Preparation method and application of protein nanocage loaded with tuna peptide

Abstract

The invention provides a preparation method and application of a protein nanocage loaded with tuna peptide. The preparation method specifically comprises the following steps: constructing and modifying an iron protein nanocage, and extracting, screening and embedding tuna active peptide. The tuna active peptides are embedded in ferulic acid modified ferritin nanocages, and are delivered into intestinal tracts in a targeted manner to react with anorexia hormone receptors, so that the generation of anorexia hormones such as cholecystokinin, glucagon-like peptide 1 and PYY is regulated, the appetite after normal diet is controlled, and the satiety of the body is enhanced; the effects of reducing food intake and reducing body weight are achieved, so that the composition plays a role in regulating obesity. Ferulic acid is utilized to modify the ferritin nanocage, carboxyl and phenolic hydroxyl groups in the ferritin nanocage react with amino terminals on the outer surface of the ferritin nanocage, so that the ferritin nanocage is endowed with a gastric juice environment resisting function, and the ferritin nanocage can stably exist in the stomach of an organism without being decomposed, so that the ferritin nanocage is decomposed in intestinal tracts and plays a role in precise targeting; and a foundation is laid for anti-obesity research.

Description

Technical Field

[0001] The invention belongs to the technical field of functional application of marine products, 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 with rich nutrients and many health benefits. It not only contains glutathione, carnosine, anserine and other peptide components that are vital to the human body, but also rich in trace elements such as zinc and selenium. These components work synergistically to maintain the healthy balance of the human body. However, in the process of tuna processing, by-products accounting for about 50-70% of the total weight will be produced, including fish scraps, viscera, minced meat, fish heads, fish skin and fish bones, etc., which cause great waste of tuna resources and negative effects on the ecological environment. Since the protein content in tuna processing by-products is as high as 80% or more, it is an ideal raw material for preparing tuna peptides. Tuna peptides have become a hot topic in scientific research due to their unique structure and diverse biological activities. They can significantly lower blood pressure, promote blood circulation, and protect cardiovascular health. At the same time, as a powerful natural antioxidant, they can remove free radicals in the body, enhance immunity, and slow down the aging process. It is particularly worth mentioning that the specific ingredients in tuna peptides have a significant effect on improving sleep quality, can regulate the biological clock, promote deep sleep, and keep modern people away from insomnia. In addition, the DHA component in tuna peptides is particularly important for brain health. It can promote the development of nerve cells, improve memory and learning ability, and is an ideal supplement for mental workers. However, there are few reports on its anti-obesity effect. Tuna peptides are a general term for different peptides composed of natural amino acids in different compositions and arrangements, ranging from dipeptides to complex linear and ring structures. Dipeptides, tripeptides and other oligopeptides are more efficiently digested and absorbed by the human body than polypeptides and amino acids of the same composition. The osmotic pressure of oligopeptides is lower than that of amino acids, and they have high permeability in the small intestine of the body and can be efficiently absorbed and utilized. However, most bioactive peptides are oligopeptides, which are easily degraded by enzymes and acidic pH in the oral cavity and gastric juice, thus losing their biological activity. 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 to be decomposed and inactivated, so the delivery system exists stably in gastric juice; in the prior art, ferritin protein nanocages 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 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 bioactive peptides, which is 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

[0003] Technical problems to be solved: In view of the above technical problems, the purpose of the present invention is to provide a method for preparing and applying a protein nanocage loaded with tuna peptides, which specifically includes the construction and modification of ferritin nanocages, and the extraction, screening and embedding of tuna active peptides. Tuna active peptides are embedded in ferritin nanocages modified with ferulic acid, targetedly transported to the intestine, 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 achieving the effect of reducing food intake and reducing weight, thereby playing a role in regulating obesity. In the present invention, ferritin nanocages are modified with ferulic acid, and the carboxyl and phenolic hydroxyl groups in ferulic acid interact with the amino end of the outer surface of ferritin nanocages, giving ferritin nanocages the function of resistant to gastric juice environment, and can stably exist in the stomach of the body without being decomposed, thereby decomposing in the intestines and playing a role in precise targeting, laying the foundation for anti-obesity research.

