Heme peptide iron hydrogel with pH response and preparation method thereof
By constructing soy protein isolate-carboxymethylchitosan-heme peptide iron complex, forming a pH-responsive hydrogel, solving the problems of iron stability of heme peptide and expensive traditional technical equipment, and achieving efficient heme Iron peptide embedding and sustained release effects.
Patent Information
- Application Number
- CN202411287776.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-14
- Publication Date
- 2024-12-13
AI Technical Summary
The prior art is difficult to effectively maintain the stability of the heme peptide iron, especially during the gastrointestinal digestion process, and traditional vacuum freeze-drying technology is expensive and equipment requirements are high.
Soy protein isolate-carboxymethyl chitosan-heme peptide iron complex was constructed by inducing cross-linking method to form a pH-responsive 3D reticular hydrogel, and the extraction rate and activity of heme peptide iron were improved by adding protective agents. , forming an embedded system with physical stability and mechanical strength.
It significantly improves the stability and bioavailability of heme peptide iron, extends the shelf life of the product, avoids oxidative losses, and achieves a sustained release effect in simulated digestion of the gastrointestinal tract.
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Figure CN119113090A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of heme peptide iron, and specifically relates to a heme peptide iron hydrogel with pH response and a preparation method thereof. Background Art
[0002] Hemoglobin is the most abundant protein in livestock and poultry blood. Making full use of hemoglobin is of great significance to improving the added value of livestock and poultry blood. Producing heme peptide iron by enzymatic hydrolysis of hemoglobin is an important application approach. Compared with pure heme, heme peptide iron has better water solubility, fewer side effects and higher bioavailability. In addition, heme peptide iron has many functional properties, especially antioxidant activity, and has received widespread attention in recent years. However, heme peptide iron is easily affected by gastric acid and proteases during gastrointestinal digestion, thereby reducing its activity. Therefore, it is very important to improve the activity stability of heme peptide iron.
[0003] Adding nitric oxide (NO) or carbon monoxide (CO) is a common method to enhance the stability of heme peptide iron. It has been reported that NO or CO can form a more stable structure with heme, thereby improving the color protection and stability of heme. However, both CO and NO are toxic and have poor protection effects, which limits their application in the preparation of heme peptide iron.
[0004] CN201810597655.X discloses a ferrous heme oligopeptide microcapsule powder and a preparation method thereof, wherein the heme peptide iron and porous starch are used as core materials, and gum arabic is used as wall materials to prepare heme peptide iron microcapsules, and the heme peptide iron microcapsules are obtained by vacuum freeze drying. However, the vacuum freeze drying technology involved in the method has high requirements for equipment and is relatively expensive.
[0005] Therefore, there is an urgent need to develop a safe and effective preparation method in the art. Summary of the invention
[0006] In view of the technical problems that the prior art cannot maintain the stability of heme peptide iron well, the present invention provides a pH-responsive heme peptide iron hydrogel and a preparation method thereof, and constructs a soy protein isolate-carboxymethyl chitosan-heme peptide iron complex delivery system by using an induced cross-linking method. The potential of the delivery system in heme peptide iron encapsulation and sustained release is evaluated by combining in vitro simulated sustained release experiments and other tests.
[0007] To achieve the above object, the present invention provides the following technical solutions:
[0008] A method for preparing a pH-responsive heme peptide iron hydrogel comprises the following steps:
[0009] (1) adding lysine and ascorbic acid to a hemoglobin solution, reacting for a period of time, adjusting the pH, performing enzymatic hydrolysis, centrifuging, taking the supernatant, and freeze-drying to obtain hemoglobin peptide iron freeze-dried powder;
[0010] (2) weighing a certain amount of soy protein isolate and dissolving it in ultrapure water, stirring it magnetically for 2 hours to completely hydrate it, heating the hydrated soy protein isolate solution in a hot water bath to obtain soy protein isolate aggregates, then rapidly cooling it to room temperature and storing it, stirring it magnetically overnight to completely hydrate it, and centrifuging to remove large particles to obtain a soy protein isolate aggregate solution;
[0011] (3) Weigh a certain amount of carboxymethyl chitosan and dissolve it in ultrapure water, sonicate, and stir to completely hydrate it;
[0012] (4) Preparation of soy protein isolate-carboxymethyl chitosan-heme peptide iron hydrogel: The heme peptide iron aqueous solution is added to the soy protein isolate aggregate solution and magnetically stirred at room temperature. Genipin is dissolved in anhydrous ethanol and added to the soy protein isolate solution under magnetic stirring conditions. Subsequently, the carboxymethyl chitosan solution is added to obtain the soy protein isolate-carboxymethyl chitosan-heme peptide iron hydrogel.
