Food-grade hydrogel constructed by freeze-thaw cycles and preparation method and application thereof

Abstract

This invention relates to a polyphenol-protein fiber hydrogel stabilized by polysaccharides, constructed based on freeze-thaw cycles, its preparation method, and its applications. The hydrogel possesses a uniform microstructure, enhanced mechanical properties, excellent biocompatibility, and stability. Through repeated freeze-thaw treatment, a stable three-dimensional network structure is formed without introducing chemical cross-linking bonds. This method is simple to operate, operates under mild conditions, and effectively avoids the toxic residues that may result from chemical cross-linking. The freeze-thaw cycle-constructed hydrogel is composed entirely of food-grade components and can significantly alleviate intestinal inflammation, providing a new method for the intervention of inflammatory bowel disease and related diseases.

Description

Technical Field

[0001] This invention relates to the field of bio-based hydrogel processing. Specifically, this invention relates to a method for preparing and applying edible bio-based hydrogels constructed based on freeze-thaw cycles. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of chronic inflammatory bowel diseases characterized by oxidative stress, overexpression of inflammatory cytokines, and impaired intestinal mucosal barrier function. Currently, clinical treatments mainly involve 5-aminosalicylic acid, corticosteroids, and anti-tumor necrosis factor preparations. However, these drugs generally suffer from significant side effects, numerous adverse reactions, and high rates of drug resistance. Therefore, seeking safe and long-term treatment intervention strategies is of great importance.

[0003] Hydrogels possess tunable network structures and the ability to encapsulate and deliver bioactive compounds, making them highly promising for oral delivery and intervention in intestinal diseases. Plant-derived polyphenols, widely found in natural products, exhibit excellent antioxidant and anti-inflammatory properties. Introducing plant-derived polyphenols into hydrogel systems holds promise for endowing materials with additional bioactive functions. However, most current composite hydrogels rely on covalent chemical cross-linking, providing high mechanical properties but also presenting issues such as cytotoxicity and biosafety, limiting their application in the food industry. Food hydrogels formed from single natural-based materials often suffer from poor mechanical strength and insufficient stability in the gastrointestinal environment. While introducing nanostructures such as protein fibers as a reinforcing framework can effectively improve the mechanical properties of food hydrogels, challenges remain in achieving uniform mixing and stable gelation of multi-component systems.

[0004] Freeze-thaw treatment is a typical physical cross-linking strategy and has become a simple and environmentally friendly method for preparing hydrogels. However, uncontrolled ice crystal growth during freezing leads to uneven gel pore size distribution, further affecting the uniformity and structural stability of the carrier.

[0005] Therefore, this invention develops a food-grade hydrogel based on freeze-thaw cycles. This method uses natural food-grade raw materials, such as edible proteins and plant-derived polyphenols, and adds polysaccharides to regulate ice crystal behavior. Through an optimized freeze-thaw cycle process, physical non-covalent cross-linking is achieved, successfully preparing a uniform and structurally stable hydrogel, providing a safer and more novel solution for alleviating intestinal inflammation. Summary of the Invention

[0006] To address the aforementioned issues, this invention utilizes freeze-thaw cycling technology to prepare a polyphenol-protein fiber hydrogel stabilized by polysaccharides. During the freezing process, the polysaccharides regulate the growth of ice crystals, resulting in a dense, uniform porous structure and enhanced mechanical properties in the gel, which also alleviates intestinal inflammation.

[0007] The purpose of this invention is to provide a physical preparation method and application of polysaccharide-stabilized polyphenol-protein fiber hydrogels.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0009] In a first aspect, the present invention provides a method for preparing polyphenol-protein fiber hydrogels based on freeze-thaw cycles and stabilized by polysaccharides, which is simple to operate and does not require the use of various complex instruments.

