Preparation method and application of intestinal adhesion delivery system based on pea 2S albumin

Abstract

The invention provides a preparation method and application of an intestinal adhesion delivery system based on pea 2S albumin. The intestinal adhesion delivery system is a protein-polyphenol compound generated by covalent binding of quinone obtained by oxidizing polyphenol through periodate resin and pea 2S albumin. According to the intestinal adhesion delivery system, cysteine-rich pea 2S albumin is used as a delivery carrier, polyphenol bioactive substances are loaded on the cysteine-rich pea 2S albumin, release of functional components in the digestion process is effectively delayed, and the gastrointestinal stability is improved. The composition is applied to the fields of inflammatory bowel diseases and intestinal infection, can target the diseased region of colitis, and effectively relieves the symptoms of colitis by enhancing the barrier function of intestinal mucosa, regulating intestinal flora and inhibiting inflammatory response. The intestinal adhesion delivery system can also deliver other bioactive substances, is applied to the fields of biological science, functional food and the like, and has good development potential.

Description

Technical Field

[0001] The present invention relates to the fields of biological science and food engineering, and in particular to a preparation method and application of a pea 2S albumin intestinal adhesive delivery system. Background Art

[0002] Polyphenol compounds are the most widely distributed class of bioactive ingredients in plants, with anti-inflammatory and antioxidant effects. Studies have found that polyphenols can act as prebiotics to regulate the balance of intestinal microecology, increase the abundance of beneficial microorganisms, and thus provide health benefits to the host. However, the structure of free polyphenols in fruits and vegetables is extremely unstable. The high reactivity of the phenolic hydroxyl group makes it susceptible to protonation by gastric acid, inducing intramolecular hydrogen bond breakage or oxidation reactions, while the hydrophobic aromatic ring is easily deprotonated under the alkaline conditions of intestinal fluid, resulting in changes in molecular conformation or even cleavage. This means that the time that polyphenol molecules actually stay in the intestine is extremely short, and the extent to which they are utilized by intestinal microorganisms as prebiotics is also significantly limited. Therefore, how to keep intact polyphenols in the intestine for a long time is a prerequisite for them to exert their prebiotic health effects. In nature, in addition to their free form, polyphenols also interact with proteins to exist in a bound form. Proteins and polyphenols often coexist in natural food raw materials, and the two can interact through non-covalent and / or covalent bonds. This interaction can protect them from oxidation and enzymatic hydrolysis during gastrointestinal digestion, making them different from traditional delivery systems. Protein-polyphenol complexes refer to the rational modification of the physical and chemical properties of polyphenols by proteins through interactions, learning from the existence form of natural polyphenols in raw materials to improve their stability, cellular uptake rate, and target-specific delivery, and to deliver as many functional components as possible, such as polyphenols, to the small intestine and colon to play the role of prebiotics. The human intestine is covered with a transparent mucus layer, which can reach a thickness of 200 to 800 μm. Mucin MUC2 is the main structural and functional component of this mucus layer. Studies have shown that mucin MUC2 contains cysteine residues at both the N and C ends, contributing a free sulfhydryl content of up to 4 to 6 μmol / g. Therefore, protein macromolecules rich in free sulfhydryl groups can adhere to the intestinal mucus layer by forming disulfide bonds with mucin MUC2, providing a possible solution to the problem of short-term retention of polyphenol delivery systems in the intestine. Pea 2S albumin is a typical thiol-rich, water-soluble globular protein with a molecular weight ranging from 4 to 80 kDa. It possesses a natural dimer domain, occupies more than two-thirds of the cysteine residues in pea proteins, and has a total thiol content of up to 40 μmol / g. Therefore, pea 2S albumin is likely to undergo covalent cross-linking with the mucin MUC2 via surface free thiol groups, thereby prolonging its intestinal adhesion. Furthermore, protein-polyphenol complexes based on pea 2S albumin may provide effective protection for polyphenols through intermolecular interactions. Therefore, constructing a pea albumin-rich free thiol delivery system is a feasible approach to enhance the stability of polyphenols and prolong their intestinal residence. Summary of the Invention

[0003] Technical Problem to Be Solved: To address the aforementioned technical problems, the present invention provides a method for preparing an intestinal adhesive delivery system based on pea 2S albumin and its application. This intestinal adhesive delivery system utilizes the cysteine-rich property of pea 2S albumin, which binds to the thiol groups of mucin MUC2 through disulfide-based interactions. This prolongs the intestinal retention of polyphenols, effectively delaying the release of functional components during digestion and improving their gastrointestinal stability. The pea 2S albumin-based intestinal adhesive delivery system prepared by the present invention can be applied to the fields of inflammatory bowel disease and intestinal infections. It can target colitis lesions and effectively alleviate colitis symptoms by enhancing intestinal mucosal barrier function, regulating intestinal flora, and inhibiting inflammatory responses. This provides a new technical solution for the treatment of intestinal diseases and the delivery of functional ingredients, and has significant clinical application value and industrial potential. The pea 2S albumin-based intestinal adhesive delivery system can adapt to the delivery needs of a variety of bioactive substances and has broad application prospects in biopharmaceuticals, functional foods, and nutritional supplements.