[0013] Wherein, in the step (1), the amount of lysine added is 1% (w / v), and the amount of ascorbic acid added is 0.08% (w / v).
[0014] Wherein, in the step (1), the reaction time is 30 min.
[0015] Wherein, in the step (2), the soy protein isolate solution is stirred in a water bath at 85° C. for 30 minutes, then rapidly cooled to room temperature, and stirred and refrigerated at 4° C. overnight.
[0016] Wherein, in the step (2), centrifugation is performed at 8000×g for 20 min to remove large particles.
[0017] Wherein, in the step (3), the mass concentration of the prepared carboxymethyl chitosan solution is 1.25% to 5%.
[0018] Wherein, in the step (4), the heme peptide iron aqueous solution is added to the soy protein isolate aggregate solution, and the final concentration of the heme peptide iron is 1 mg / mL.
[0019] Wherein, in the step (4), Genipin is dissolved in anhydrous ethanol and added to the soy protein isolate solution under magnetic stirring conditions, and the final concentration of Genipin is 0.2% (w / v).
[0020] Wherein, in the step (4), the carboxymethyl chitosan solution is added in a volume ratio of 15:4.
[0021] Compared with the prior art, the present invention has the following beneficial effects:
[0022] The present invention adds a protective agent and uses an induced cross-linking method for embedding, that is, a protective agent is added during enzymatic hydrolysis, and the yield of heme peptide iron is increased by 89.64%, reaching 93.02%, and the activity of heme peptide iron is maintained. Soybean protein isolate and carboxymethyl chitosan are added to form a pH-responsive 3D mesh hydrogel polymerization system to embed the heme peptide iron. The gel formed by natural proteins and polysaccharides has physical stability and mechanical strength, which can prevent the heme peptide iron from being adversely affected by environmental conditions during storage and processing, and has a sustained release effect during simulated digestion of the gastrointestinal tract.
[0023] The present invention provides a new method for the research and development of new heme peptide iron products, which can overcome the defect that heme peptide iron itself is easily oxidized, effectively prevent the loss of heme peptide iron during processing, improve its stability and convenience to a certain extent, greatly extend the shelf life of the product, and significantly improve its bioavailability, which has important social and economic value. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 This is the effect of different protective agents on the iron yield of heme peptide.
[0025] Figure 2 Flow chart for the preparation of soy protein isolate-carboxymethyl chitosan-heme peptide iron hydrogel.
[0026] Figure 3 This is the appearance picture of samples M1, M2 and M3.
[0027] Figure 4 This is the in vitro release curve of sample M1 under different pH environments.
[0028] Figure 5 This is the in vitro release curve of sample M2 under different pH environments.
[0029] Figure 6 This is the in vitro release curve of sample M3 under different pH environments.
[0030] Figure 7 The microscopic morphology characteristics of sample M3 under different pH environments.
[0031] Figure 8 In vitro release curves of samples M1, M2 and M3 in a simulated gastrointestinal environment. DETAILED DESCRIPTION
[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0033] Example 1
[0034] like Figure 2 As shown, a method for preparing a pH-responsive heme peptide iron hydrogel comprises the following specific steps:
[0035] (1) Thawed red blood cells were mixed with water at a ratio of 10:13 (w / w), and 1% lysine and 0.08% ascorbic acid were added. After 30 minutes of reaction, the pH of the mixture was adjusted to 10 with 5M NaOH, and alkaline protease was added. Incubate at 45°C for 5 hours and heat at 80°C for 10 minutes to inactivate the alkaline protease. Centrifuge at 5000 r / min for 10 minutes, take the supernatant, and freeze-dry it for further operation.
[0036] (2) Dissolve soy protein isolate powder in ultrapure water at room temperature (25°C) to prepare an 8% (w / v) soy protein isolate solution, stir it magnetically for 2 hours to fully hydrate it, then stir the soy protein isolate solution in a water bath at 85°C for 30 minutes, then quickly cool it to room temperature, and stir and refrigerate it at 4°C overnight to ensure that the protein is completely dissolved, and centrifuge it at 8000×g for 20 minutes to remove large particles, thereby obtaining a soy protein isolate solution.