[0010] Includes the following steps:

[0011] 1) Dissolve the food-derived protein in ultrapure water and hydrate it overnight, then adjust the pH to 1.9-2.1 to obtain solution A;

[0012] 2) Heat and stir solution A at 85-95℃ for 6-10 hours to induce protein fibrillation, thus obtaining solution B;

[0013] 3) Dissolve dextran and plant-derived polyphenols in a buffer solution to obtain solution C;

[0014] 4) Mix equal volumes of solution B and solution C, and stir thoroughly to obtain solution D;

[0015] 5) Solution D was subjected to freeze-thaw treatment, including freezing at -20°C and thawing at room temperature, with a cycle time of 12-12 hours. Different cycles were performed depending on the experiment to obtain the hydrogel constructed based on the freeze-thaw cycle.

[0016] Secondly, the present invention provides a polysaccharide-stabilized polyphenol-protein fiber hydrogel constructed through freeze-thaw cycles, which has a uniform microstructure, enhanced mechanical properties, excellent biocompatibility and stability.

[0017] Thirdly, the present invention provides the application of the polysaccharide-stabilized polyphenol-protein fiber hydrogel constructed based on freeze-thaw cycles in alleviating intestinal inflammation.

[0018] The beneficial technical effects of this invention are as follows:

[0019] 1) This invention provides a polyphenol-protein fiber hydrogel, which is constructed based on freeze-thaw cycles and introduces dextran to regulate the growth of ice crystals during the freezing process.

[0020] 2) The increased dextran content significantly improved the gel's resistance to repeated freeze-thaw cycles, further promoting the formation of a denser and more uniform porous structure, resulting in a uniform microstructure, enhanced mechanical properties, and stability. Furthermore, the gel was prepared entirely from food-grade raw materials, thus exhibiting excellent biocompatibility.

[0021] 3) Applying the dextran-polyphenol-protein fiber hydrogel constructed by freeze-thaw cycle to DSS-induced acute colitis in mice can effectively alleviate intestinal inflammation, providing a new non-drug food intervention method for alleviating this type of intestinal disease. Attached Figure Description

[0022] The present invention will now be described and explained in more detail with reference to the accompanying drawings, wherein:

[0023] Figure 1 The results show a comparison of the gelation properties of the hydrogel based on freeze-thaw cycles prepared in Example 1 of the present invention with those of the hydrogel without freeze-thaw cycles.

[0024] Figure 2 The characterization results of the freeze-thaw cycle-based hydrogel prepared in Example 1 of the present invention are shown, wherein A: freezing process and ice crystal formation state of the hydrogel; B: cryo-scanning electron microscopy image; C: mechanical strength.

[0025] Figure 3 The biocompatibility of the freeze-thaw cycle-based hydrogel prepared in Example 1 of the present invention is shown. Green represents living cells, and DH-F(0D), DH-F(15D), and DH-F(35D) represent freeze-thaw hydrogels prepared from different concentrations of dextran, respectively.

[0026] Figure 4 The following figures illustrate the alleviating effect of oral gavage administration of the freeze-thaw cycle-based hydrogel on DSS-induced acute colitis in mice, as described in Example 2. A: Body weight change; B: Disease Activity Index (DAI); C: Colon length analysis; (The experimental results in Figures A and C are expressed as mean ± standard deviation (Mean ± SD). Data were analyzed using GraphPad Prism 10.0 software. p < 0.05 was considered statistically significant. Significant differences between groups are indicated by different letters in the figures.) D: Normal control group (NC); DSS-induced group; Colonic sections of mice treated with the freeze-thaw cycle-based hydrogel (DH-F) group stained with hematoxylin and eosin (H&E). Detailed Implementation

[0027] Some specific embodiments of the invention are now described for illustrative purposes and not for limitation.

[0028] According to a first aspect, the present invention provides a method for preparing a polyphenol-protein fiber hydrogel based on freeze-thaw cycles and stabilized by polysaccharides, which is simple to operate and does not require the use of various complex instruments. It contains 5-20 mg / mL of dietary protein fiber, 150-300 mg / mL of dextran, and 1-40 mg / mL of plant-derived polyphenols.