[0004] Technical solution: A pea 2S albumin-based intestinal adhesive delivery system uses pea 2S albumin as a delivery carrier and loads polyphenol-based bioactive substances. A method for preparing an intestinal adhesive delivery system based on pea 2S albumin comprises the following steps: S1. Preparation of pea 2S albumin solution: Dissolve pea 2S albumin powder in water and hydrate overnight at low temperature; S2. Preparation of periodic acid resin: Weigh ion exchange resin and periodic acid powder and dissolve them in water. After stirring at room temperature, rinse with ultrapure water four times, then rinse with tetrahydrofuran and diethyl ether twice respectively. Use nitrogen blowdown to remove diethyl ether to obtain periodic acid resin; S3. Preparation of polyphenol quinone solution: Weigh polyphenol powder and fully dissolve it in water. Use nitrogen blowdown to remove air from the solution. Then, add excess periodic acid resin to the polyphenol solution. After sealed and shaken for oxidation, filter and remove the resin immediately to obtain a polyphenol quinone solution after polyphenol oxidation. S4. Preparation of an intestinal adhesive delivery system: Mix the pea 2S albumin solution and the polyphenolquinone solution, stir in a dark and oxygen-free environment, remove unreacted compounds from the reaction mixture using a dialysis bag, and freeze-dry the dialyzed solution to obtain an intestinal adhesive delivery system based on pea 2S albumin. Preferably, the content of periodic acid in the periodic acid resin in step S2 is 1.30-1.40 mmol / g. Furthermore, the content of periodic acid in the periodic acid resin in step S2 is 1.39 mmol / g. Preferably, the polyphenols in step S3 include anthocyanins, chlorogenic acid, quercetin, caffeic acid and resveratrol. Preferably, the concentration of the polyphenol solution in step S3 is 0.1 to 1.0 mmol / L. Furthermore, the concentration of the polyphenol solution in step S3 is 1.0 mmol / L. Preferably, the molar ratio of the polyphenol solution to periodic acid in step S3 is 1:10-40. Furthermore, the molar ratio of the polyphenol solution to periodic acid in step S3 is 1:10. Preferably, the volume ratio of the pea 2S albumin solution to the polyphenolquinone solution in step S4 is 1 to 3:1. Furthermore, in step S4, the volume ratio of the pea 2S albumin solution to the polyphenolquinone solution is 1:1. Preferably, the stirring speed in step S4 is 100 to 500 rpm; and the stirring time is 1 to 5 hours. Application of the above-mentioned pea 2S albumin intestinal adhesive delivery system in the preparation of drugs for inflammatory bowel disease and intestinal infection. The application of the above-mentioned pea 2S albumin intestinal adhesive delivery system in the preparation of drugs for enhancing intestinal mucosal barrier function, regulating intestinal flora and inhibiting inflammatory response. Beneficial effects: 1. The pea 2S albumin-based intestinal adhesive delivery system prepared by the present invention effectively delays the release of polyphenols during digestion. Compared with free polyphenols, the covalent polyphenol form bound to pea 2S albumin improves the gastrointestinal stability of polyphenols. 2. In the intestinal adhesive delivery system prepared by the present invention, the cysteine of pea 2S albumin forms a disulfide bond with the sulfhydryl group of mucin MUC2, thereby giving the delivery system excellent intestinal adhesion, effectively increasing the intestinal retention time of polyphenols to 24 hours, which is conducive to their prebiotic effect. 3. The intestinal adhesive delivery system prepared by this invention enhances the therapeutic efficacy of polyphenols for colitis. Compared with free polyphenols, the covalently bound polyphenols to pea 2S albumin more effectively ameliorated DSS-induced colon shortening and cecal shrinkage in mice, and increased the concentration of the anti-inflammatory cytokine IL-10 in mice. 4. The intestinal adhesive delivery system prepared by the present invention uses pea 2S albumin as a delivery carrier. It is a pure natural, non-toxic and harmless plant-derived substance and conforms to the concept of green and healthy. 5. The intestinal adhesive delivery system prepared by the present invention uses pea 2S albumin as a delivery carrier and can be used to deliver other bioactive substances. It has good development potential in the fields of biological sciences and functional foods. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 These are sample images of 2S-C3G complexes formed with pea 2S albumin at different concentrations prepared in Example 1, Comparative Example 1, and Comparative Example 2; Figure 2 The C3G binding rates after forming 2S-C3G complexes with pea 2S albumin at different concentrations prepared in Example 1, Comparative Example 1, and Comparative Example 2; Figure 3 C3G content in the supernatant during in vitro digestion of 2S-C3G complexes formed by different concentrations of C3G prepared in Example 1, Comparative Example 1, and Comparative Example 2 with pea 2S albumin; Figure 4 This is the SDS-PAGE electrophoresis pattern of the 2S-C3G complex prepared in Example 1 and pea 2S albumin during in vitro digestion; Figure 5 The particle size distribution changes of the 2S-C3G complex and pea 2S albumin prepared in Example 1 during gastric digestion; Figure 6 The changes in the adhesion amount of 2S-C3G, 7S-C3G, and 11S-C3G complexes formed by different albumins and C3G prepared in Example 1, Comparative Example 3, and Comparative Example 4 to mucin; Figure 7Characterization of the diffusion movement of 2S-C3G, 7S-C3G, and 11S-C3G complexes formed by different albumins prepared