[0037] (3) A certain amount of carboxymethyl chitosan was weighed and dissolved in ultrapure water to prepare a carboxymethyl chitosan solution with a concentration of 1.25%.
[0038] (4) Preparation of soy protein isolate-carboxymethyl chitosan-heme peptide iron hydrogel: The heme peptide iron lyophilized powder was added to the soy protein isolate aggregate solution, and the final heme peptide iron reached a final concentration of 1 mg / mL, and magnetically stirred at room temperature for 2 h. The Genipin solution was added to the soy protein isolate solution to reach a final concentration of 0.2% (w / v). Subsequently, the carboxymethyl chitosan solution was added at a volume ratio of 15:4 and stirred for 6 h. The mixed solution was then quickly poured into a vial (diameter 27.5 mm, height 57 mm), and cured at 4°C for 24 h to finally obtain the heme peptide iron hydrogel sample M1.
[0039] (5) The obtained sample M1 was stored at room temperature for further analysis. Figure 3 shown.
[0040] Example 2
[0041] like Figure 2 As shown, a method for preparing a pH-responsive heme peptide iron hydrogel comprises the following specific steps:
[0042] (1) Thawed red blood cells were mixed with water at a ratio of 10:13 (w / w), and 1% lysine and 0.08% ascorbic acid were added. After 30 minutes of reaction, the pH of the mixture was adjusted to 10 with 5M NaOH, and alkaline protease was added. Incubate at 45°C for 5 hours and heat at 80°C for 10 minutes to stop the enzyme activity. Centrifuge at 5000 r / min for 10 minutes, take the supernatant, and freeze-dry it for further operation.
[0043] (2) Dissolve soy protein isolate powder in ultrapure water at room temperature (25°C) to prepare an 8% (w / v) soy protein isolate solution, stir it magnetically for 2 hours to fully hydrate it, then stir the soy protein isolate solution in a water bath at 85°C for 30 minutes, then quickly cool it to room temperature, and stir and refrigerate it at 4°C overnight to ensure that the protein is completely dissolved, and centrifuge it at 8000×g for 20 minutes to remove large particles, thereby obtaining a soy protein isolate solution.
[0044] (3) A certain amount of carboxymethyl chitosan was weighed and dissolved in ultrapure water to prepare a carboxymethyl chitosan solution with a concentration of 2.5%.
[0045] (4) Preparation of soy protein isolate-carboxymethyl chitosan-heme peptide iron hydrogel: The heme peptide iron lyophilized powder was added to the soy protein isolate aggregate solution, and the final heme peptide iron reached a final concentration of 1 mg / mL, and magnetic stirring was carried out at room temperature for 2 hours. The Genipin solution was added to the soy protein isolate solution to reach a final concentration of 0.2% (w / v). Subsequently, the carboxymethyl chitosan solution was added at a volume ratio of 15:4 and stirred for 6 hours, and then the mixed solution was quickly poured into a vial (diameter 27.5 mm, height 57 mm), and cured at 4°C for 24 hours to finally obtain the heme peptide iron hydrogel sample M2.
[0046] (5) The obtained sample M2 was stored at room temperature for further analysis. Figure 3 shown.
[0047] Example 3
[0048] like Figure 2 As shown, a method for preparing a pH-responsive heme peptide iron hydrogel comprises the following specific steps:
[0049] (1) Thawed red blood cells were mixed with water at a ratio of 10:13 (w / w), and 1% lysine and 0.08% ascorbic acid were added. After 30 minutes of reaction, the pH of the mixture was adjusted to 10 with 5M NaOH, and alkaline protease was added. Incubate at 45°C for 5 hours and heat at 80°C for 10 minutes to stop the enzyme activity. Centrifuge at 5000 r / min for 10 minutes, take the supernatant, and freeze-dry it for further operation.
[0050] (2) Dissolve soy protein isolate powder in ultrapure water at room temperature (25°C) to prepare an 8% (w / v) soy protein isolate solution, stir it magnetically for 2 hours to fully hydrate it, then stir the soy protein isolate solution in a water bath at 85°C for 30 minutes, then quickly cool it to room temperature, and stir and refrigerate it at 4°C overnight to ensure that the protein is completely dissolved, and centrifuge it at 8000×g for 20 minutes to remove large particles, thereby obtaining a soy protein isolate solution.
