Food self-healing hydrogel as well as preparation method and application thereof

Abstract

The invention relates to food self-healing hydrogel as well as a preparation method and application thereof. The food self-healing hydrogel has excellent dynamic functional mechanical properties such as mechanical property matching with biological tissues, self-healing property, stability in a physiological environment and the like. All the materials used in the invention are food-grade materials, the food self-healing hydrogel can significantly relieve intestinal inflammation, and a new non-drug food intervention method is provided for relieving diseases such as intestinal inflammation. In addition, the method for preparing the food self-healing hydrogel and the preparation conditions thereof are mild, the operation is simple, and various complex instruments are not needed.

Description

Technical Field

[0001] The present invention relates to the field of biobased hydrogel processing. Specifically, the present invention relates to a preparation method and application of a food self-healing hydrogel. Background Art

[0002] Microbial drug resistance caused by drug abuse has become one of the major crises in the global public health field in the 21st century. In addition, drug abuse also brings serious environmental pollution, threatening human health and the ecosystem. In this context, it is urgent to explore non-drug intervention strategies.

[0003] Oral administration is the most convenient and acceptable way to take therapeutic and bioactive substances. Hydrogels have become promising oral drug delivery materials due to their viscoelastic properties. However, hydrogels based on biomaterials are easily damaged during use due to their inherent low Young's modulus and the complex loading and dynamic environment in the human body (especially in the digestive tract), which severely limits their long-term reliable application.

[0004] As a potential biomaterial, self-healing hydrogels can dynamically and spontaneously transform between solid and liquid states during the peristalsis of the digestive tract to avoid possible life-threatening side effects such as intestinal obstruction. However, the safety assessment of the materials for constructing self-healing hydrogels and their occurrence mechanisms remains a major challenge.

[0005] Therefore, the present invention has developed a completely food-grade self-healing hydrogel constructed through multiple completely spontaneous physical non-covalent interactions, providing a safer and more innovative solution for alleviating intestinal inflammation. Summary of the Invention

[0006] The present invention aims to develop food self-healing hydrogels and expand their uses.

[0007] Therefore, according to the first aspect of the present invention, a food self-healing hydrogel has excellent dynamic functional mechanical properties such as mechanical property matching with biological tissues, self-healing characteristics, and stability in the physiological environment.

[0008] According to the second aspect of the present invention, there is provided a method for preparing the above-mentioned food self-healing hydrogel, the preparation conditions of which are mild, the operation is simple, and various complex instruments are not required. It is characterized by including the following steps:

[0009] 1) Dissolve a food-derived protein in ultrapure water, adjust the pH to the range of 1.9 - 2.1 with 2M HCl after overnight hydration to obtain solution A;

[0010] 2) Place solution A in an environment with a temperature in the range of 85 - 95 °C, heat and stir it to fibrillate the protein, and centrifuge to obtain the supernatant after cooling to obtain solution B;

[0011] 3) Dissolve α-glucan and small molecule polyphenols in a buffer solution to obtain solution C;

[0012] 4) Add natural metal-containing protein to solution C to obtain solution D;

[0013] 5) Dropwise add solution D into solution B, stir well and mix evenly, and incubate in an environment at 37 °C for 3-7 days to obtain the food self-healing hydrogel.

[0014] According to the third aspect of the present invention, there is provided the use of the food self-healing hydrogel according to the first aspect of the present invention for relieving intestinal inflammation, providing a new non-drug food intervention method for relieving diseases such as intestinal inflammation. Description of the Drawings

[0015] The present invention will be described and explained in more detail below with reference to the drawings, wherein:

[0016] Figure 1 Shows the characterization results of the food self-healing hydrogel prepared in Example 1, wherein, A: Formation of the hydrogel (where the concentration of lysozyme protein fibers is 10 mg / mL, the concentration of α-glucan is 250 mg / mL, the concentration of EGCG is 5 mg / mL, from left to right, the concentration of horse spleen ferritin is 0.16, 0.32, 1.6, 3.2 mg / mL); B: Mechanical strength (where the concentration of lysozyme protein fibers is 10 mg / mL, the concentration of α-glucan is 250 mg / mL, the concentration of EGCG is 5 mg / mL, the concentration of horse spleen ferritin is 1.6 mg / mL); C: Large shear recovery test analysis (same sample as in B); D: Self-healing test (same sample as in B); E: In vitro simulated gastrointestinal digestion image (same sample as in B).