[0004] Technical solution: A method for preparing a protein nanocage loaded with tuna peptides, comprising the following steps: 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 0.1-1M 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 0.1-1M NaOH solution, adjust the pH to 7.0-8.0, maintain for 1-2 hours, and dialyze 3 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 0.1-1M HCl solution, incubate at 37°C for 2-6 hours, and dialyze 3 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, dialyze 3 times to obtain protein nanocages loaded with tuna peptide. Furthermore, in step 4, the volume ratio of the modified ferritin nanocage solution to the tuna peptide is (100-400):1. Furthermore, the extraction and screening method of tuna peptides comprises the following steps: 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; 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. The tuna minced meat enzymatic hydrolysate was centrifuged at 8000-10000 r / min and 4°C for 15-20 min to obtain a supernatant; S4. The supernatant is subjected to ultrafiltration separation, evaporation concentration, and freeze-drying to obtain tuna peptides of different molecular weights; S5. Carry out cell experiments on tuna peptides of different molecular weights, evaluate the secretion level of anorexia hormone, and screen for highly active tuna peptides. Further, the conditions for enzymatic hydrolysis in step S2 are: alkaline protease activity 200,000 U / g, alkaline protease addition amount 2500-4500 U / g, enzymatic hydrolysis temperature 45-55°C, and enzymatic hydrolysis time 4-6h. 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. 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 components with molecular weights <3 kDa, 3-5 kDa and >5 kDa. Application of the protein nanocage loaded with tuna peptide prepared by any of the preparation methods described above in foods, health products and medicines for improving obesity. Beneficial effects: 1. The tuna peptides 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 digestive tract of the body 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. 2. In the present invention, protein nanocages are used for targeted delivery of tuna peptides, which are accurately transported to the intestinal tract to exert their effects. In addition, 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 to form more stable interactions, further enhancing the structural stability of the ferritin nanocages. At the same time, the close combination between the inside and outside of the ferritin nanocages is promoted to form a more compact and stable nanostructure, which can stably exist 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, and further delivered to the intestines of the body to bind to the corresponding receptors to exert their effects, thereby achieving the purpose of targeted release. 3. The present invention can not only provide a reference for further research on tuna peptides, but also be beneficial to the market development and utilization of tuna peptides, improve the level of deep processing and high-value utilization of tuna peptides, make full use of abundant marine protein resources, show great potential in the field of nutrition and health, and provide a new direction for the innovation and development of the bio-peptide functionalization industry, showing great application potential and value. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The changes of J1, J2 and J3 in Example 1 on the secretion of CCK, PYY and GLP-1 by STC-1 cells; Figure 2 The inhibitory effects of J1, J2, and J3 on triglyceride in mouse 3T3-L1 cells in Example 1; 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; Figure 4 The 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; Figure 5 The inhibitory effects of the simulated intestinal digestion components of Example 10 and Comparative Examples 1-4 on intracellular triglycerides in mouse 3T3-L1 cells. DETAILED DESCRIPTION The present invention will be further described below in conjunction with embodiments, which are explanations of the present invention and are not limited to the following embodiments: Materials used in the present invention: human heavy chain ferritin (molecular weight of about 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. Reagents used in the present invention: Induced differentiation solution A solution (200 μL, 50 μL and 1 mL of insulin solution, dexamethasone solution and 3-isobutyl-1-methylxanthine solution were taken respectively and added to 98.75 mL of DMEM high-glucose culture medium containing 10% (v / v) fetal bovine serum and 1% (v / v) double