in Example 1, Comparative Example 3, and Comparative Example 4 and C3G in the mucus layer; Figure 8 Adhesion properties of 2S-C3G, 7S-C3G, and 11S-C3G complexes formed with C3G by different albumins prepared in Example 1, Comparative Example 3, and Comparative Example 4 in different intestinal segments; Figure 9 3D images of the penetration of 2S-C3G, 7S-C3G, and 11S-C3G complexes formed by different albumins prepared in Example 1, Comparative Example 3, and Comparative Example 4 with C3G in the isolated intestinal mucus layer of rats; Figure 10 The distribution of the 2S-C3G complex prepared in Example 1 in the entire digestive tract of mice at different digestion times; Figure 11 The weight loss and disease activity index of colitis in the mice of Example 2, Comparative Example 5, Comparative Example 6, Comparative Example 7, and Comparative Example 8 during DSS treatment; Figure 12 These are representative images of the overall appearance of the intestinal tissues of mice in Example 2, Comparative Example 5, Comparative Example 6, Comparative Example 7, and Comparative Example 8; Figure 13 The concentration levels of inflammatory factors in the colon of mice in Example 2, Comparative Example 5, Comparative Example 6, Comparative Example 7, and Comparative Example 8; Figure 14 The genus levels of the mouse intestinal flora in Example 2, Comparative Example 5, Comparative Example 6, Comparative Example 7, and Comparative Example 8 are shown. DETAILED DESCRIPTION The present invention will be further described below in conjunction with examples, which are provided to explain the present invention and are not limited to the following examples: Example 1 This example is a method for preparing an intestinal adhesive delivery system 2SC-1.0 with an anthocyanin concentration of 1.0 mmol / L, comprising the following steps: S1. Prepare pea 2S albumin solution: Dissolve pea 2S albumin powder (5 mg / mL, w / v) in aqueous solution and hydrate overnight at 4°C for 12 h. S2. Prepare periodic acid resin: Dissolve 18 g of Amberlyst A26 resin and 9.9 g of periodic acid powder in 50 mL of water and stir at room temperature for 1–2 h. Rinse the mixture four times with 200 mL of ultrapure water, then twice with 200 mL of tetrahydrofuran and 100 mL of diethyl ether. Remove the diethyl ether using a nitrogen purge apparatus to obtain periodic acid resin (1.39 mmol of periodic acid per gram of periodic acid resin). S3. Prepare polyphenolquinone C3GQ solution: Weigh cyanidin-3-O-glucoside (C3G) to prepare a 1.0 mmol / L C3G solution. Dissolve C3G completely in a nitrogen purge for 10 minutes to remove air from the solution. Add periodic acid resin (C3G:periodic acid molar ratio of 1:10) to the C3G solution. Shake and oxidize the mixture in a sealed container for 30 seconds, then immediately filter to remove the resin and obtain the C3GQ solution. S4. Preparation of intestinal adhesive delivery system: Pea 2S albumin solution and C3GQ solution were mixed in a volume ratio of 1:1, and stirred at 300 rpm for 4 hours at room temperature, protected from light, and isolated from oxygen. The reaction mixture was filtered through a dialysis bag (7kDa) to remove unreacted compounds, and the dialyzed solution was freeze-dried for 48 hours to obtain a pea 2S albumin-anthocyanin complex (2S-C3G-1.0, abbreviated as 2SC-1.0) with an anthocyanin concentration of 1.0 mmol / L, which was the intestinal adhesive delivery system. The 2S protein that did not react with C3GQ was set as the control sample (abbreviated as 2S). The appearance of each sample is as follows: Figure 1 shown. Comparative Example 1 The difference between this comparative example and Example 1 is that the anthocyanin concentration in this comparative example is 0.3 mmol / L, the prepared pea 2S albumin-anthocyanin complex is 2SC-0.3, and the appearance of the sample is as follows: Figure 1 shown. Comparative Example 2 The difference between this comparative example and Example 1 is that the anthocyanin concentration in this comparative example is 0.6 mmol / L, the prepared pea 2S albumin-anthocyanin complex is 2SC-0.6, and the appearance of the sample is as follows: Figure 1 shown. Index determination 1. Determination of C3G Content in 2S-C3G (including Example 1, Comparative Example 1, and Comparative Example 2): C3G content was determined using the Folin-Ciocalteu method. 1 mL of Folin-Ciocalteu reagent and 3 mL of 20% w / v Na2CO3 solution were sequentially added to 1 mL of 2S-C3G solution (2 mg / mL) and thoroughly mixed using an oscillator. The mixture was placed in a 50°C water bath and reacted for 30 minutes in the dark. The absorbance of the reaction was measured at 760 nm, and a standard curve was constructed to calculate the C3G content (mg / g) in the sample. Depend on Figure 2 It can be seen that the ability of pea 2S albumin to bind to C3G increases with the increase of C3G concentration. When the C3G concentration is 1 mmol / L, the binding amount of C3G in the 2S-C3G complex (Example 1) is the largest, which is 42.97 mg / g. 2. Stability testing of the delivery system (1) In vitro simulated digestion experiment: The INFOGEST 2.0 method was used to evaluate the digestion stability of the 2S-C3G complex. The electrolyte stock solutions of simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared according to Table 1. Before the gastrointestinal digestion experiment, 0.2 g of the powder sample was dispersed in 20 mL of SGF (pH 3.0), and then pepsin (2000 U / mL) and CaCl2 solution were added immediately. The conical flask was then placed in a constant temperature water bath shaker, maintained at a speed of 130 rpm and a temperature of 37°C, and the gastric stage digestion lasted for 2 