Lysozyme-xanthan gum conjugate nanoparticle for improving bioavailability of pleurotus eryngii immunoactive peptide and preparation method of lysozyme-xanthan gum conjugate nanoparticle

Abstract

The invention discloses a lysozyme-xanthan gum conjugate nano-particle for improving bioavailability of pleurotus eryngii immune active peptide, which is a nano-particle loaded with pleurotus eryngii immune active peptide prepared by taking lysozyme, xanthan gum and pleurotus eryngii immune active peptide as raw materials through a Maillard reaction controllable damp-heat method. Wherein the amino acid sequence of the pleurotus eryngii immune active peptide is LLGVD. By combining the mild and controllable Maillard reaction with the ultrafiltration purification technology, high-proportion and high-activity loading of the pleurotus eryngii immunocompetent peptide is realized under the mild condition, and the structural integrity and immunomodulatory activity of the pleurotus eryngii immunocompetent peptide are effectively maintained; wide application prospects are realized in the aspect of developing functional foods or related products for improving low immunity. The physical stability, the digestion resistance and the potential oral bioavailability of the active peptide are remarkably improved, and the whole process is safe, green and relatively simple and has good amplification potential.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and particularly relates to lysozyme-xanthan gum conjugate nanoparticles for improving the bioavailability of Pleurotus eryngii immune active peptides and a preparation method thereof. Background Art

[0002] King oyster mushrooms have a protein content of up to 28.1% and are rich in 17 amino acids, including eight essential amino acids. Research has found that peptides obtained through proteolysis of King oyster mushrooms can modulate host immune responses by interacting with immune cells. Researchers isolated and purified a peptide fraction from King oyster mushrooms using enzymatic hydrolysis, ultrafiltration, and gel chromatography. This peptide fraction activated the TLR2 / 4 signaling pathway in mouse Ana-1 macrophages and induced the cells to secrete TNF-α and IL-6, thereby enhancing phagocytic function and exhibiting significant immunomodulatory activity (Sun, er. al. Antioxidant, antitumor, and immunostimulatory activities of the polypeptide from Pleurotus eryngii mycelium. International Journal of Biological Macromolecules, 2017, 97:323-330.). However, King oyster mushroom immune-active peptides must be administered orally, digested and absorbed in the gastrointestinal tract, and then transported through the systemic circulation to various organs, tissues, and cells. They can only exert their optimal physiological functions when they accumulate to a certain concentration. During this process, factors such as changes in gastrointestinal pH, hydrolysis by digestive enzymes, and intestinal absorption barriers affect the stability of King Oyster Mushroom immune active peptides, thereby reducing their bioavailability and causing their physiological effects in the body to be unable to fully manifest, thereby limiting the widespread application of King Oyster Mushroom immune active peptides in the food industry.

[0003] Delivery systems such as lipid-based carriers, emulsions, and biopolymer nanoparticles are widely used to enhance the oral bioavailability of peptides. However, each of these delivery systems has limitations. For example, phospholipids, the primary component of liposomes, are susceptible to the effects of gastric acid, digestive enzymes, and bile salts during digestion, leading to decreased activity of the encapsulated peptide. Emulsions, as thermodynamically unstable systems, can experience droplet aggregation or fusion during long-term storage, leading to peptide leakage or uneven distribution. In recent years, biopolymer nanoparticles designed based on protein-polysaccharide interactions have attracted considerable attention due to their low toxicity, reproducibility, biodegradability, and biocompatibility. Patent CN113416350A discloses a method for modifying whey protein onto the surface of ε-polylysine-gum arabic nanoparticles via cross-linking with transglutaminase. However, this method suffers from high enzyme costs and low economic efficiency for large-scale production. Furthermore, excessive cross-linking can increase the carrier's rigidity, thereby impairing peptide release.