[0051] (3) A certain amount of carboxymethyl chitosan was weighed and dissolved in ultrapure water to prepare a carboxymethyl chitosan solution with a concentration of 5%.
[0052] (4) Preparation of soy protein isolate-carboxymethyl chitosan-heme peptide iron hydrogel: The heme peptide iron lyophilized powder was added to the soy protein isolate aggregate solution, and the final heme peptide iron reached a final concentration of 1 mg / mL, and magnetic stirring was carried out at room temperature for 2 h. The Genipin solution was added to the soy protein isolate solution to reach a final concentration of 0.2% (w / v). Subsequently, the carboxymethyl chitosan solution was added at a volume ratio of 15:4 and stirred for 6 h, and then the mixed solution was quickly poured into a vial (diameter 27.5 mm, height 57 mm), and cured at 4 ° C for 24 h to finally obtain the heme peptide iron hydrogel sample M3.
[0053] (5) The obtained sample M3 was stored at room temperature for further analysis. Figure 3 shown.
[0054] In order to highlight the beneficial effects of the present invention, the following comparative example experiments are given.
[0055] Comparative Example 1
[0056] (1) Thawed red blood cells were mixed with water at a ratio of 10:13 (w / w), the pH of the mixture was adjusted to 10 with 5M NaOH, and alkaline protease was added. Incubate at 45°C for 5 h, and heat at 80°C for 10 min to stop the enzyme activity. Centrifuge at 5000 r / min for 10 min, take the supernatant, and freeze-dry for further operation.
[0057] (2) Dissolve soy protein isolate powder in ultrapure water at room temperature (25°C) to prepare an 8% (w / v) soy protein isolate solution, stir it magnetically for 2 hours to fully hydrate it, then stir the soy protein isolate solution in a water bath at 85°C for 30 minutes, then quickly cool it to room temperature, and stir and refrigerate it at 4°C overnight to ensure that the protein is completely dissolved, and centrifuge it at 8000×g for 20 minutes to remove large particles, thereby obtaining a soy protein isolate solution.
[0058] (3) A certain amount of carboxymethyl chitosan was weighed and dissolved in ultrapure water to prepare a carboxymethyl chitosan solution with a concentration of 2.5%.
[0059] (4) Preparation of soy protein isolate-carboxymethyl chitosan-heme peptide iron hydrogel: The heme peptide iron lyophilized powder was added to the soy protein isolate aggregate solution, and the final heme peptide iron reached a final concentration of 1 mg / mL, and magnetic stirring was carried out at room temperature for 2 h. The Genipin solution was added to the soy protein isolate solution to reach a final concentration of 0.2% (w / v). Subsequently, the carboxymethyl chitosan solution was added at a volume ratio of 15:4 and stirred for 6 h, and then the mixed solution was quickly poured into a vial (diameter 27.5 mm, height 57 mm), and cured at 4 ° C for 24 h to finally obtain the heme peptide iron hydrogel sample M4.
[0060] (5) The obtained sample M4 was stored at room temperature for further analysis.
[0061] The difference between Comparative Example 1 and Example 2 is that no protective agent is added during the preparation of heme peptide iron. Figure 1 As shown, compared with the sample M2 obtained in Example 2, the extraction rate of heme peptide iron is 49.05%, which is lower.
[0062] Comparative Example 2
[0063] (1) Thawed red blood cells were mixed with water at a ratio of 10:13 (w / w), 1% lysine was added, and the mixture was reacted for 30 min. The pH of the mixture was adjusted to 10 with 5 M NaOH, and alkaline protease was added. The mixture was incubated at 45°C for 5 h and heated at 80°C for 10 min to stop the enzyme activity. The supernatant was centrifuged at 5000 r / min for 10 min and freeze-dried for further operation.
[0064] (2) Dissolve soy protein isolate powder in ultrapure water at room temperature (25°C) to prepare an 8% (w / v) soy protein isolate solution, stir it magnetically for 2 hours to fully hydrate it, then stir the soy protein isolate solution in a water bath at 85°C for 30 minutes, then quickly cool it to room temperature, and stir and refrigerate it at 4°C overnight to ensure that the protein is completely dissolved, and centrifuge it at 8000×g for 20 minutes to remove large particles, thereby obtaining a soy protein isolate solution.
[0065] (3) A certain amount of carboxymethyl chitosan was weighed and dissolved in ultrapure water to prepare a carboxymethyl chitosan solution with a concentration of 2.5%.
