a laser scattering particle size analyzer.

[0139] (2) Encapsulation efficiency

[0140] 0.2 mL of Examples 2-11 and Comparative Examples 1-4 were placed in the top cannula of an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, and centrifuged at 3500 rpm for 20 min in a refrigerated centrifuge (4° C.) to separate free peptides. The top cannula of the centrifuge tube was removed, the liquid in the tube was aspirated and diluted with deionized water, and then measured by the bicinchoninic acid method (BCA method). The encapsulation efficiency equation is as follows: Encapsulation efficiency (%) = (W 总 -W 游离 ) / W游离 ×100%

[0141] Table 1 Average particle size and encapsulation efficiency of Examples 2-11 and Comparative Examples 1-4

[0142]

[0143]

[0144] As can be seen from Table 1, the average particle size and encapsulation efficiency of Examples 2-11 are both higher than those of Comparative Examples 1-4, indicating that compared with genipin and sodium tripolyphosphate modification, the carboxyl and hydroxyl groups in ferulic acid can undergo cross-linking reactions with the amino and thiol groups on the ferritin nanocages, forming more stable interactions, further enhancing the structural stability of the ferritin nanocages, and promoting close bonding between the interior and exterior of the ferritin nanocages, forming a more compact and stable nanostructure, and improving the encapsulation efficiency of the loaded component J1. In addition, ferulic acid, as a naturally occurring phenolic acid, has better biocompatibility and food safety.

[0145] (3) In vitro simulated gastrointestinal digestion test

[0146] Simulated gastric digestion: Example 9 and Comparative Examples 1-4 were placed in simulated gastric fluid with a pH of 1.5 and dissolved evenly. The mixture was incubated at 37°C in a shaker at a speed of 150 rpm for 2 h to obtain gastric digestive fluid. The level of intestinal hormone secretion was detected, and the stability of Example 10 and Comparative Examples 1-4 in the stomach was evaluated.

[0147] Simulated intestinal digestion: The gastric digestive fluid of Example 10 and Comparative Examples 1-4 was placed in a simulated intestinal fluid with a pH of 7.5 and dissolved evenly. The mixture was incubated at 37°C in a shaker at a speed of 150 rpm for 2 h, the enzyme was inactivated in a boiling water bath for 5 min, and the mixture was rapidly cooled and centrifuged at 9000 rpm for 15 min. The supernatant was collected to obtain intestinal digestive fluid. The level of intestinal hormone secretion was detected, and the stability of Example 10 and Comparative Examples 1-4 in the intestine was evaluated.

[0148] Evaluation of intestinal hormone secretion: STC-1 cells were seeded in 24-well plates and cultured at 37°C for 24 h. The cells were washed twice with HBSS buffer (pH 7.4) to remove the culture medium and incubated in a culture incubator with buffer containing gastric digestive juice and intestinal digestive juice, respectively, for 4 h. The cell supernatant was collected for analysis using a CCK detection kit, a GLP-1 detection kit, and a PYY detection kit.

[0149] Depend on Figure 3It can be seen that the simulated gastric fluid components of Comparative Examples 1 and 4 promoted the secretion of CCK, GLP-1 and PYY by STC-1 cells, while Comparative Examples 2 and 3 promoted secretion to a certain extent, but Example 9 had no significant promoting effect. This indicates that when simulating gastric digestion in vitro, Comparative Examples 1 and 4 were decomposed by pepsin in the simulated gastric fluid and under the conditions of gastric fluid pH 1.5, thereby releasing the tuna peptide J1 component. Therefore, the application of the simulated gastric fluid components of Comparative Examples 1 and 4 to STC-1 cells can promote the secretion of CCK, GLP-1 and PYY. At the same time, Comparative Examples 2 and 3 were decomposed to a certain extent and released the tuna peptide J1 component, but Example 9 was able to exist stably in the simulated gastric fluid.

[0150] Depend on Figure 4 It can be seen that the simulated intestinal fluid component of Example 9 promotes the secretion of CCK, GLP-1 and PYY by STC-1 cells, and Figure 1 The effects of the J1 component are similar, but since J1 has been released in the simulated gastric fluid in Comparative Examples 1 and 4, there is no secretion-promoting effect on STC-1 cells in the simulated intestinal digestion. Comparative Examples 2 and 3, in which ferritin nanocages are modified with genipin and sodium tripolyphosphate, respectively, have lower secretion-promoting effects on STC-1 cells than Example 9, indicating that ferulic acid modification imparts good gastric fluid stability to ferritin nanocages. In addition, as the pH of ferulic acid increases from the low pH of simulated gastric fluid to the pH of simulated intestinal fluid, the ferulic acid structure on the ferritin nanocage becomes active, allowing enzymes and other substances in the simulated intestinal fluid to penetrate, promoting the degradation of the ferulic acid-modified ferritin nanocages and releasing the J1 component, which further acts on STC-1 cells to promote the secretion of CCK, GLP-1, and PYY.