[0004] The protein-polysaccharide covalent complex formed by the controlled moist heat method based on the Maillard reaction has a wide range of stability under environmental stresses such as high temperature, acid, alkali and high ion concentration. Researchers successfully prepared nanoparticles by heat-induced self-assembly (90°C) of lysozyme and xanthan gum under alkaline conditions (pH 12.0), and achieved efficient encapsulation of rice selenoproteins, which remained stable in pH 3.0-7.0 and 0-200 mmol / L sodium chloride solutions (Encapsulation of selenium-containing peptides in xanthan gum-lysozyme nanoparticles as a powerful gastrointestinal delivery system, Food Research International, 2022, 156, 111351). However, since the electrostatic interactions that stabilize nanoparticles are sensitive to extreme pH and high ion concentrations, the stability of the assembly may be reduced or even disintegrate. Summary of the Invention

[0005] The inventors isolated and purified a peptide, LLGVD, from Pleurotus eryngii that exhibits immunomodulatory activity against RAW 264.7 cells. To overcome the instability of peptide-loaded protein / polysaccharide complexes formed through non-covalent interactions and the low bioavailability of Pleurotus eryngii immunoreactive peptides, the core objective of the present invention is to provide lysozyme-xanthan gum conjugate nanoparticles loaded with Pleurotus eryngii immunoreactive peptides, prepared via a Maillard reaction controlled moist heat method. These nanoparticles enhance the stability of the encapsulated target peptide at pH 2.0-12.0 and high ion concentrations, in simulated in vitro gastrointestinal environments, and in Caco-2 cell monolayers (simulating human small intestinal epithelial cells). This enables the stable delivery of Pleurotus eryngii immunoreactive peptides, significantly improving their bioavailability and potentially enabling their application as functional food additives.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A lysozyme-xanthan gum conjugate nanoparticle for improving the bioavailability of King Oyster Mushroom immune active peptide. The nanoparticle, loaded with King Oyster Mushroom immune active peptide, is prepared using lysozyme, xanthan gum and King Oyster Mushroom immune active peptide as raw materials through a Maillard reaction controlled moist heat method.

[0008] The amino acid sequence of the Pleurotus eryngii immune active peptide is LLGVD, and its structure is shown in Formula I:

[0009]

[0010] Preferably, the lysozyme-xanthan gum conjugate nanoparticles are prepared by the following method: dissolving lysozyme and xanthan gum in ultrapure water, sequentially performing hydration, wet-heat reaction, and centrifuging using an ultrafiltration centrifuge tube to obtain a lysozyme-xanthan gum conjugate solution; mixing the King Oyster Mushroom immune active peptide with the lysozyme-xanthan gum conjugate solution, stirring at room temperature, and freeze-drying to obtain King Oyster Mushroom immune active peptide-loaded lysozyme-xanthan gum conjugate nanoparticles.

[0011] A method for preparing lysozyme-xanthan gum conjugate nanoparticles for improving the bioavailability of Pleurotus eryngii immunoreactive peptides comprises the following steps:

[0012] Step (1), dissolving lysozyme and xanthan gum in ultrapure water, stirring at room temperature until completely dissolved, hydrating, and then performing a wet heat reaction. After the reaction is completed, centrifuging using an ultrafiltration centrifuge tube, collecting a sample in the inner tube, and obtaining a lysozyme-xanthan gum conjugate solution;

[0013] Step (2): adding the Pleurotus eryngii immune active peptide to the lysozyme-xanthan gum conjugate solution, stirring continuously at room temperature, and freeze-drying to obtain the Pleurotus eryngii immune active peptide-loaded lysozyme-xanthan gum conjugate nanoparticles.

[0014] In step (1), the mass ratio of lysozyme to xanthan gum is 1:2 to 1:4, preferably 1:2.5 to 1:4, and more preferably 1:3.

[0015] The lysozyme and xanthan gum are dissolved in ultrapure water, and the total concentration of the lysozyme and xanthan gum is 3% (w / v).

[0016] The stirring time is 1 to 5 hours.

[0017] The hydration temperature is 4° C. and the hydration time is 12 h.

[0018] The temperature of the wet heat reaction is 70 to 90° C., preferably 80° C.; the time of the wet heat reaction is 1 to 9 hours, preferably 6 to 8 hours, more preferably 7 hours.

[0019] The ultrafiltration membrane of the ultrafiltration centrifuge tube has a molecular weight cutoff of 1 kDa to 10 kDa; the centrifugation time is 10 to 30 minutes, and the centrifugation speed is 4000 to 8000 rpm.

[0020] In step (2), the mass ratio of King Oyster Mushroom immune active peptide to lysozyme is 1:1 to 5:1, preferably 1:3.

[0021] The stirring time is 1 to 5 hours.

[0022] The stirring speed is 1000-2000 rpm.