[0066] (4) Preparation of soy protein isolate-carboxymethyl chitosan-heme peptide iron hydrogel: The heme peptide iron lyophilized powder was added to the soy protein isolate aggregate solution, and the final heme peptide iron reached a final concentration of 1 mg / mL, and magnetic stirring was carried out at room temperature for 2 hours. The Genipin solution was added to the soy protein isolate solution to reach a final concentration of 0.2% (w / v). Subsequently, the carboxymethyl chitosan solution was added at a volume ratio of 15:4 and stirred for 6 hours, and then the mixed solution was quickly poured into a vial (diameter 27.5 mm, height 57 mm), and cured at 4°C for 24 hours to finally obtain the heme peptide iron hydrogel sample M5.
[0067] (5) The obtained sample M5 was stored at room temperature for further analysis.
[0068] The difference between Comparative Example 2 and Example 2 is that only 1% lysine was added during the preparation of heme peptide iron. Figure 1 As shown, compared with the sample M2 obtained in Example 2, the extraction rate of heme peptide iron is 89.27%, and the extraction rate needs to be improved.
[0069] Comparative Example 3
[0070] (1) Thawed red blood cells were mixed with water at a ratio of 10:13 (w / w), and 0.08% ascorbic acid was added. After reacting for 30 minutes, the pH of the mixture was adjusted to 10 with 5M NaOH, and alkaline protease was added. Incubate at 45°C for 5 hours, and heat at 80°C for 10 minutes to stop the enzyme activity. Centrifuge at 5000 r / min for 10 minutes, take the supernatant, and freeze-dry it for further operation.
[0071] (2) Dissolve soy protein isolate powder in ultrapure water at room temperature (25°C) to prepare an 8% (w / v) soy protein isolate solution, stir it magnetically for 2 hours to fully hydrate it, then stir the soy protein isolate solution in a water bath at 85°C for 30 minutes, then quickly cool it to room temperature, and stir and refrigerate it at 4°C overnight to ensure that the protein is completely dissolved, and centrifuge it at 8000×g for 20 minutes to remove large particles, thereby obtaining a soy protein isolate solution.
[0072] (3) A certain amount of carboxymethyl chitosan was weighed and dissolved in ultrapure water to prepare a carboxymethyl chitosan solution with a concentration of 2.5%.
[0073] (4) Preparation of soy protein isolate-carboxymethyl chitosan-heme peptide iron hydrogel: The heme peptide iron lyophilized powder was added to the soy protein isolate aggregate solution, and the final heme peptide iron reached a final concentration of 1 mg / mL, and magnetic stirring was carried out at room temperature for 2 h. The Genipin solution was added to the soy protein isolate solution to reach a final concentration of 0.2% (w / v). Subsequently, the carboxymethyl chitosan solution was added at a volume ratio of 15:4 and stirred for 6 h, and then the mixed solution was quickly poured into a vial (diameter 27.5 mm, height 57 mm), and cured at 4 ° C for 24 h to finally obtain the heme peptide iron hydrogel sample M6.
[0074] (5) The obtained sample M6 was stored at room temperature for further analysis.
[0075] The difference between Comparative Example 3 and Example 2 is that only 0.08% ascorbic acid was added during the preparation of heme peptide iron. Figure 1 As shown, compared with the sample M2 obtained in Example 2, the extraction rate of heme peptide iron is 83.44%, and the extraction rate needs to be improved.
[0076] Comparative Example 4
[0077] (1) Thawed red blood cells were mixed with water at a ratio of 10:13 (w / w), and 1% lysine and 0.08% ascorbic acid were added. After 30 minutes of reaction, the pH of the mixture was adjusted to 10 with 5M NaOH, and alkaline protease was added. Incubate at 45°C for 5 hours and heat at 80°C for 10 minutes to stop the enzyme activity. Centrifuge at 5000 r / min for 10 minutes, take the supernatant, and freeze-dry it for further operation.
[0078] (2) Dissolve soy protein isolate powder in ultrapure water at room temperature (25°C) to prepare an 8% (w / v) soy protein isolate solution, stir it magnetically for 2 hours to fully hydrate it, then stir the soy protein isolate solution in a water bath at 85°C for 30 minutes, then quickly cool it to room temperature, and stir and refrigerate it at 4°C overnight to ensure that the protein is completely dissolved, and centrifuge it at 8000×g for 20 minutes to remove large particles, thereby obtaining a soy protein isolate solution.