[0151] Depend on Figure 5 It can be seen that the simulated intestinal fluid digestion component of Example 9 can inhibit the production of triglycerides in mouse 3T3-L1 cells, and has an anti-obesity effect. Therefore, it can be targeted and delivered to the intestinal part of the body, decomposed by the rich enzyme system and intestinal microorganisms in the body's intestine, and act on the CCK, GLP-1 and PYY receptors in the body's intestine, promoting the secretion of CCK, GLP-1 and PYY, reducing the intake of food other than daily diet, and reducing the accumulation of triglycerides in the body, thereby achieving the purpose of regulating obesity.

[0152] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, use the methods and technical contents disclosed above to make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing protein nanocages loaded with tuna peptides, characterized in that: The following steps are involved: Step 1. Weigh human heavy chain ferritin, add water to dissolve evenly, and prepare a human heavy chain ferritin aqueous solution with a concentration of 250-1000 μg / mL; Step 2. Add 0.1-1 M HCl solution to the human heavy chain ferritin aqueous solution, adjust the pH to 2.0-2.8, maintain for 15-20 minutes, then add 0.1-1 M NaOH solution, adjust the pH to 7.0-8.0, maintain for 1-2 hours, and dialyze three times to obtain a ferritin nanocage solution; Step 3. Add 0.2-1.0% ferulic acid to the ferritin nanocage solution, adjust the pH to 2.0-2.8 with 0.1-1 M HCl solution, incubate at 37°C for 2-6 hours, and dialyze three times to obtain a modified ferritin nanocage solution; Step 4. Take the modified ferritin nanocage solution and add HCl solution to adjust the pH to 2.0-2.8, slowly add tuna peptide, keep at room temperature for 15-20 minutes, then adjust the pH to 7.0-8.0 with NaOH solution, keep for 1-2 hours, and dialyze 3 times to obtain protein nanocages loaded with tuna peptide.

2. The method for preparing a tuna peptide-loaded protein nanocage according to claim 1, characterized in that: In step 4, the volume ratio of the modified ferritin nanocage solution to the tuna peptide is (100-400):

1.

3. The method for preparing a tuna peptide-loaded protein nanocage according to claim 1, characterized in that: The extraction and screening method of tuna peptides comprises the following steps: S1. Take the tuna minced meat scraps, add water at a material-liquid ratio of 1:(2-4) and blend evenly to prepare a tuna minced meat homogenate; S2. The pH of the tuna minced meat homogenate was adjusted to 9-11, and alkaline protease was added to the tuna minced meat homogenate for enzymatic hydrolysis, and then the enzyme was inactivated to obtain a tuna minced meat hydrolysate; S3. Centrifuge the tuna minced meat enzymatic hydrolysate at 8000-10000 rpm and 4°C for 15-20 min to obtain the supernatant. S4. The supernatant was subjected to ultrafiltration separation, evaporation concentration, and freeze-drying to obtain tuna peptides of different molecular weights; S5. Conduct cell experiments with tuna peptides of different molecular weights to evaluate the secretion level of anorectic hormones and screen for highly active tuna peptides.

4. The method for preparing a tuna peptide-loaded protein nanocage according to claim 3, characterized in that: The enzymatic hydrolysis conditions in step S2 are: alkaline protease activity of 200,000 U / g, alkaline protease addition amount of 2500-4500 U / g, enzymatic hydrolysis temperature of 45-55° C., and enzymatic hydrolysis time of 4-6 hours.

5. The method for preparing a tuna peptide-loaded protein nanocage according to claim 3, characterized in that: The conditions for inactivating the enzyme in step S2 are: an enzyme inactivation temperature of 90-95° C. and an enzyme inactivation time of 10-20 min.

6. The method for preparing a tuna peptide-loaded protein nanocage according to claim 3, characterized in that: The ultrafiltration separation conditions in step S4 are as follows: the supernatant is ultrafiltered step by step through 3 kDa and 5 kDa ultrafiltration membranes to retain supernatant ultrafiltration fractions with molecular weights <3 kDa, 3-5 kDa, and >5 kDa.

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