[0023] Another object of the present invention is to provide the use of the lysozyme-xanthan gum conjugate nanoparticles in the preparation of health products or medicines that help enhance immunity.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention utilizes safe and natural raw materials (lysozyme and xanthan gum) and, through a mild and controllable Maillard reaction combined with ultrafiltration purification technology, efficiently constructs a nanocarrier with superior performance. It can also achieve high-proportion and high-activity loading of King Oyster Mushroom immune active peptides under mild conditions, effectively maintaining the structural integrity and immunomodulatory activity of King Oyster Mushroom immune active peptides, and has broad application prospects in the development of functional foods or related products for improving low immunity.

[0026] (2) The present invention not only significantly improves the physical stability, digestion resistance and potential oral bioavailability of the active peptide, but also the entire process is safe, green, relatively simple and has good scalability potential.

[0027] (3) The present invention provides a promising nano-solution for the efficient application of Pleurotus eryngii immune active peptides (and other similar bioactive peptides) in functional foods, health products or drug delivery systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The grafting degree of lysozyme-xanthan gum conjugate prepared at different reaction times (A), reaction temperatures (B) and lysozyme / xanthan gum mass ratios (C).

[0029] Figure 2 Transmission electron microscopy of lysozyme-xanthan gum conjugate nanoparticles loaded with Pleurotus eryngii immune active peptide.

[0030] Figure 3 The effects of different pH and sodium chloride concentrations on the particle size of lysozyme-xanthan gum conjugate nanoparticles loaded with Pleurotus eryngii immune active peptides.

[0031] Figure 4 The loading rate and encapsulation rate of Pleurotus eryngii active peptide in lysozyme nanoparticles, lysozyme-xanthan gum mixture nanoparticles and lysozyme-xanthan gum conjugate nanoparticles.

[0032] Figure 5 The particle size and potential of lysozyme nanoparticles, lysozyme-xanthan gum mixture nanoparticles and lysozyme-xanthan gum combination nanoparticles.

[0033] Figure 6 The bioavailability of King Oyster Mushroom immune active peptide, lysozyme nanoparticles, lysozyme-xanthan gum mixture nanoparticles and lysozyme-xanthan gum conjugate nanoparticles after in vitro digestion and Caco-2 cell transport.

[0034] Figure 7 The immunomodulatory activity of lysozyme nanoparticles, lysozyme-xanthan gum mixture nanoparticles, and lysozyme-xanthan gum conjugate nanoparticles after in vitro digestion and cellular transport. DETAILED DESCRIPTION

[0035] The amino acid sequence of the King Oyster Mushroom immune active peptide is LLGVD, and its structure is shown in Formula I:

[0036]

[0037] Example 1

[0038] 1. Effect of different reaction time, reaction temperature and reaction ratio on the grafting degree of lysozyme-xanthan gum conjugate

[0039] The grafting degree characterizes the degree of covalent bonding between lysozyme and xanthan gum, and its value is positively correlated with the stability of lysozyme-xanthan gum nanoparticles. Therefore, precise control of reaction conditions (including reaction time, temperature, and mass ratio) is key to obtaining highly stable nanoparticles.

[0040] Effect of reaction time: Lysozyme and xanthan gum were mixed in a mass ratio of 1:3 and dissolved in ultrapure water (the total concentration of lysozyme and xanthan gum was 3%, w / v, i.e., g / mL); stirred at room temperature for 2 h until completely dissolved, and then stored at 4°C overnight for sufficient hydration; the above mixed solution was placed in an 80°C water bath for 1, 3, 5, 7, and 9 h, respectively, to obtain a lysozyme-xanthan gum conjugate.

[0041] Effect of reaction temperature: Lysozyme and xanthan gum were mixed in a mass ratio of 1:3 and dissolved in ultrapure water (the total concentration of lysozyme and xanthan gum was 3%, w / v). The mixture was stirred at room temperature for 2 h until completely dissolved and then stored at 4°C overnight for full hydration. The mixed solution was placed in a water bath at 60, 70, 80, and 90°C for 7 h, respectively, to obtain a lysozyme-xanthan gum conjugate.

[0042] Effect of reaction ratio: Lysozyme and xanthan gum were mixed at a mass ratio of 1:1, 1:2, 1:3, 1:4, and 1:5, respectively, and dissolved in ultrapure water (the total concentration of lysozyme and xanthan gum was 3%, w / v). The mixture was stirred at room temperature for 2 h until completely dissolved, and then stored at 4°C overnight for sufficient hydration. The mixed solution was placed in an 80°C water bath for 7 h to obtain a lysozyme-xanthan gum conjugate.