antibody); Induced differentiation solution B solution (200 μL of insulin solution was taken and dissolved in 98.8 mL of DMEM high-glucose culture medium containing 10% (v / v) fetal bovine serum and 1% (v / v) double antibody) Example 1 A method for extracting and screening tuna peptides comprises the following steps: S1. Take 100g of tuna minced meat scraps, add 400g of water and mix evenly to obtain a tuna minced meat homogenate; 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 the tuna minced meat was enzymatically hydrolyzed at 55° C. for 5 h, and then the enzyme was inactivated at 95° C. for 15 min to obtain a tuna minced meat enzymatic hydrolyzate; S3. The tuna minced meat enzymatic hydrolysate was centrifuged at 10000 r / min and 4°C for 20 min to obtain a supernatant; S4. The supernatant is separated by ultrafiltration through 5kDa and 3kDa ultrafiltration membranes step by step, and the supernatant ultrafiltration fractions with molecular weights <3kDa, 3-5kDa and >5kDa are retained, and then evaporated, concentrated and freeze-dried to obtain tuna peptides with molecular weights <3kDa, 3-5kDa and >5kDa; S5. Tuna peptides with molecular weights <3kDa, 3-5kDa and >5kDa were used for in vitro cell experiments to evaluate the secretion level of anorexigenic hormones and screen for highly active tuna peptides; Among them, the tuna peptide with a molecular weight of <3kDa was recorded as J1, the tuna peptide with a molecular weight of 3-5kDa was recorded as J2, and the tuna peptide with a molecular weight of >5kDa was recorded as J3. Performance Testing (1) STC-1 cell in vitro experiments STC-1 cell culture: STC-1 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin and 1% NEAA, and stored in a 5% CO2 humidified atmosphere at 37°C. When the confluence reached 80-90%, trypsinization and subculture were routinely performed, and STC-1 cells of 10-20 generations were used. 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 at pH 7.4 to remove the culture medium and incubated in an incubator with buffer containing J1, J2, and J3 for 2 h. The cell supernatants were collected for analysis and measured using CCK detection kits, GLP-1 detection kits, and PYY detection kits. 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 level of secretion promotion because the molecular weight of J1 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. (2) Evaluation of anti-obesity effects in vitro Cell culture: Mouse 3T3-L1 cells were cultured in high-glucose DMEM medium containing 10% (v / v) newborn calf serum. When the cells grew to the logarithmic phase, they were inoculated 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 48 hours. The cells were divided into 5 groups (including blank group, model group, J1 group, J2 group, and J3 group). Target component culture: J1, J2, and J3 groups were replaced with induction differentiation solution A containing J1, J2, and J3 components for 60 hours, and then replaced with induction differentiation solution B containing the corresponding J1, J2, and J3 components for 60 hours, and then replaced with DMEM medium containing 10% (v / v) fetal bovine serum and the same concentration of the same peptide for 72 hours. The blank group was replaced with DMEM medium containing 10% (v / v) newborn calf serum instead of the induction differentiation solution, and the model group was replaced with culture medium instead of peptide solution. Triglyceride detection: Use a triglyceride kit to measure the triglyceride content in cells. Depend on Figure 2It can be seen that J1, J2, and J3 can all act on mouse 3T3-L1 cells to inhibit the formation of triglycerides, and J1 has the highest inhibitory effect, indicating that the molecular active peptides produced by tuna after enzymatic hydrolysis have the effect of regulating obesity. Combined with the in vitro test of STC-1 cells, it is shown that tuna peptides promote the intestinal secretion of anorexigenic hormones, 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. Example 2 A method for preparing a modified ferritin nanocage loaded with highly active tuna peptides comprises the following steps: Step 1. Weigh 250 μg of human heavy chain ferritin, add 1 mL of water to dissolve evenly, and prepare a human heavy chain ferritin aqueous solution with a concentration of 250 μg / mL; Step 2. Slowly add 1M HCl solution to the human heavy chain ferritin aqueous solution, adjust the pH to 2.0, maintain for 15 minutes, then add 0.1M NaOH solution, adjust the pH to 7.4, maintain for 2 hours, and dialyze three times to obtain a ferritin nanocage solution; 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, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution. 