[0017] Figure 2 Shows the alleviating effect of oral gavage of the food self-healing hydrogel on DSS-induced acute colitis mice in Example 2, wherein, A: Body weight change; B: Change in the content of tumor necrosis factor-α in serum; C: Change in the content of interleukin-1β in serum; D: Change in the content of interleukin-6 in serum; E: Change in the content of interferon-γ in serum (the experimental results of the above Figures A-E are expressed as mean ± standard deviation (Mean ± SD), and the data is analyzed using IBM SPSS Statistics software, p < 0.05 is considered to be statistically significant); F: Hematoxylin and eosin (H&E) stained section image of the colon of mice in the normal control (NC) group; G: H&E stained section image of mice in the DSS group; H: H&E stained section image of mice in the food self-healing hydrogel treatment group. Detailed Embodiments

[0018] Some specific embodiments of the present invention are described for illustrative purposes and not for limitation.

[0019] Therefore, according to a first aspect, the present invention provides a food self-healing hydrogel, characterized in that it has excellent dynamic functional mechanical properties such as mechanical property matching with biological tissues, self-healing characteristics, and stability in a physiological environment. It comprises: 5 - 20 mg / mL of food-derived protein fibers, 150 - 300 mg / mL of α-glucan, 1 - 40 mg / mL of small molecule polyphenols, and 0.1 - 5 mg / mL of natural metal-containing proteins.

[0020] Preferably, the food-derived protein is a natural food protein, without particular limitation, and can be, for example, lysozyme protein, β-lactoglobulin, soy protein, etc.

[0021] Preferably, the relative molecular mass of the α-glucan is not less than 450,000.

[0022] Preferably, the small molecule polyphenols are food polyphenols, such as epigallocatechin gallate (EGCG), gallic acid, tannic acid, procyanidins, etc.

[0023] Preferably, the natural metal-containing protein is a food protein, such as horse spleen ferritin, ceruloplasmin, laccase, etc.

[0024] According to some embodiments, the food self-healing hydrogel is composed of lysozyme protein fibers, α-glucan, EGCG, and horse spleen ferritin.

[0025] According to a second aspect, the present invention provides a method for preparing the above-mentioned food self-healing hydrogel, characterized by comprising:

[0026] 1) Dissolve the food-derived protein in ultrapure water, adjust the pH to the range of 1.9 - 2.1 with 2M HCl after overnight hydration to obtain solution A;

[0027] 2) Place solution A in an environment with a temperature in the range of 85 - 95 °C, heat and stir to make the protein fibrillate, cool down and centrifuge to obtain the supernatant to get solution B;

[0028] 3) Dissolve α-glucan and small molecule polyphenols in a buffer solution to obtain solution C;

[0029] 4) Add the natural metal-containing protein to solution C to obtain solution D;

[0030] 5) Dropwise add solution D into solution B, stir well and mix evenly, and incubate in an environment of 37 °C for 3 - 7 days to obtain the food self-healing hydrogel.

[0031] Preferably, the content of the food-derived protein in Solution A is 10-40 mg / mL.

[0032] Preferably, the content of the food-derived protein fiber in Solution B is 10-40 mg / mL.

[0033] Preferably, the content of α-glucan in Solution C is 300-600 mg / mL.

[0034] Preferably, the content of polyphenols in Solution C is 2-80 mg / mL.

[0035] Preferably, the content of the natural metal-containing protein in Solution D is 0.2-10 mg / mL.

[0036] According to the third aspect, there is provided the use of the food self-healing hydrogel according to the first aspect of the present invention for relieving intestinal inflammation, providing a new non-drug food intervention method for relieving diseases such as intestinal inflammation.

[0037] The inventors found that the food self-healing hydrogel according to the first aspect of the present invention can effectively relieve intestinal inflammation symptoms.

[0038] In the present application, the descriptions of each feature can be combined with each other without contradiction and all fall within the scope claimed in the present application.

[0039] As used herein, the terms "comprising" and "including" cover the case of further comprising or including other elements not explicitly mentioned and the case consisting of the mentioned elements.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. When the definition of a term in this specification conflicts with the meaning commonly understood by those skilled in the art to which the present invention belongs, the definition herein shall prevail.

[0041] Unless otherwise specified, all numerical values expressing amounts of ingredients etc. used in the specification and claims are understood to be modified by the term "about". Therefore, unless there is a contrary indication, the numerical parameters set forth herein are approximate values that can vary according to the desired properties required.

[0042] Examples

[0043] The following will further illustrate the concept and the resulting technical effects of the present invention in conjunction with examples and drawings, so that those skilled in the art can fully understand the purpose, features and effects of the present invention. Those skilled in the art will understand that the examples herein are for illustrative purposes only and the scope of the present invention is not limited thereto.