[0029] Preferably, the food-derived protein is a natural food protein, without particular limitation, such as lysozyme protein, whey protein, mung bean protein, etc.

[0030] Preferably, the relative molecular mass of the dextran is not less than 450,000.

[0031] Preferably, the polyphenols are plant-derived polyphenol compounds, such as epigallocatechin (EGC), catechin gallate (ECG), epigallocatechin gallate (EGCG), resveratrol, leucine, ferulic acid, chlorogenic acid, rutin, rosmarinic acid, myricetin, thearubigin, curcumin, anthocyanins, cyanidin, pelargonidin, gallic acid, ellagic acid, tannic acid, vanillic acid, syringic acid, p-coumaric acid, naringenin, sennaol, hesperidin, quercetin, baicalin, apigenin, luteolin, mangiferin, etc.

[0032] According to some embodiments, the polysaccharide-polyphenol-protein fiber hydrogel constructed by the freeze-thaw cycle is composed of lysozyme protein fibers, dextran, and EGCG.

[0033] According to a second aspect, the present invention provides a method for preparing the above-mentioned hydrogel based on freeze-thaw cycles, wherein the hydrogel has a uniform microstructure, enhanced mechanical properties, stability, and excellent biocompatibility. It is characterized by comprising:

[0034] 1) Dissolve the food-derived protein in ultrapure water and hydrate it overnight, then adjust the pH to 1.9-2.1 to obtain solution A;

[0035] 2) Heat and stir solution A at 85-95℃ for 6-10 hours to induce protein fibrillation, thus obtaining solution B;

[0036] 3) Dissolve dextran and plant-derived polyphenols in a buffer solution to obtain solution C;

[0037] 4) Mix equal volumes of solution B and solution C, and stir thoroughly to obtain solution D;

[0038] 5) The solution D was subjected to freeze-thaw treatment, including freezing at -20℃ and thawing at room temperature, with a cycle time of 12-12h. Different cycles were performed depending on the experiment to obtain the hydrogel constructed based on the freeze-thaw cycle.

[0039] Preferably, the content of the food-derived protein in solution A is 10-40 mg / mL.

[0040] Preferably, the content of the dietary protein fiber in solution B is 10-40 mg / mL.

[0041] Preferably, the content of dextran in solution C is 300-600 mg / mL.

[0042] Preferably, the polyphenol content in solution C is 2-80 mg / mL.

[0043] According to a third aspect, the present invention provides that the dextran-polyphenol-protein fiber hydrogel constructed by the freeze-thaw cycle is applied to DSS-induced acute colitis in mice to alleviate intestinal inflammation.

[0044] The descriptions of each feature in this application can be combined with each other as long as they do not contradict each other, and all of them fall within the scope of protection claimed in this application.

[0045] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any discrepancy between the definitions of terms in this specification and their commonly understood meaning by one of ordinary skill in the art to which this invention pertains, the definitions set forth herein shall prevail.

[0046] Unless otherwise stated, all numerical values ​​of the quantities of expressed components, etc., used in the specification and claims are to be understood as being modified by the term "about". Therefore, unless otherwise indicated, the numerical parameters set forth herein are approximate values ​​that can be varied to obtain the desired performance as needed.

[0047] Example

[0048] The following will further illustrate the concept and technical effects of the present invention with reference to embodiments and accompanying 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 embodiments described herein are for illustrative purposes only, and the scope of the present invention is not limited thereto.

[0049] Example 1

[0050] Prepare freeze-thaw food hydrogels as follows:

[0051] Dissolve 1g of lysozyme protein in 49mL of ultrapure water and hydrate overnight. Then, adjust the pH of the protein solution to 2.0 to obtain solution A;

[0052] Solution A was placed in a 90°C environment and stirred at 300 rpm for 8 hours to induce protein fibrillation, resulting in solution B.