[0079] (3) A certain amount of carboxymethyl chitosan was weighed and dissolved in ultrapure water to prepare a carboxymethyl chitosan solution with a concentration of 2.5%.
[0080] (4) Preparation of soy protein isolate-carboxymethyl chitosan-heme peptide iron hydrogel: The heme peptide iron lyophilized powder was added to the soy protein isolate aggregate solution, and the final heme peptide iron reached a final concentration of 1 mg / mL, and magnetic stirring was carried out at room temperature for 2 h. The Genipin solution was added to the soy protein isolate solution to reach a final concentration of 0.1% (w / v). Subsequently, the carboxymethyl chitosan solution was added at a volume ratio of 15:4 and stirred for 6 h, and then the mixed solution was quickly poured into a vial (diameter 27.5 mm, height 57 mm), and cured at 4 ° C for 24 h to finally obtain the heme peptide iron hydrogel sample M7.
[0081] (5) The obtained sample M7 was stored at room temperature for further analysis.
[0082] The difference between Comparative Example 4 and Example 2 is that the final content of Genipin in the polymer solution is 0.1% (w / v). Compared with the sample M2 obtained in Example 2, the hydrogel structure is weaker and easier to break due to the lower crosslinking agent content.
[0083] Comparative Example 5
[0084] (1) Thawed red blood cells were mixed with water at a ratio of 10:13 (w / w), and 1% lysine and 0.08% ascorbic acid were added. After 30 minutes of reaction, the pH of the mixture was adjusted to 10 with 5M NaOH, and alkaline protease was added. Incubate at 45°C for 5 hours and heat at 80°C for 10 minutes to stop the enzyme activity. Centrifuge at 5000 r / min for 10 minutes, take the supernatant, and freeze-dry it for further operation.
[0085] (2) Dissolve soy protein isolate powder in ultrapure water at room temperature (25°C) to prepare a 6% (w / v) soy protein isolate solution, stir it magnetically for 2 hours to fully hydrate it, then stir the soy protein isolate solution in a water bath at 85°C for 30 minutes, then quickly cool it to room temperature, and stir and refrigerate it at 4°C overnight to ensure that the protein is completely dissolved, and centrifuge it at 8000×g for 20 minutes to remove large particles, thereby obtaining a soy protein isolate solution.
[0086] (3) A certain amount of carboxymethyl chitosan was weighed and dissolved in ultrapure water to prepare a carboxymethyl chitosan solution with a concentration of 2.5%.
[0087] (4) Preparation of soy protein isolate-carboxymethyl chitosan-heme peptide iron hydrogel: The heme peptide iron lyophilized powder was added to the soy protein isolate aggregate solution, and the final heme peptide iron reached a final concentration of 1 mg / mL, and magnetic stirring was carried out at room temperature for 2 h. The Genipin solution was added to the soy protein isolate solution to reach a final concentration of 0.2% (w / v). Subsequently, the carboxymethyl chitosan solution was added at a volume ratio of 15:4 and stirred for 6 h, and then the mixed solution was quickly poured into a vial (diameter 27.5 mm, height 57 mm), and cured at 4 ° C for 24 h to finally obtain the heme peptide iron hydrogel sample M8.
[0088] (5) The obtained sample M8 was stored at room temperature for further analysis.
[0089] The difference between Comparative Example 5 and Example 2 is that the mass fraction of soy protein isolate in the polymer solution is 6%, and the final content of soy protein isolate in the hydrogel obtained is 4.5%. Compared with sample M2 obtained in Example 2, due to the lower soy protein isolate content, the stability of the hydrogel structure is poor and it is easy to break.
[0090] Comparative Example 6
[0091] (1) Thawed red blood cells were mixed with water at a ratio of 10:13 (w / w), and 1% lysine and 0.08% ascorbic acid were added. After 30 minutes of reaction, the pH of the mixture was adjusted to 10 with 5M NaOH, and alkaline protease was added. Incubate at 45°C for 5 hours and heat at 80°C for 10 minutes to stop the enzyme activity. Centrifuge at 5000 r / min for 10 minutes, take the supernatant, and freeze-dry it for further operation.