h. When the gastric stage digestion was completed, the pH value of the digestate was adjusted to 7.0, and then an equal amount of SIF and pancreatin (100 U / mL) were added, and the intestinal stage digestion was carried out under the same conditions for 2 h. The supernatants of the digests at 5 min, 30 min, 60 min, 90 min, 120 min, 150 min, 180 min, 210 min, and 240 min were collected and added with anhydrous methanol (the volume ratio of digest to anhydrous methanol was 1:2). The supernatants were centrifuged at 12000 rpm for 10 min, and the C3G content in the supernatants was determined by the Folin-phenol method and analyzed. Table 1 Preparation method of simulated gastric juice and intestinal juice (500 mL) Depend on Figure 3 The dynamic release of C3G in the samples during digestion can be seen. The amount of anthocyanin released decreases with increasing C3G addition to the complex, indicating that the complex formed by high-concentration C3G and 2S albumin can better protect anthocyanins. After 4 hours of gastrointestinal digestion, the 2SC-1.0 sample (Example 1) had the lowest anthocyanin release rate, only 71%. The anthocyanin release rate in the 2SC-0.3 sample (Comparative Example 1) was 77%, and the anthocyanin release rate in the 2SC-0.6 sample (Comparative Example 2) was 85%, demonstrating that 2SC-1.0 (Example 1) has the strongest protective effect on C3G and that the complex has the highest gastrointestinal stability. (2) SDS-PAGE electrophoresis experiment during in vitro digestion: SDS-PAGE electrophoresis experiment was performed under reducing conditions. The sample was digested for 120 min (end of the gastric digestion stage) and 240 min (end of the intestinal digestion stage). The digestate was taken out and immediately placed in a -80°C refrigerator for pre-freezing, and then freeze-dried. The sample was dissolved in a loading buffer (containing β-mercaptoethanol) to make the final protein concentration reach 10 mg / mL. The sample was mixed and boiled for 5 min. After cooling to room temperature, it was centrifuged at 10,000 rpm and 20°C for 3 min. 15 μL of supernatant and 5 μL of protein marker (10-180 kDa) were taken and added to the pre-prepared acrylamide gel. A voltage of 100 V was continuously applied during the electrophoresis experiment, and then Coomassie Brilliant Blue R250 was used for staining for 30 min at the end of the electrophoresis. It was then decolorized until the sample bands were clear, and finally photographed and analyzed using a gel imager. By Figure 4 As can be seen, compared to the product of 2S albumin after gastric digestion, the digestion product produced by the 2SC-1.0 sample (Example 1) has a darker band color. Furthermore, it can be observed that the polymer band produced by the 2SC-1.0 sample (Example 1) near 100 kDa still exists after gastric digestion, indicating that 2SC-1.0 (Example 1) can effectively resist hydrolysis by pepsin. As the intestinal digestion stage progresses, the protein continues to be hydrolyzed into smaller peptide chains, mainly concentrated around 25 kDa and 15 kDa. After the same digestion time, the band around 15 kDa of the 2SC-1.0 sample (Example 1) is darker, indicating that the structure of 2SC-1.0 is less affected by the digestion process. Overall, compared with 2S albumin, the 2SC-1.0 sample (Example 1) has a higher resistance to digestion. 3. Particle Size Distribution Changes During In Vitro Digestion: Digestates were collected at 30 and 120 minutes of digestion and particle size was determined using the wet-phase module of a dynamic light scattering analyzer. Testing was performed only when the shading coefficient of each sample stabilized at 0.3 ± 0.05. The D(4,3) data were used to plot particle size changes during sample digestion. Depend on Figure 5 As can be seen, at 30 minutes of digestion, the particle size of the 2SC-1.0 sample (Example 1) was larger than that of 2S albumin. After 120 minutes of digestion, the scattering intensity of small particles in all samples gradually increased during digestion, indicating that large particles were gradually broken down into small particles by proteases. At the end of gastric digestion, the 2SC-1.0 sample (Example 1) still contained a relatively large number of large particles, and the scattering intensity of particles larger than 100 μm increased significantly, indicating that the 2SC-1.0 sample (Example 1) is more resistant to digestion in the stomach than the 2S albumin sample. Based on the above experimental results, it was found that the 2SC-1.0 sample (Example 1) had higher in vitro digestion stability. In subsequent experiments, a sample group with a C3G addition concentration of 1.0 mmol / L was selected to explore the intestinal adhesion properties of the 2SC complex. Comparative Example 3 The difference between this comparative example and Example 1 is that the protein in this comparative example is pea 7S protein, and the prepared pea 7S albumin-anthocyanin complex is 7S-C3G, abbreviated as 7SC. Comparative Example 4 The difference between this comparative example and Example 1 is that the protein in this comparative example is pea 11S protein, and the prepared pea 11S albumin-anthocyanin complex is 11S-C3G, abbreviated as 11SC. Intestinal adhesion test of 2SC complex delivery system 7SC and 11SC were set as control samples to investigate the intestinal adhesion properties of the 2SC complex. Index determination 1. Interaction between Complexes and Mucins: Changes in Mucin Adhesion Amount: 0.6 mL of mucin solution (1 mg / mL, w / v) and 0.6 mL of each of the three complex sample solutions (3 mg / mL, w / v) were dissolved in PBS buffer (pH 7.4), mixed, and incubated at 37°C for 1 