[0053] Dissolve 500 mg / mL dextran and 10 mg / mL EGCG in 1 mL of Bis-Tris buffer to obtain solution C;

[0054] Mix 500 μL of solution B with 500 μL of solution C in equal volumes, and stir thoroughly to obtain solution D;

[0055] Solution D was subjected to freeze-thaw treatment, including freezing at -20°C and thawing at room temperature, with a cycle time of 12–12 hours. Different cycles were performed depending on the experiment, resulting in the above-mentioned structure based on freeze-thaw cycles.

[0056] Solution D was subjected to freeze-thaw cycles, including freezing at -20°C and thawing at room temperature, with a cycle time of 12–12 hours. Different cycles were performed depending on the experiment to obtain the hydrogel constructed based on freeze-thaw cycles. The concentrations of lysozyme protein cellulose were 10 mg / mL, dextran was 250 mg / mL, and EGCG was 5 mg / mL.

[0057] Characterization

[0058] (1) The macroscopic stability of the gel was evaluated using the inversion method. After different freeze-thaw cycles, the bottles containing the hydrogel samples were inverted to determine whether the samples could maintain their structure.

[0059] from Figure 1 It was observed that hydrogels without dextran lost their structural integrity after one freeze-thaw cycle; hydrogels containing 15% dextran failed after 12 freeze-thaw cycles; and hydrogels containing 25% and 35% dextran remained stable after 50 freeze-thaw cycles. The addition of dextran can increase the viscosity of the continuous phase, promote the formation of more hydrogen bonds between protein fibers and polyphenols, and mitigate the damage caused by repeated freeze-thaw cycles.

[0060] (2) Hydrogel freezing process and ice crystal formation state

[0061] The freezing process of the hydrogel precursor solution was recorded using optical imaging.

[0062] from Figure 2 A shows that as the concentration of dextran increases, the ice crystals formed in the hydrogel system are arranged more tightly, and the system is more uniform.

[0063] (3) Cryo-scanning electron microscopy (Cryo-SEM)

[0064] The microstructure of the hydrogel was observed using a cryo-scanning electron microscope equipped with a Quorum PP3010T sample transport channel. The hydrogel sample (approximately 20 μL) was placed on a copper support and rapidly frozen in liquid nitrogen (-210 °C). Subsequently, the sample was etched at -140 °C to expose the fresh fracture surface, sublimated at this temperature for 10 min, and finally sputtered with gold (10 mA, 90 s) at -175 °C. The sample was then placed on the cold stage of the scanning electron microscope via a cryo-transfer system for observation. The cryo-scanning electron microscopy of the hydrogel constructed based on freeze-thaw cycles is shown below. Figure 2 As shown in B.

[0065] from Figure 2 B. Cryo-SEM images show that after freeze-thaw treatment, the hydrogel exhibits an enhanced uniform microstructure, the hydrogel skeleton becomes denser, and the thickness increases significantly.

[0066] (4) Determination of mechanical strength

[0067] The temperature was set to 25℃, the shear strain Y was fixed at 0.1%, and the angular frequency varied within the range of 0.1–10 Rad / s. The changes in the elastic modulus (G′) and loss modulus (G″) of the hydrogel were measured using a frequency scanning method. Mechanical strength analyses of hydrogels without freeze-thaw cycles and hydrogels constructed based on freeze-thaw cycles are as follows: Figure 2 As shown in C and 2D.

[0068] from Figure 2 C- and 2D analyses of the elastic modulus (G′) and loss modulus (G″) of the hydrogels revealed that the loss modulus (G″) of the untreated hydrogel system was higher than the storage modulus (G′), while the storage modulus (G′) of the hydrogel system after freeze-thaw treatment was higher than the loss modulus (G″), indicating a normal gelation state.

[0069] (5) Determination of biocompatibility

[0070] Hydrogels prepared based on freeze-thaw cycles were dissolved in culture medium at a ratio of 1:200 and co-incubated with THP-1 and Caco-2 cells for 24 h. The biocompatibility of the hydrogels was then determined using the CCK-8 assay and Calcein-AM / PI staining. Results are as follows: Figure 3 As shown, green represents living cells.