[0092] (2) Dissolve soy protein isolate powder in ultrapure water at room temperature (25°C) to prepare an 8% (w / v) soy protein isolate solution, stir it magnetically for 2 hours to fully hydrate it, then stir the soy protein isolate solution in a water bath at 85°C for 30 minutes, then quickly cool it to room temperature, and stir and refrigerate it at 4°C overnight to ensure that the protein is completely dissolved, and centrifuge it at 8000×g for 20 minutes to remove large particles, thereby obtaining a soy protein isolate solution.
[0093] (3) A certain amount of carboxymethyl chitosan was weighed and dissolved in ultrapure water to prepare a carboxymethyl chitosan solution with a concentration of 0.25%.
[0094] (4) Preparation of soy protein isolate-carboxymethyl chitosan-heme peptide iron hydrogel: The heme peptide iron lyophilized powder was added to the soy protein isolate aggregate solution, and the final heme peptide iron reached a final concentration of 2 mg / mL, and magnetic stirring was carried out at room temperature for 2 hours, and the Genipin solution was added to the soy protein isolate solution to reach a final concentration of 0.2% (w / v). Subsequently, the carboxymethyl chitosan solution was added at a volume ratio of 15:4 and stirred for 6 hours, and then the mixed solution was quickly poured into a vial (diameter 27.5 mm, height 57 mm), and cured at 4°C for 24 hours to finally obtain the heme peptide iron hydrogel sample M9.
[0095] (5) The obtained sample M9 was stored at room temperature for further analysis.
[0096] The difference between Comparative Example 6 and Example 2 is that the initial concentration of the carboxymethyl chitosan solution in the polymer solution is 0.25%. Compared with the sample M2 obtained in Example 2, due to the lower carboxymethyl chitosan content, the hydrogel structure is weaker and the release rate of heme peptide iron is too fast.
[0097] In vitro drug release study and result analysis:
[0098] (1) pH controlled release in vitro
[0099] The in vitro release experiment of soy protein isolate-carboxymethyl chitosan-heme peptide iron hydrogel samples (M1, M2, M3) in different pH media was performed as follows:
[0100] The samples (M1, M2, M3) were placed in 50 mL of solution (pH 2, 5.5, 7.4, 10, respectively) and left to stand at a constant temperature of 37 ° C. 1 mL of the release solution was taken at a certain time interval and then replaced with a blank solution to maintain the original volume. The concentration of heme peptide iron in the release solution was determined by UV-visible spectroscopy. Sample M3 treated with different pH values was freeze-dried, and the effect of different pH treatments on the microstructure of sample M3 was observed under a scanning electron microscope (Regulus 8230, Hitachi, Japan) at a magnification of 500 times.
[0101] The release effect of samples under different pH environments can reflect the pH sensitivity of the composite hydrogel. Figure 4-6 The influence of different pH environments on the microstructure of sample M3 is shown in Figure 7 shown.
[0102] Figure 4The release curves of sample M1 in solutions with pH values of 2, 5.5, 7.4, and 10 are shown. It can be seen that there are significant differences in the cumulative release rate and release rate of heme peptide iron under different pH environments. At pH 2, the release amount of heme peptide iron is low. In the initial release period (0-8h), under the conditions of pH values of 7.4 and 10, the release rate of heme peptide iron is fast. As the release process proceeds, the cumulative release amount of heme peptide iron reaches a state of equilibrium, indicating that the soy protein isolate-carboxymethyl chitosan-heme peptide iron hydrogel is completely degraded. Figure 5 and 6 These are the release curves of samples M2 and M3 in solutions with pH values of 2, 5.5, 7.4, and 10, respectively. Their release patterns are similar to that of M1.
[0103] Figure 7 The influence of pH 2, 5.5, 7.4, and 10 on the micromorphology of sample M3 is shown. The microstructure of the hydrogel will affect its performance. It can be seen that the hydrogel has a continuous and stable network structure under different pH conditions. When the pH is 2, the network structure of sample M3 is denser, more compact, and has smaller pores. When the pH is 5.5 and 7.4, the structure of sample M3 shows relatively loose and smaller pores, and the pores are evenly distributed. When the pH is 10, the structure of sample M3 shows a large number of voids. The loose and porous structure of the hydrogel can provide a large space for drug storage and is more conducive to drug delivery.