hour. After incubation, the mixed samples were centrifuged at 10,000 rpm for 15 minutes, and the supernatant was collected. The absorbance of the supernatant sample at 251 nm was measured using a UV spectrophotometer. A mucin standard curve (0.05-0.5 mg / mL) was also established. The mucin adhesion rate (%) was calculated using the following formula: Mucin adhesion rate (%) = [(M0-Ms) / M0] × 100 Where M0 is the initial mucin concentration and Ms is the mucin concentration in the supernatant after incubation. Depend on Figure 6 It can be seen that among the three composite samples, the 2SC sample (Example 1) has the highest mucin adhesion rate, which is 58.6%. The 7SC sample (Comparative Example 3) and the 11SC sample (Comparative Example 4) are 40.8% and 51.6%, respectively. This result confirms that due to the high thiol content in pea 2S protein, the interaction with mucin is stronger. 2. Diffusion of the complexes in the mucus layer: 50 μL of FITC dye solution (1 mg / mL DMSO) was added to 1 mL of the three complex solutions (3 mg / mL, w / v). The mixture was gently stirred and reacted at 4°C in the dark for 6 h. NH4Cl (final concentration 50 mM) was then added and incubated at 4°C for 2 h to terminate the reaction. Unbound FITC was removed by dialysis, and the FITC-labeled complexes were collected for subsequent experimental analysis. Approximately 70 mg of intestinal mucus was plated onto the center of a confocal microplate dish (35 mm diameter) and equilibrated at 37°C for 30 min. Subsequently, 10 μL of FITC-labeled complex sample (1 mg / mL, w / v) was dripped onto the small intestine, and the dish was incubated at 37°C for 1 h. After the reaction, an inverted high-throughput super-resolution fluorescence confocal microscope was used to capture the movement trajectory of the complex in the mucus layer, and the trajectory map was analyzed using ImageJ software. Finally, the center of mass coordinates of each single sample collected were converted into mean square displacement (MSD) and calculated according to the following formula: MSD=[x(t+τ)-x(t)] 2 +[y(t+τ)-y(t)] 2 Wherein, x and y represent the horizontal coordinate and vertical coordinate of the sample in the two-dimensional plane at time t, respectively, and τ represents the exposure time or time interval. Depend on Figure 7 It can be seen from the motion trajectory diagram that the three complex samples move in the range of 1μm to 3μm, indicating that after interacting with mucin, they are captured by the network structure of the mucus layer and their movement in the mucus layer is restricted. In particular, the motion trajectory of the 2SC sample (Example 1) is a cluster at 20s, indicating that 2SC has the strongest interaction with mucin and is easily captured in the mucus layer and is not easy to penetrate and diffuse, thereby effectively increasing its retention time in the intestine. At the same time, the mean square displacement of the sample is calculated to determine its movement and diffusion behavior in the mucus. The results show that on a time scale of 2s, the MSD values of the 2SC sample (Example 1), the 7SC sample (Comparative Example 3), and the 11SC sample (Comparative Example 4) are 0.03, 0.08, and 0.10μm, respectively. 2 The MSD values of the samples at all time scales were ranked from highest to lowest: 11SC > 7SC > 2SC. A larger MSD value indicates more pronounced movement of the complex within the mucus layer. The 2SC sample (Example 1) had the smallest MSD value, indicating that it could form more intermolecular disulfide bonds with mucin, thereby enhancing adhesion. 3. Adhesion of the Complexes to Small Intestinal Tissue: 7-8 week old female Sprague-Dawley rats weighing 200-220 g were acclimated under standard pathogen-free (SPF) conditions for one week. Prior to the experiment, the rats were fasted overnight (without water withdrawal). After the fasting period, the rats were sacrificed, and three sections of the small intestine (duodenum, jejunum, and ileum) were removed. Each section was cut into 6 cm segments, and the residual digestive matter in the intestine was slowly flushed (without disrupting the mucus layer) using Krebs-Ringer buffer (preheated to 37°C). One end of the intestinal segment was tied with medical cotton thread, and 1.5 mL of the three complex solutions (3 mg / mL, w / v) was injected into the intestine. The other end of the intestinal segment was tied with medical cotton thread. The intestinal segment tied at both ends was placed in Krebs-Ringer buffer at 37°C and placed in a 10 mL centrifuge tube. The mixture was shaken and incubated for 2 h to allow the complex to adhere spontaneously. 0.5 mL of the intestinal and external sample solutions were taken to determine the C3G concentration. The final intestinal adhesion amount adhered to the intestine was calculated according to the following formula: Intestinal adhesion amount (%) = [(C 初 -C 内 -C 外 ) / C 初 ]×100 Among them, C 初 is the initial sample anthocyanin concentration; C 内 is the anthocyanin concentration in the intestinal sample after the reaction; C 外 Anthocyanin concentrations in the reaction and sample external solutions. Depend on Figure 8 It can be seen that the adhesion rates of the 2S-C3G sample (Example 1) in the duodenum, jejunum, and ileum are 42%, 65%, and 66%, respectively, which are significantly higher than those of the 7S-C3G sample (Comparative Example 3) and the 11S-C3G sample (Comparative Example 4). The results show that the 2S-C3G sample (Example 1) has good intestinal adhesion properties and effectively increases the residence time of C3G in the small intestine. 