[0071] from Figure 3 It can be observed that there is no significant difference in cell viability between the hydrogel-treated group and the control group, indicating its excellent biocompatibility.

[0072] Example 2

[0073] This embodiment investigated the alleviating effect of a freeze-thaw cycle-constructed, dextran-stabilized EGCG-lysozyme protein fiber hydrogel on DSS-induced acute colitis in mice.

[0074] The experiment was approved by the Animal Experiment Center of Nanjing Agricultural University [SYXK(Jiangsu)2011-0037] and strictly adhered to national guidelines for laboratory animal welfare and ethics. Twenty-four 5-week-old SPF-grade male C57BL / 6J mice (weighing 19-21g) were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. The mice were housed at a temperature of 25℃ and humidity of 75%, with a 12-hour light-dark cycle. Mice had free access to sterile water and standard laboratory feed. After a 7-day acclimatization period, the mice were randomly divided into three groups: normal control group (NC), DSS model group (DSS), and DSS + dextran-stabilized polyphenol-lysozyme protein fiber hydrogel treatment group (DH-F). For the first 7 days, the NC group received distilled water, while the other two groups had free access to a distilled water solution containing 2.5% (w / v) DSS to induce acute colitis. Subsequently, mice were divided into the DH-F group (DSS) and the DSS+gamma-to-gamma-thaw cycle-induced dextran-stabilized polyphenol-lysozyme protein fiber hydrogel treatment group (DH-F). For the first 7 days, the NC group was given distilled water, while the other two groups had free access to a distilled water solution containing 2.5% (w / v) DSS to induce acute colitis. Subsequently, DH-F group mice were administered 200 μL of DH-F hydrogel by gamma daily for 7 consecutive days. Simultaneously, the NC and DSS groups were given an equal volume of distilled water. During the experiment, mouse body weight, food / water consumption, fecal characteristics, and rectal bleeding were monitored daily. After the experiment, mice were anesthetized with 2% isoflurane, and blood was collected via cardiac puncture. Blood samples were allowed to stand at room temperature for 4 hours, then centrifuged at 3000 rpm for 20 minutes at 4°C to separate serum, and stored at -80°C for further analysis. The cecum to anus segment of the intestine was harvested and its length measured. After rinsing with physiological saline, the colon was longitudinally opened and divided into two parts. One portion was fixed in 4% paraformaldehyde for histological examination; the other portion was stored at -80°C for subsequent experiments.

[0075] Characterization

[0076] (1) Monitoring of mouse weight change

[0077] The mice were weighed and their weight recorded daily during the experiment. The results are as follows: Figure 4 As shown in Figure A.

[0078] from Figure 4As shown in Figure A, both the DSS group and the DH-F group experienced a decrease in body weight after DSS treatment. After DSS treatment was stopped, and the mice were administered the freeze-thaw cycled hydrogel via gavage, their body weight gradually recovered, eventually reaching a level close to that of the NC group. However, until the end of the intervention, the body weight of the DSS group remained significantly lower than that of the NC and DH-F groups.

[0079] (2) Disease Activity Index (DAI) score

[0080] The disease activity index of mice was comprehensively evaluated based on changes in body weight, fecal characteristics, and bleeding during the experiment. The results are as follows: Figure 4 As shown in B.

[0081] from Figure 4 As can be seen from B, both the DSS group and the DH-F group showed an increase in DAI scores in the first 7 days. From day 7 to day 14, after hydrogel gavage intervention, the DAI score of the DH-F group was significantly reduced compared with the DSS group, and dropped to a level close to that of the NC group.

[0082] (3) Colon length analysis

[0083] A segment of intestine from the cecum to the anus was harvested and its length measured. The results are as follows: Figure 4 As shown in Figure C. Compared with the NC group, the DSS treatment group showed significant colonic shortening, while the DH-F intervention alleviated this symptom and partially restored colonic length.