[0104] (2) In vitro release in simulated gastrointestinal environment
[0105] In order to simulate the release behavior of soy protein isolate-carboxymethyl chitosan-heme peptide iron hydrogel in the gastrointestinal environment, the samples (M1, M2, M3) were first placed in a phosphate buffer solution with a pH of 1.2, and a constant temperature oscillator (37°C, 150rpm) was used to simulate gastrointestinal digestion, and a simulated in vitro release experiment was carried out. After 2 hours, the release medium was replaced with a phosphate buffer solution with a pH of 7.4 to continue the release.
[0106] In order to explore the possibility of soy protein isolate-carboxymethyl chitosan-heme peptide iron hydrogel as an oral drug delivery system, the dynamic release behavior of the samples in a simulated gastrointestinal environment was studied. Figure 8It can be seen that the release rate of the sample is slow under the environment of pH 1.2, and the cumulative release amounts of samples M1, M2 and M3 in 2h are 8.9μg / mL, 7.73μg / mL and 7.76μg / mL respectively. When the release medium changes to pH 7.4, the heme peptide iron is released rapidly and reaches equilibrium after 3 hours. Obviously, the pH value of the release medium has a significant effect on the release behavior of the heme peptide iron, and acidic and neutral conditions can control the release of the heme peptide iron in the micellar hydrogel. Therefore, the pH-sensitive composite hydrogel of the present invention is expected to become a pH-responsive oral drug release system.
[0107] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A method for preparing a pH-responsive heme peptide iron hydrogel, characterized in that: The following steps are involved: (1) adding lysine and ascorbic acid to a hemoglobin solution, reacting for a period of time, adjusting the pH, performing enzymatic hydrolysis, centrifuging, taking the supernatant, and freeze-drying to obtain hemoglobin peptide iron freeze-dried powder; (2) weighing a certain amount of soy protein isolate and dissolving it in ultrapure water, stirring it to fully hydrate it, heating the hydrated soy protein isolate solution in a hot water bath to obtain soy protein isolate aggregates, then rapidly cooling it to room temperature and storing it, magnetically stirring it overnight to fully hydrate it, and centrifuging to remove large particles to obtain a soy protein isolate aggregate solution; (3) Weigh a certain amount of carboxymethyl chitosan and dissolve it in ultrapure water, sonicate, and stir to completely hydrate it; (4) Preparation of soy protein isolate-carboxymethyl chitosan-heme peptide iron hydrogel: add heme peptide iron lyophilized powder to the soy protein isolate aggregate solution and stir magnetically at room temperature; dissolve genipin in anhydrous ethanol and add it to the soy protein isolate solution under magnetic stirring conditions; then add carboxymethyl chitosan solution to obtain soy protein isolate-carboxymethyl chitosan-heme peptide iron hydrogel.
2. The method for preparing the pH-responsive heme peptide iron hydrogel according to claim 1, characterized in that: In the step (1), the amount of lysine added is 1% w / v, and the amount of ascorbic acid added is 0.08% w / v.
3. The method for preparing the pH-responsive heme peptide iron hydrogel according to claim 1, characterized in that: In the step (1), the reaction time is 30 min.
4. The method for preparing the pH-responsive heme peptide iron hydrogel according to claim 1, characterized in that: In the step (2), the soy protein isolate solution is stirred in a water bath at 85° C. for 30 minutes, then rapidly cooled to room temperature, and stirred and refrigerated at 4° C. overnight.
5. The method for preparing the pH-responsive heme peptide iron hydrogel according to claim 1, characterized in that: In the step (2), centrifugation was performed at 8000×g for 20 min to remove large particles.
6. The method for preparing the pH-responsive heme peptide iron hydrogel according to claim 1, characterized in that: In the step (3), the mass concentration of the prepared carboxymethyl chitosan solution is 1.25% to 5%.
7. The method for preparing the pH-responsive heme peptide iron hydrogel according to claim 6, characterized in that: In the step (4), the heme peptide iron lyophilized powder is added to the soy protein isolate aggregate solution, and the final concentration of the heme peptide iron is 1 mg / mL.
8. The method for preparing the pH-responsive heme peptide iron hydrogel according to claim 7, characterized in that: In the step (4), Genipin is dissolved in anhydrous ethanol and added to the soy protein isolate solution under magnetic stirring, and the final concentration of Genipin is 0.2% w / v.
9. The method for preparing the pH-responsive heme peptide iron hydrogel according to claim 8, characterized in that: In the step (4), the carboxymethyl chitosan solution is added at a volume ratio of 15:
4.
10. The pH-responsive heme peptide iron hydrogel prepared by the preparation method according to any one of claims 1 to 9.