4. Penetration of the Complexes in Intestinal Mucus: 1 mL of each of the three complex solutions (3 mg / mL) was injected into the rat colon. The colon was then tied at both ends and placed in Krebs-Ringer buffer for incubation at 37°C for 1 hour. Following incubation, one end of the colon was cut and the intestinal fluid discarded. A small section of the colon was cut longitudinally and plated flat in the center of a small confocal dish (35 mm diameter). 20 μL of Alexa 555 wheat germ agglutinin was then added for staining for 30 minutes. After staining, the sample was scanned along the z-axis using a two-photon confocal microscope to observe the penetration of the complexes in the colonic mucus. Depend on Figure 9It can be seen that the three complexes are labeled with fluorescein isothiocyanate (FITC) and appear green in the figure; the mucin in the mucus layer is labeled with Alexa 555 wheat germ agglutinin, and the result is red. This experiment was filmed by inversion, so the closer the green-labeled complex is to the lower layer, the easier it is to penetrate into the mucus layer. As a result, it can be clearly observed that the effect of the 2S-C3G sample (Example 1) retained in intestinal mucus is better than that of the 7S-C3G sample (Comparative Example 3) and the 11S-C3G sample (Comparative Example 4). The 2S-C3G complex acts on mucin, and most of the green particles and red mucin are at the same depth and retained in the mucus layer, which inhibits the penetration of the 2S-C3G complex in the mucus layer and is more easily captured by the mucus layer network structure, so that it can be retained in the mucus layer. 5.2S-C3G intestinal adhesion properties in vivo: The distribution of 2S-C3G complex in the gastrointestinal tract after oral administration to mice was observed using animal in vivo fluorescence imaging technology. The experiment was conducted on 8-week-old C57BL / 6J male mice. First, the 2S-C3G complex was labeled with Nile blue and then gavaged with 0.2 mL of the labeled 2S-C3G complex at 25 mg per kg of body weight (the concentration of the 2S-C3G complex was 3 mg / mL). The mice were killed by cervical dislocation at 1 hour, 3 hours, 6 hours, 12 hours, and 24 hours after oral administration. The complete digestive tract tissue of the mice was removed, and the entire digestive tract was imaged using an animal in vivo fluorescence imager. Depend on Figure 10 It can be seen that after 1 hour of digestion, most of the samples are still retained in the stomach, showing a large fluorescence intensity. As the digestion time advances, after 3 hours and 6 hours of digestion, the samples gradually reach the intestine, and it is observed that the 2S-C3G sample (Example 1) is mostly concentrated in the jejunum and ileum, with increased adhesion in the intestine, and the adhesion amount in the ileum is the largest. As the digestion time further increases, the fluorescence intensity of the sample in the digestive tract gradually weakens. After 24 hours of digestion, the covalently modified sample also has weak fluorescence. This is due to the intestinal alkaline pH environment, which is conducive to the activation of thiols and the formation of disulfide bonds, and the secondary oxidation of anthocyanins can also cross-link mucins, so it can stay in the intestine for a longer time. In summary, the above results show that the 2S-C3G complex covalently modified with pea 2S albumin as a carrier used in the present invention can effectively enhance the resistance of C3G to the digestive process and improve its stability. In addition, the intestinal adhesion properties of pea 2S albumin can prolong the retention time of C3G in the intestine, and the adhesion effect is significantly stronger than that of pea 7S and 11S proteins. Example 2 This example shows the effect of the pea 2S protein-anthocyanin complex on alleviating colitis in mice, which is the 2S-C3G intervention group (2SC+DSS). Comparative Example 5 The difference between this comparative example and Example 4 is that this comparative example is a CT control group without any intervention measures. Comparative Example 6 The difference between this comparative example and Example 4 is that this comparative example is a DSS group in which DSS-induced colitis in mice was detected. Comparative Example 7 The difference between this comparative example and Example 4 is that this comparative example is an anthocyanin pure product intervention group (C3G+DSS). Comparative Example 8 The difference between this comparative example and Example 4 is that this comparative example is a 2S protein intervention group (2S+DSS). Verification of the effect of the delivery system in alleviating colitis in mice Mice were randomly divided into 5 groups, each with 10 mice. They were a CT control group (Comparative Example 5) without any intervention measures, a DSS group (Comparative Example 6) in which colitis was induced in mice using DSS, an anthocyanin pure intervention group C3G+DSS (Comparative Example 7), a 2S protein intervention group 2S+DSS (Comparative Example 8), and a complex 2S-C3G intervention group 2SC+DSS (Example 2). Among them, the C3G dosage of the anthocyanin pure intervention group (Comparative Example 7) was 30 mg / kg / day, the 2S-C3G complex group (Example 2) was prepared at the same dosage as the anthocyanin concentration contained in the sample, and the 2S protein intervention group (Comparative Example 8) was prepared at the same 2S protein concentration as the 2S-C3G group. The mice were intervened by gavage for 21 days. The CT control group (Comparative Example 5) and the DSS group (Comparative Example 6) were fed normally. Starting on day 21, mice in the DSS group (Comparative Example 6) and the intervention groups (Example 2, Comparative