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 min, then adjust the pH to 7.4 with 1 M NaOH solution, keep for 2 h, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptides. Example 3 A method for preparing a modified ferritin nanocage loaded with highly active tuna peptides comprises the following steps: Step 1. Weigh 500 μg of human heavy chain ferritin, add 1 mL of water to dissolve evenly, and prepare a human heavy chain ferritin aqueous solution with a concentration of 500 μg / mL; Step 2. Slowly add 1M HCl solution to the human heavy chain ferritin aqueous solution, adjust the pH to 2.5, maintain for 20 minutes, then add 0.1M NaOH solution, adjust the pH to 7.0, maintain for 2 hours, and dialyze three times to obtain a ferritin nanocage solution; 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, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution. 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 min, then adjust the pH to 7.4 with 1 M NaOH solution, keep for 2 h, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptides. Example 4 A method for preparing a modified ferritin nanocage loaded with highly active tuna peptides comprises the following steps: Step 1. Weigh 750 μg of human heavy chain ferritin, add 1 mL of water to dissolve evenly, and prepare a human heavy chain ferritin aqueous solution with a concentration of 750 μg / mL; Step 2. Slowly add 1M HCl solution to the human heavy chain ferritin aqueous solution, adjust the pH to 2.0, maintain for 20 minutes, then add 0.1M NaOH solution, adjust the pH to 7.0, maintain for 2 hours, and dialyze three times to obtain a ferritin nanocage solution; 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, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution. 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 min, then adjust the pH to 7.4 with 1 M NaOH solution, keep for 2 h, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptides. Example 5 A method for preparing a modified ferritin nanocage loaded with highly active tuna peptides comprises the following steps: Step 1. Weigh 1000 μg of human heavy chain ferritin, add 1 mL of water to dissolve evenly, and prepare a human heavy chain ferritin aqueous solution with a concentration of 1000 μg / mL; Step 2. Slowly add 1M HCl solution to the human heavy chain ferritin aqueous solution, adjust the pH to 2.4, maintain for 15 minutes, then add 0.1M NaOH solution, adjust the pH to 7.4, maintain for 2 hours, and dialyze three times to obtain a ferritin nanocage solution; 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, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution. 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 min, then adjust the pH to 7.4 with 1 M NaOH solution, keep for 2 h, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptides. Example 6 A method for preparing a modified ferritin nanocage loaded with highly active tuna peptides comprises the following steps: Step 1. Weigh 1000 μg of human heavy chain ferritin, add 1 mL of water to dissolve evenly, and prepare a human heavy chain ferritin aqueous solution with a concentration of 1000 μg / mL; Step 2. Slowly add 1M HCl solution to the human heavy chain ferritin aqueous solution, adjust the pH to 2.0, maintain for 15 minutes, then add 0.1M NaOH solution, adjust the pH to 7.4, maintain for 2 hours, and dialyze three times to obtain a ferritin nanocage solution; 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, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution. 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 min, then adjust the pH to 7.4 with 1 M NaOH solution, keep for 2 h, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptides. Example 7 A method for preparing a modified ferritin nanocage loaded with highly active tuna peptides comprises the following steps: Step 1. Weigh 1000 μg of human heavy chain ferritin, add 1 mL of water to dissolve evenly, and prepare a human heavy chain ferritin aqueous solution with a concentration of 1000 μg / mL; Step 2. Slowly add 1M HCl solution to the human heavy chain ferritin aqueous solution, adjust the pH to 2.0, maintain for 20 minutes, then add 0.1M NaOH solution, adjust the pH to 7.4, maintain for 2 hours, and dialyze three times to obtain a ferritin nanocage solution; 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, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution. 