[0043] The o-phthalaldehyde method was used to study the effects of different wet heat reaction time, temperature, and mass ratio of lysozyme to xanthan gum on the lysozyme-xanthan gum conjugate. Figure 1 As shown in the figure, with the increase of reaction time, reaction temperature, and the mass ratio of lysozyme to xanthan gum, the grafting degree of the lysozyme-xanthan gum conjugate showed a trend of first increasing and then decreasing. When the lysozyme / xanthan gum mass ratio was 1:2-1:4, the reaction time was 6-8h, and the reaction temperature was 70-90℃, the grafting degree could be maintained at a high level. When the mass ratio was 1:3 and the reaction was carried out in a water bath at 80℃ for 7h, the grafting rate reached the highest value, which was (45.68%±1.69%).

[0044] Therefore, the optimal preparation process of lysozyme-xanthan gum conjugate was determined as follows: the mass ratio of lysozyme to xanthan gum was 1:3, and the reaction was carried out in a water bath at 80°C for 7 hours.

[0045] Example 2

[0046] A method for preparing lysozyme-xanthan gum conjugate nanoparticles for improving the bioavailability of Pleurotus eryngii immune active peptides comprises the following steps:

[0047] Step (1), lysozyme and xanthan gum are mixed in a mass ratio of 1:3, dissolved in ultrapure water (the total concentration of lysozyme and xanthan gum is 3%, w / v), stirred at room temperature for 2 hours until completely dissolved, and then stored at 4°C overnight to achieve full hydration; the hydrated mixed solution is placed in an 80°C water bath for 7 hours to obtain a lysozyme-xanthan gum conjugate;

[0048] Step (2): transferring the lysozyme-xanthan gum conjugate to an ultrafiltration centrifuge tube with a molecular weight cutoff value of 1 kDa, centrifuging at 4000 rpm for 30 minutes, and collecting the sample in the inner tube to obtain a lysozyme-xanthan gum conjugate solution; mixing the lysozyme-xanthan gum conjugate solution and the Pleurotus eryngii immune active peptide according to a mass ratio (m / m) of lysozyme to Pleurotus eryngii immune active peptide of 1:3, stirring continuously at 1000 rpm for 5 hours at room temperature to achieve loading, and freeze-drying to obtain lysozyme-xanthan gum conjugate nanoparticles loaded with Pleurotus eryngii immune active peptide. Figure 2 This is a transmission electron microscope image of lysozyme-xanthan gum conjugate nanoparticles loaded with King Oyster Mushroom immune active peptides. The nanoparticles are spherical and evenly dispersed under the microscopic view.

[0049] Comparative Example 1

[0050] Preparation of lysozyme nanoparticles loaded with King Oyster Mushroom immune active peptide: lysozyme (3%, w / v) was dissolved in ultrapure water; stirred at room temperature for 2 hours to completely dissolve, and then stored at 4°C overnight for full hydration; the mixed solution was placed in an 80°C water bath for 7 hours, transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 1 kDa, centrifuged at 4000 rpm for 30 minutes, and the sample in the inner tube was collected; the sample in the inner tube and the King Oyster Mushroom immune active peptide were mixed at a mass ratio of lysozyme to King Oyster Mushroom immune active peptide of 1:3, stirred continuously at 1000 rpm at room temperature for 5 hours, and freeze-dried to obtain lysozyme nanoparticles loaded with King Oyster Mushroom immune active peptide.

[0051] Comparative Example 2

[0052] Preparation of lysozyme-xanthan gum mixture nanoparticles loaded with King Oyster Mushroom immune active peptide: lysozyme and xanthan gum were mixed in a mass ratio of 1:3 and dissolved in ultrapure water (the total concentration of the lysozyme / xanthan gum mixture was 3%, w / v); stirred at room temperature for 2 hours to completely dissolve, and then stored at 4°C overnight to fully hydrate; the above solution was transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 1 kDa, centrifuged at 4000 rpm for 30 minutes, and the sample in the inner tube, i.e., the lysozyme-xanthan gum mixture, was collected; the lysozyme-xanthan gum mixture and the King Oyster Mushroom immune active peptide were mixed in a mass ratio of lysozyme to King Oyster Mushroom immune active peptide of 1:3 (m / m), stirred continuously at 1000 rpm at room temperature for 5 hours, and freeze-dried to obtain lysozyme-xanthan gum mixture nanoparticles loaded with King Oyster Mushroom immune active peptide.