Example 7, and Comparative Example 8) were given free access to 2.5% DSS water for 7 days. On day 28, blood was drawn from the eyeballs of the mice and they were sacrificed by cervical dislocation. Serum, colon tissue, and intestinal contents were collected from each group and stored in a -80°C freezer until further use. Some colon tissue was placed in a tissue fixative for further processing. Index determination 1. Disease Activity Index Assessment: The body weight of mice was recorded daily starting from the onset of DSS induction, and the Disease Activity Index (DAI) was assessed on the final day. The DAI is an indicator of colitis severity, including weight loss, bloody stools, and health status. The DAI score is shown in Table 2. Table 2 Disease Activity Index Score Depend on Figure 11It can be seen that starting from the 4th day after the induction of DSS water, the body weights of mice in the 2SC+DSS group (Example 2), the DSS group (Comparative Example 6), the C3G+DSS group (Comparative Example 7) and the 2S+DSS group (Comparative Example 8) decreased to varying degrees compared with the normal diet group (Comparative Example 5). The body weight of the DSS group (Comparative Example 6) decreased to 78% of the initial body weight on the 7th day of DSS induction, while the weight loss of mice in the intervention groups (Example 2, Comparative Example 7, Comparative Example 8) improved, and the disease activity index was lower than that of the DSS group (Comparative Example 6). It can be seen that the 2S-C3G complex group (Example 2) has the most obvious effect on improving colitis. 2. Colon Tissue Analysis: Mice were sacrificed by cervical dislocation, and colon tissues from each group of mice were collected and placed on the same plane for observation. Depend on Figure 12 It can be seen that the colon tissue of mice in the normal diet group (Comparative Example 5) is normal, without congestion and edema, the length of the colon tissue is between 7.5 and 8 cm, and there are formed fecal particles in the intestine. In contrast, the colon of mice in the DSS group (Comparative Example 6) is congested and its length is significantly shortened to about 5 cm. There is no formed feces in the intestine and the cecum is wrinkled. Compared with the DSS group (Comparative Example 6), the shortening of the colon and the wrinkling of the cecum of mice in all intervention groups (Example 2, Comparative Example 7, Comparative Example 8) are significantly improved, among which the improvement effect of the 2S-C3G group (Example 2) is the most obvious. In summary, the 2S-C3G complex can effectively alleviate the clinical symptoms of DSS-induced colitis in mice. 3. Analysis of inflammatory factors in the colon: Part of the mouse colon tissue was homogenized by adding 100-200 μL RIPA protein lysis buffer and then fully lysed by ultrasonic treatment for 10 minutes. The supernatant was collected for determination. Figure 13 It can be seen that the concentration of the anti-inflammatory cytokine IL-10 in mice in the DSS group (Comparative Example 6) was significantly reduced by about 50%, while the 2S-C3G intervention group (Example 2) can significantly increase the content of IL-10 in the intestinal tissue of colitis mice, which is beneficial to restore the intestinal barrier function of colitis mice. Compared with the normal group, the concentration of the proinflammatory cytokine IFN-γ in the colon tissue of the DSS group (Comparative Example 6) showed a significant upward trend. This shows that DSS induction causes damage to the colon tissue and increases the secretion of proinflammatory factors. After intervention with the 2S-C3G complex, the concentration of IFN-γ in the colon can be significantly reduced. 4. Intestinal microbial analysis: 16S rRNA high-throughput sequencing was used to analyze changes in mouse intestinal microbiota, primarily including the effects of the 2S-C3G complex on the composition and structure of the intestinal flora. Depend on Figure 14It can be seen that the composition structure of the intestinal flora can be analyzed from the differences in microorganisms at the genus level. For example, Dubosiella and Akkermansia are both probiotics that can inhibit colitis and improve mucosal barrier damage in different ways. Compared with the DSS group (Comparative Example 6), the relative abundance of Dubosiella and Akkermansia in the intestinal flora of mice in the 2S-C3G group (Example 2) after feeding the 2S-C3G complex increased by 2.1 times and 7.6 times, respectively. The results show that the relative abundance of the above two probiotics in the 2S-C3G complex intervention group showed an increasing trend, enabling them to be significantly enriched in colitis mice. In summary, the 2S-C3G complex covalently modified with pea 2S albumin as a carrier adopted in the present invention has increased intestinal adhesion and can effectively alleviate the physiological symptoms of inflammation in DSS colitis mice. It can regulate the intestinal environment by promoting the secretion of anti-inflammatory cytokines and increasing the relative abundance of beneficial bacteria, thereby improving colitis. The covalently modified samples are significantly different from the samples fed with C3G alone (Comparative Example 7) and 2S alone (Comparative Example 8). The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, use the methods and technical contents disclosed above to make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A pea 2S albumin-based intestinal adhesive delivery system, characterized by: The intestinal adhesive delivery system uses pea 2S albumin as a delivery carrier and loads polyphenol bioactive substances.