[0084] (4) Histopathological studies

[0085] Collected colon tissue was fixed for 24 hours, dehydrated using a gradient ethanol series, and then embedded in paraffin. (4 μm) sections were then stained with H&E. Histological structures were examined and photographed under a light microscope to assess histological characteristics. Results are as follows: Figure 4 As shown in D.

[0086] H&E staining of colon sections as follows Figure 4 The D-scan showed that the colonic structure in the NC group was intact, the glands were neatly arranged, the mucosa was intact, and there was no obvious inflammatory cell infiltration. In contrast, the DSS group showed severe histological damage, including epithelial rupture, gland destruction or loss, and extensive inflammatory cell infiltration. These pathological changes were significantly alleviated after DH-F intervention.

[0087] The foregoing descriptions are merely exemplary embodiments or examples of the present invention and are not intended to limit the invention. Those skilled in the art will recognize that the present invention can be modified and varied in many ways. Any modifications, equivalent substitutions, 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 freeze-thaw cycled polysaccharide-polyphenol-protein fiber hydrogel, characterized in that, It possesses a uniform microstructure, enhanced mechanical properties, and excellent biocompatibility and stability. It contains: 5-20 mg / mL of dietary protein fiber, 150-300 mg / mL of α-glucan, and 1-40 mg / mL of plant-derived polyphenols.

2. The food grade hydrogel based on freeze-thaw cycles construction according to claim 1, characterized in that, The dietary protein fiber is lysozyme protein fiber.

3. The food-grade hydrogel constructed based on freeze-thaw cycles according to any one of claims 1 or 2, characterized in that, The plant-derived polyphenolic compounds are one or more of the following catechin compounds: epigallocatechin (EGC), catechin gallate (ECG), epigallocatechin gallate (EGCG), resveratrol, leucine, ferulic acid, chlorogenic acid, rutin, rosmarinic acid, myricetin, thearubigin, curcumin, anthocyanins, cyanidin, pelargonidin, gallic acid, ellagic acid, tannic acid, vanillic acid, syringic acid, p-coumaric acid, naringenin, sennaol, hesperidin, quercetin, baicalin, apigenin, luteolin, mangiferin, etc.

4. A method for preparing freeze-thaw cycle food-grade hydrogels according to any one of claims 1-3, characterized in that, include: 1) Dissolve the dietary protein in ultrapure water and hydrate overnight, then adjust the pH to 1.9-2.1 to obtain solution A; 2) Heat and stir solution A at 85-95℃ for 6-10 hours to induce protein fibrillation, thus obtaining solution B; 3) Dissolve dextran and plant-derived polyphenols in a buffer solution to obtain solution C; 4) Mix equal volumes of solution B and solution C, and stir thoroughly to obtain solution D; 5) Perform freeze-thaw treatment on solution D, including freezing at -20℃ and thawing at room temperature, with a cycle time of 12-12h. Depending on the experiment, different cycles were performed to obtain the freeze-thaw cycled food hydrogels described above.

5. The method according to claim 4, characterized in that, The content of dietary protein in solution A is 10-40 mg / mL.

6. The method according to claim 4 or 5, characterized in that, The content of dietary protein fiber in solution B is 10-40 mg / mL.

7. The method according to any one of claims 4-6, characterized in that, The content of dextran in solution C is 300-600 mg / mL.

8. The method according to any one of claims 4-7, characterized in that, The content of plant-derived polyphenols in solution C is 2-80 mg / mL.

9. The method according to any one of claims 4-8, characterized in that, The hydrogel is constructed based on freeze-thaw cycles.

10. The method according to any one of claims 4-9, characterized in that, The food-derived protein is lysozyme protein.

11. The method according to any one of claims 4-10, characterized in that, The dietary protein fiber is lysozyme protein fiber.

12. The method according to any one of claims 4-11, characterized in that, The plant-derived polyphenol is EGCG.

13. Use of the freeze-thaw cycled food hydrogel according to any one of claims 1-3 for relieving intestinal inflammation.

Images