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 min, then adjust the pH to 7.4 with 1 M NaOH solution, keep for 2 h, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptides. Example 8 A method for preparing a modified ferritin nanocage loaded with highly active tuna peptides comprises the following steps: Step 1. Weigh 1000 μg of human heavy chain ferritin, add 1 mL of water to dissolve evenly, and prepare a human heavy chain ferritin aqueous solution with a concentration of 1000 μg / mL; Step 2. Slowly add 1M HCl solution to the human heavy chain ferritin aqueous solution, adjust the pH to 2.4, maintain for 20 minutes, then add 0.1M NaOH solution, adjust the pH to 8.0, maintain for 2 hours, and dialyze three times to obtain a ferritin nanocage solution; 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, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution. 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 min, then adjust the pH to 7.4 with 1 M NaOH solution, keep for 2 h, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptides. Example 9 A method for preparing a modified ferritin nanocage loaded with highly active tuna peptides comprises the following steps: Step 1. Weigh 1000 μg of human heavy chain ferritin, add 1 mL of water to dissolve evenly, and prepare a human heavy chain ferritin aqueous solution with a concentration of 1000 μg / mL; Step 2. Slowly add 1M HCl solution to the human heavy chain ferritin aqueous solution, adjust the pH to 2.0, maintain for 15 minutes, then add 0.1M NaOH solution, adjust the pH to 7.0, maintain for 2 hours, and dialyze three times to obtain a ferritin nanocage solution; 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, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution. 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 min, then adjust the pH to 7.4 with 1 M NaOH solution, keep for 2 h, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptides. Example 10 A method for preparing a modified ferritin nanocage loaded with highly active tuna peptides comprises the following steps: Step 1. Weigh 1000 μg of human heavy chain ferritin, add 1 mL of water to dissolve evenly, and prepare a human heavy chain ferritin aqueous solution with a concentration of 1000 μg / mL; Step 2. Slowly add 1M HCl solution to the human heavy chain ferritin aqueous solution, adjust the pH to 2.4, maintain for 20 minutes, then add 0.1M NaOH solution, adjust the pH to 8.0, maintain for 2 hours, and dialyze three times to obtain a ferritin nanocage solution; 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, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution. 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 min, then adjust the pH to 7.4 with 1 M NaOH solution, keep for 2 h, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptides. Embodiment 11 A method for preparing a modified ferritin nanocage loaded with highly active tuna peptides comprises the following steps: Step 1. Weigh 1000 μg of human heavy chain ferritin, add 1 mL of water to dissolve evenly, and prepare a human heavy chain ferritin aqueous solution with a concentration of 1000 μg / mL; Step 2. Slowly add 1M HCl solution to the human heavy chain ferritin aqueous solution, adjust the pH to 2.4, maintain for 20 minutes, then add 0.1M NaOH solution, adjust the pH to 8.0, maintain for 2 hours, and dialyze three times to obtain a ferritin nanocage solution; 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, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution. 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 min, then adjust the pH to 7.4 with 1 M NaOH solution, keep for 2 h, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptides. Comparative Example 1 The difference between this comparative example and Example 9 is that no ferulic acid modification was used. A method for preparing a ferritin nanocage loaded with a high secretion level of tuna peptide comprises the following steps: Step 1. Weigh 1000 μg of human heavy chain ferritin, add 1 mL of water to dissolve evenly, and prepare a human heavy chain ferritin aqueous solution with a concentration of 1000 μg / mL; Step 2. Slowly add 1M HCl solution to the human heavy chain ferritin aqueous solution, adjust the pH to 2.0, maintain for 15 minutes, then add 0.1M NaOH solution, adjust the pH to 7.0, maintain for 2 hours, and dialyze three times to obtain a ferritin nanocage solution; 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, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptides. Comparative Example 2 The difference between this comparative example and Example 9 is that genipin is used for modification. A method for preparing a modified ferritin nanocage loaded with highly active tuna peptides comprises the following steps: Step 1. Weigh 1000 μg of human heavy chain ferritin, add 1 mL of water to dissolve evenly, and prepare a human heavy chain ferritin aqueous solution with a concentration of 1000 μg / mL; Step 2. Slowly add 1M HCl solution to the human heavy chain ferritin aqueous solution, adjust the pH to 2.0, maintain for 15 minutes, then add 0.1M NaOH solution, adjust the pH to 7.0, maintain for 2 hours, and dialyze three times to obtain a ferritin nanocage solution; 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, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution. 