[0044] Example 1

[0045] Prepare a food self-healing hydrogel as follows:

[0046] Dissolve 1 g of lysozyme protein in 49 mL of ultrapure water. After overnight hydration, adjust the pH to 2.0 using 2 M HCl to obtain Solution A;

[0047] Place Solution A in an environment at 90 °C and heat with stirring (8 h, 300 rpm) to fibrillate the protein. After cooling, centrifuge (4 °C, 20 min, 3000 rpm) to obtain the supernatant and get Solution B;

[0048] Dissolve 500 mg / mL of α-dextran and 10 mg / mL of EGCG in 1 mL of Bis-tris buffer to obtain Solution C;

[0049] Add 100 μL of horse spleen ferritin with a concentration of 32 mg / mL to Solution C to obtain Solution D;

[0050] Dropwise add 500 μL of Solution D into 500 μL of Solution B, stir well and mix evenly, and incubate in an environment at 37 °C for 7 days to obtain 1 mL of food self-healing hydrogel. Among them, the concentration of lysozyme protein fiber is 10 mg / mL, the concentration of α-dextran is 250 mg / mL, the concentration of EGCG is 5 mg / mL, and the concentration of horse spleen ferritin is 1.6 mg / mL.

[0051] Characterize

[0052] (1) Inversion experiment of the hydrogel

[0053] Invert the incubated hydrogel onto a flat surface and observe its gel formation, as Figure 1 shown in A.

[0054] From Figure 1 A, it can be observed that the hydrogel does not fall off after inversion. In addition, the hydrogel shows a transparent and clear macroscopic morphology, indicating the formation of the hydrogel.

[0055] (2) Determination of mechanical strength

[0056] Use an MCR302 intelligent advanced rheometer to detect the mechanical strength of the prepared hydrogel. Use a clamp with a diameter of 25 mm (PP25), set the gap d to 0.103 mm, and set the test temperature to 25 °C. Fix the measured shear strain γ at 0.1%, and the angular frequency varies in the range of 0.1 - 10 Rad / s. Use the frequency sweep method to measure the changes in the elastic modulus (G’) and loss modulus (G”) of the hydrogel. The mechanical strength analysis of the food self-healing hydrogel is as Figure 1 shown in B.

[0057] FromFigure 1 From the analysis results of the elastic modulus (G’) and loss modulus (G”) of the hydrogel, it can be seen that the elastic modulus of the food self-healing hydrogel is close to 10 3 Pa, reaching a strength comparable to that of biological tissues.

[0058] (3) Determination of self-healing performance

[0059] The self-healing ability of the hydrogel was analyzed using an MCR302 intelligent advanced rheometer. During the test, the hydrogel was sealed in a closed container to prevent dehydration. The high-efficiency rheological recovery performance of the hydrogel was demonstrated by a continuous step strain test. A continuous step strain scan of 0.1% for recovery and 100% for destruction was performed, and the continuous step changes in the oscillatory strain between 0.1% and 100% were measured at the same frequency (1 Hz). The oscillatory strain changes alternated between 0.1% recovery (held for 160 s) and 100% destruction (held for 160 s), and a total of three cycle tests were carried out, recording the changes in the elastic modulus (G’) and loss modulus (G”).

[0060] In addition, the self-healing performance was further determined by observing the cutting and recombination of the hydrogel under environmental conditions without external stimuli. Briefly, the hydrogel was divided into two parts, and then by bringing them close to each other to observe whether they could quickly form a whole.

[0061] The determination results of the self-healing performance of the food self-healing hydrogel are as Figure 1 shown in Figure 1 Figures C and

[0062] D. Figure 1 As can be observed from 3 Figure C, after applying a 100% stress to cause destruction, the food self-healing hydrogel can quickly recover to the initial strength level within dozens of seconds. It can still quickly reach a value close to 10

[0063] From Figure 1 the self-healing test experimental results in Figure D, it shows that the food self-healing hydrogel can be cut into two completely independent pieces. By bringing them close to each other, they can quickly reconnect and self-repair. This once again confirms the self-healing performance of the food self-healing hydrogel.