2. The method for preparing an intestinal adhesive delivery system based on pea 2S albumin according to claim 1, characterized in that: The following steps are involved: S1. Preparation of pea 2S albumin solution: Dissolve pea 2S albumin powder in water and hydrate overnight at low temperature; S2. Preparation of periodic acid resin: Weigh ion exchange resin and periodic acid powder and dissolve in water, stir at room temperature, rinse with ultrapure water, and then rinse with tetrahydrofuran and diethyl ether, respectively. Use nitrogen blower to remove diethyl ether to obtain periodic acid resin. S3. Preparation of polyphenol quinone solution: After the polyphenol powder is fully dissolved in water, the air in the solution is removed by nitrogen blowing. Then, an excess of periodate resin is added to the polyphenol solution. After the sealed oscillation oxidation, the resin is immediately filtered to remove the resin, thereby obtaining a polyphenol quinone solution after oxidation; S4. Preparation of an intestinal adhesive delivery system: Mix the pea 2S albumin solution and the polyphenolquinone solution, stir in a dark and oxygen-free environment, remove unreacted compounds from the reaction mixture using a dialysis bag, and freeze-dry the dialyzed solution to obtain an intestinal adhesive delivery system based on pea 2S albumin.

3. The preparation method according to claim 2, wherein: The content of periodic acid in the periodic acid resin in step S2 is 1.30-1.40 mmol / g.

4. The preparation method according to claim 2, wherein: The polyphenols in step S3 include anthocyanins, chlorogenic acid, quercetin, caffeic acid and resveratrol.

5. The preparation method according to claim 2, wherein: The concentration of the polyphenol solution in step S3 is 0.1-1.0 mmol / L.

6. The preparation method according to claim 2, wherein: The molar ratio of the polyphenol solution to periodic acid in step S3 is 1:10-40.

7. The preparation method according to claim 2, characterized in that: In step S4, the volume ratio of the pea 2S albumin solution to the polyphenolquinone solution is 1 to 3:1; the stirring speed is 100 to 500 rpm; and the stirring time is 1 to 5 hours.

8. Use of the pea 2S albumin-based intestinal adhesive delivery system according to claim 1 in the preparation of drugs for inflammatory bowel disease and intestinal infection.

9. The use according to claim 7, characterized in that: The drug is a drug that can target the lesion site of colitis.

10. The use according to claim 7, characterized in that: The drug is a drug that can effectively relieve the symptoms of colitis by enhancing the intestinal mucosal barrier function, regulating intestinal flora and inhibiting inflammatory response.

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