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 min, then adjust the pH to 7.4 with 1 M NaOH solution, keep for 2 h, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptides. Comparative Example 3 The difference between this comparative example and Example 9 is that sodium tripolyphosphate is used for modification. A method for preparing a modified ferritin nanocage loaded with highly active tuna peptides comprises the following steps: Step 1. Weigh 1000 μg of human heavy chain ferritin, add 1 mL of water to dissolve evenly, and prepare a human heavy chain ferritin aqueous solution with a concentration of 1000 μg / mL; Step 2. Slowly add 1M HCl solution to the human heavy chain ferritin aqueous solution, adjust the pH to 2.0, maintain for 15 minutes, then add 0.1M NaOH solution, adjust the pH to 7.0, maintain for 2 hours, and dialyze three times to obtain a ferritin nanocage solution; 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, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain a modified ferritin nanocage solution. 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 min, then adjust the pH to 7.4 with 1 M NaOH solution, keep for 2 h, dialyze three times in pH 8.0, 25 mM Tris-HCl buffer to obtain modified ferritin nanocages loaded with high secretion levels of tuna peptides. Comparative Example 4 The difference between this comparative example and Example 9 is that the delivery system is replaced by liposomes instead of ferritin nanocages. A method for preparing liposomes loaded with highly active tuna peptides comprises the following steps: Step 1. Weigh 20 mg of soybean lecithin and 4 mg of cholesterol, 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 obtain liposomes loaded with high secretion levels of tuna peptides. Performance Testing (1) Average particle size 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. (2) Encapsulation efficiency 0.2 mL of Examples 2-11 and Comparative Examples 1-4 were placed in the top sleeve of an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, placed in a refrigerated centrifuge (4° C.) and centrifuged at 3500 rpm for 20 min to separate free peptides, the top sleeve 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% Table 1 Average particle size and encapsulation efficiency of Examples 2-11 and Comparative Examples 1-4 As can be seen from Table 1, the average particle size and encapsulation efficiency of Examples 2-11 are higher than those of Comparative Examples 1-4, indicating that compared with the modification of genipin and sodium tripolyphosphate, the carboxyl and hydroxyl groups in ferulic acid can undergo cross-linking reactions with the amino and thiol groups on the ferritin nanocages to form more stable interactions, further enhancing the structural stability of the ferritin nanocages, while promoting close bonding between the inside and outside of the ferritin nanocages to form a more compact and stable nanostructure, thereby improving the encapsulation efficiency of the loaded component J1. Moreover, ferulic acid, as a naturally occurring phenolic acid, has better biocompatibility and food safety. (3) In vitro simulated gastrointestinal digestion test 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, and incubated at 37°C for 2 h in a shaker with a rotation speed of 150 rpm to obtain gastric digestive fluid, and the level of intestinal hormone secretion was detected to evaluate the stability of Example 10 and Comparative Examples 1-4 in the stomach. Simulated intestinal digestion: The gastric digestive juice of Example 10 and Comparative Examples 1-4 was placed in a simulated intestinal juice with a pH of 7.5 and dissolved evenly, incubated at 37°C for 2 h in a shaker with a rotation speed of 150 rpm, inactivated the enzyme in a boiling water bath for 5 min, cooled rapidly, centrifuged at 9000 rpm for 15 min, and the supernatant was taken to obtain intestinal digestive juice, and the intestinal hormone secretion level was detected to evaluate the stability of Example 10 and Comparative Examples 1-4 in the intestine. 