[0064] (4) In vitro simulated gastrointestinal digestion

[0065] Weigh an appropriate amount of pepsin and dissolve it in ultrapure water. After adjusting the pH, prepare simulated gastric juice (pH 2.0) at a concentration of 3.2 mg / mL. Dissolve trypsin in Bis-Tris buffer. After adjusting the pH, prepare simulated intestinal juice (pH 7.0) at a concentration of 1.6 mg / mL. Inject 1 mL of the α-dextran-stabilized lysozyme protein fiber-ferritin-polyphenol chelated quaternary food hydrogel sample (lysozyme protein fiber concentration is 1 wt%, α-dextran concentration is 25 wt%, EGCG concentration is 0.5 wt%, ferritin concentration is 0.16 wt%) into 4 mL of simulated gastric juice, and shake it in a water bath at 37 °C for 2 h. After sucking off the simulated gastric juice, add 9 mL of simulated intestinal juice and continue to culture it in a water bath at 37 °C for 4 h. All samples were shaken in a water bath at a speed of 120 r / min to simulate gastrointestinal peristalsis. Take pictures to record the hydrogel state at 0, 0.5, 1, and 2 h of simulated gastrointestinal digestion. The appearance state of the food self-healing hydrogel in the in vitro simulated gastrointestinal digestive juice is as Figure 1 shown in Figure E.

[0066] As Figure 1 observed from Figure E, the hydrogel maintained its complete morphology in the simulated gastric juice environment at pH 2.0; in the simulated intestinal juice environment at pH 7.0, the food self-healing hydrogel slightly deformed but still could aggregate together, showing good stability in the physiological environment.

[0067] Example 2

[0068] This example studies the alleviating effect of the α-dextran-stabilized lysozyme protein fiber-templated polyphenol-ferritin chelated food self-healing hydrogel on DSS-induced acute colitis in mice.

[0069] The mice were 5-week-old male C57BL / 6J mice, weighing 18-20 g (Shanghai SLAC Laboratory Animal Co., Ltd.). Approved by the Animal Experiment Center of Nanjing Agricultural University (SYXK (Jiangsu) 2011-0037), they conformed to the national experimental animal welfare guidelines and animal experiment ethics. The breeding environment temperature of the mice was maintained at 25 °C, the humidity was controlled at 75%, and the light was a 12 h light / dark cycle. The mice could freely drink sterile water and feed. First, the mice were adaptively bred for 7 days. Then, the mice were divided into the following three groups: (I) blank control group (NC group); (II) DSS model group (DSS group); (III) DSS-induced + intragastric administration of α-dextran-stabilized lysozyme protein nanofibril templated ferritin-polyphenol EGCG chelated food self-healing hydrogel group (0.5E-0.16F group). Among them, there were 6 mice in each group. 2.5 wt% DSS was added to the drinking water and orally administered for 7 days to induce acute colitis. From the 8th day to the 14th day, the corresponding hydrogel (200 μL / d) was intragastrically administered to the mice in the 0.5E-0.16F group. On the 8th-14th day, the mice in the NC group and the DSS group were intragastrically administered an equal amount of sterile water. The body weight, blood loss, fecal consistency, and food and water intake of the mice were recorded daily as indicators for daily clinical evaluation. Before the mice died, they were fully anesthetized by inhaling 2% isoflurane. Before the end of the experiment, blood was collected by cardiac puncture. After the plasma was stored at room temperature for 4 h, it was centrifuged (4 °C, 3000 rpm, 20 min), and the obtained serum was placed at -80 °C for standby. After the colon was washed with normal saline, it was longitudinally cut in half: one half was frozen in liquid nitrogen and then stored at -80 °C, and the other half was placed in a freeze-drying tube and fixed with 4% paraformaldehyde for histopathological examination.

[0070] Characterize

[0071] (1) Monitoring of mouse body weight changes

[0072] During the experiment, the body weight of the mice was weighed and recorded daily, and the recording results were as Figure 2 shown in

[0073] As Figure 2 shown in A, it can be observed that all DSS-treated groups showed weight loss. With the cessation of DSS treatment, after intragastric administration of the food self-healing hydrogel, the body weight of the mice gradually recovered and finally approached the body weight of the control (NC) group, reaching a comparable level. Conversely, the body weight of the mice in the DSS group did not show an upward trend until the end of the intervention and was significantly (p < 0.05) lower than that of the NC and intragastric administration of food self-healing hydrogel groups.

[0074] (2) Determination of inflammatory factors in serum

[0075] The contents of TNF-α, IL-1β, IL-6 and IFN-γ in the serum were detected using an ELISA kit according to the steps in the relevant instructions, and the analysis results are as Figure 2 shown in B-E.