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 at pH 7.4 to remove the culture medium, and incubated in an incubator with buffer containing gastric digestive juice and intestinal digestive juice for 4 h, respectively. The cell supernatant was collected for analysis and measured using CCK detection kit, GLP-1 detection kit and PYY detection kit. Depend on Figure 3It can be seen that the simulated gastric juice components of Comparative Examples 1 and 4 promote the secretion of CCK, GLP-1 and PYY by STC-1 cells, while Comparative Examples 2 and 3 promote secretion to a certain extent, but Example 9 has no significant promoting effect, indicating that when simulating gastric digestion in vitro, Comparative Examples 1 and 4 are decomposed under the conditions of pepsin in simulated gastric juice and gastric juice pH 1.5, thereby releasing the tuna peptide J1 component. Therefore, the simulated gastric juice components of Comparative Examples 1 and 4 are applied to STC-1 cells to promote the secretion of CCK, GLP-1 and PYY. At the same time, Comparative Examples 2 and 3 are decomposed to a certain extent to release the tuna peptide J1 component, but Example 9 can stably exist in simulated gastric juice. 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 juice in Comparative Examples 1 and 4, they have no secretion-promoting effect on STC-1 cells in the simulated intestinal digestion. Comparative Examples 2 and 3 use genipin and sodium tripolyphosphate to modify the ferritin nanocages, respectively, and their secretion-promoting effects on STC-1 cells are lower than that in Example 9, indicating that ferulic acid modification gives the ferritin nanocages good gastric juice stability, and as the pH of ferulic acid increases from the low pH of simulated gastric juice to the pH of simulated intestinal juice, the ferulic acid structure on the ferritin nanocage becomes active, and enzymes and other substances in the simulated intestinal juice begin to penetrate, promoting the degradation of the ferulic acid-modified ferritin nanocages, releasing the J1 component, which further acts on STC-1 cells to promote the secretion of CCK, GLP-1 and PYY. 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 in 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, promote the secretion of CCK, GLP-1 and PYY, reduce the intake of food other than daily diet, and reduce the accumulation of triglycerides in the body, thereby achieving the purpose of regulating obesity. The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing a protein nanocage loaded with tuna peptide, 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 0.1-1M 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 0.1-1M NaOH solution, adjust the pH to 7.0-8.0, maintain for 1-2 hours, and dialyze 3 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 0.1-1M HCl solution, incubate at 37°C for 2-6 hours, and dialyze 3 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, dialyze 3 times to obtain protein nanocages loaded with tuna peptide.

2. The method for preparing a protein nanocage loaded with tuna peptide 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 protein nanocage loaded with tuna peptide 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 according to the material-liquid ratio of 1: (2-4) and evenly blend to obtain 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. The tuna minced meat enzymatic hydrolysate was centrifuged at 8000-10000 r / min and 4°C for 15-20 min to obtain a supernatant; S4. The supernatant is subjected to ultrafiltration separation, evaporation concentration, and freeze-drying to obtain tuna peptides of different molecular weights; S5. Carry out cell experiments with tuna peptides of different molecular weights to evaluate the secretion level of anorexic hormones and screen for highly active tuna peptides.

4. The method for preparing a protein nanocage loaded with tuna peptide according to claim 3, characterized in that: The conditions for enzymatic hydrolysis 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 h.

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

6. The method for preparing a protein nanocage loaded with tuna peptide 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 3kDa and 5kDa ultrafiltration membranes to intercept supernatant ultrafiltration components with molecular weights <3kDa, 3-5kDa and >5kDa.

7. Use of the protein nanocage loaded with tuna peptide prepared according to the preparation method of claims 1-6 in foods, health products and medicines for improving obesity.

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