[0076] Figure 2 As shown in B-E, it can be observed that DSS treatment caused an increase in four pro-inflammatory cytokines, namely tumor necrosis factor-α, interleukin-1β, interleukin-6 and interferon-γ, in the serum. Treatment with the food self-healing hydrogel could significantly (p < 0.05) reduce the levels of tumor necrosis factor-α ( Figure 2 B), interleukin-1β ( Figure 2 C), interleukin-6 ( Figure 2 D) and interferon-γ ( Figure 2 E) and decreased to a level comparable to that of the NC group.

[0077] (3) Histopathological study

[0078] After removing the Swiss roll-shaped colon tissue soaked in 4% paraformaldehyde fixative, it was dehydrated and cleared. Then, the cleared tissue was infiltrated with wax and embedded in a wax block and cut into sections with a thickness of 4-6 μm. After dewaxing, the sections were stained step by step with the staining solutions in the H&E staining kit, dehydrated, and finally the sections were taken out, quickly air-dried and made into mounted slides for subsequent photographing and observation. The results are as Figure 2 shown in F-H.

[0079] The histological examination of the colon tissue sections is as Figure 2 shown in F-H. In the NC group, the goblet cells in the colon were arranged neatly and were abundant, there were crypts of normal size, and no histological lesions were observed ( Figure 2 F). In the DSS group, the colon was severely fibrotic, goblet cells were not visible, crypts were lost, and a large number of inflammatory factors infiltrated ( Figure 2 G). After intragastric administration of the food self-healing hydrogel, the level of goblet cells basically returned to normal, the crypts were clear, and the infiltration of inflammatory factors was also significantly reduced ( Figure 2 H).

[0080] The above only describes the exemplary embodiments or examples of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can be variously modified and changed. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention are included within the scope of the claims of this application.

Claims

1. A food self-healing hydrogel, characterized in that, It has excellent dynamic functional mechanical properties such as mechanical property matching with biological tissues, self-healing characteristics, and stability in physiological environment. It contains: 5-20 mg / mL of food-derived protein fibers, 150-300 mg / mL of α-glucan, 1-40 mg / mL of small molecule polyphenols, and 0.1-5 mg / mL of natural metal-containing protein.

2. The food self-healing hydrogel according to claim 1, wherein The food-derived protein fiber is lysozyme protein fiber.

3. The self-healing hydrogel for food according to any one of claims 1-2, characterized in that, The small molecule polyphenol is EGCG.

4. The food self-healing hydrogel according to any one of claims 1-3, characterized in that, The natural metal-containing protein is horse spleen ferritin.

5. A method for preparing the food self-healing hydrogel according to any one of claims 1-4, characterized in that, It includes: 1) Dissolve the food-derived protein in ultrapure water, adjust the pH to the range of 1.9-2.1 with 2M HCl after overnight hydration to obtain solution A; 2) Place solution A in an environment with a temperature in the range of 85-95 °C, heat and stir to make the protein fibrillate, centrifuge after cooling to obtain the supernatant, and obtain solution B; 3) Dissolve α-glucan and small molecule polyphenols in a buffer solution to obtain solution C; 4) Add natural metal-containing protein to solution C to obtain solution D; 5) Dropwise add solution D into solution B, stir well to mix evenly, and incubate in an environment at 37 °C for 3-7 days to obtain the food self-healing hydrogel.

6. The method according to claim 5, wherein The content of the food-derived protein in solution A is 10-40 mg / mL.

7. The method according to claim 5 or 6, characterized in that, The content of the food-derived protein fiber in solution B is 10-40 mg / mL.

8. The method according to any one of claims 5 to 7, characterized in that, The content of α-glucan in solution C is 300-600 mg / mL.

9. The method according to any one of claims 5 - 8, characterized in that The content of the small molecule polyphenol in solution C is 2-80 mg / mL.

10. The method according to any one of claims 5-9, characterized in that, The content of the natural metal-containing protein in solution D is 0.2-10 mg / mL.

11. The method according to any one of claims 5-10, characterized in that, In step 5), first add solution B to a container, and then dropwise add the same volume of solution D into it.

12. The preparation method according to any one of claims 5-11, characterized in that, The food-derived protein is lysozyme protein.

13. The preparation method according to any one of claims 5-12, characterized in that, The food-derived protein fiber is lysozyme protein fiber.

14. The preparation method according to any one of claims 5-13, characterized in that, The small molecule polyphenol is EGCG.

15. The preparation method according to any one of claims 5-14, characterized in that, The natural metal-containing protein is horse spleen ferritin.

16. Use of the food self-healing hydrogel according to any one of claims 1-4 for relieving intestinal inflammation.

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