[0053] Example 3

[0054] 1. Particle size of lysozyme-xanthan gum conjugate nanoparticles loaded with Pleurotus eryngii immune active peptide at pH 2.0-12.0 and different concentrations of sodium chloride

[0055] pH: In the dark, at room temperature, adjust the pH of 10 mL of the nanoparticle aqueous solution to 2.0, 4.0, 6.0, 8.0, 10.0, or 12.0 with 0.1 mol / L hydrochloric acid or 0.1 mol / L sodium hydroxide aqueous solution and let it stand at room temperature for 30 min.

[0056] Sodium chloride: 10 mL of nanoparticle aqueous solution (pH 7.0) was mixed with equal volumes of sodium chloride solutions with concentrations of 200, 320, 440, 560, 680, and 800 mmol / L, respectively, and allowed to stand at room temperature for 30 min.

[0057] The particle size of the lysozyme-xanthan gum conjugate nanoparticles loaded with Pleurotus eryngii immune active peptide treated with different pH and different concentrations of sodium chloride (Example 2) was measured using a laser particle size analyzer.

[0058] like Figure 3 As shown in the figure, under the conditions of pH 2.0 to 12.0 and different concentrations of sodium chloride, the particle size of the nanoparticles did not change significantly, indicating that the nanoparticles have good stability in different pH environments and high ionic strength conditions.

[0059] 2. Loading rate and entrapment rate of Pleurotus eryngii active peptide in lysozyme nanoparticles, lysozyme-xanthan gum mixture nanoparticles, and lysozyme-xanthan gum conjugate nanoparticles

[0060] The encapsulation efficiency and loading rate of Pleurotus eryngii active peptide in lysozyme nanoparticles (Comparative Example 1), lysozyme-xanthan gum mixture nanoparticles (Comparative Example 2) and lysozyme-xanthan gum conjugate nanoparticles (Example 2) were determined by high performance liquid chromatography. Figure 4 As shown in the results, compared with lysozyme nanoparticles loaded with Pleurotus eryngii immune active peptide and lysozyme-xanthan gum mixture nanoparticles, lysozyme-xanthan gum conjugate nanoparticles showed the highest loading rate and encapsulation efficiency, which were 3.83% ± 0.39% and 60.43% ± 2.39%, respectively.

[0061] 3. Particle size and potential of lysozyme nanoparticles, lysozyme-xanthan gum mixture nanoparticles, and lysozyme-xanthan gum conjugate nanoparticles

[0062] The particle size and potential of lysozyme nanoparticles (Comparative Example 1), lysozyme-xanthan gum mixture nanoparticles (Comparative Example 2) and lysozyme-xanthan gum conjugate nanoparticles (Example 2) were measured using a laser particle size analyzer. The results are as follows: Figure 5 As shown in the results, it was found that the particle size of the lysozyme-xanthan gum conjugate nanoparticles was the smallest among all groups (180.561±10.247 nm), and the absolute value of the ζ-potential reached 35.52±4.034 mV, indicating that its stability was higher than that of other nanoparticles.

[0063] 4. Bioavailability of Pleurotus eryngii immunoreactive peptides from lysozyme nanoparticles, lysozyme-xanthan gum mixture nanoparticles, and lysozyme-xanthan gum conjugate nanoparticles after in vitro digestion and Caco-2 cell transport

[0064] Lysozyme nanoparticles (Comparative Example 1), lysozyme-xanthan gum mixture nanoparticles (Comparative Example 2), lysozyme-xanthan gum conjugate nanoparticles (Example 2), and free Pleurotus eryngii immune active peptide were taken and ultrapure water was used to prepare a nanoparticle solution or a free Pleurotus eryngii immune active peptide solution with a concentration of 0.5 mg / mL of Pleurotus eryngii immune active peptide.

[0065] In vitro simulated digestion: Artificial gastric fluid (SGF, sterile, Beijing Solaibao Technology Co., Ltd.) was mixed with an equal volume of a 0.5 mg / mL peptide nanoparticle solution or a free Pleurotus eryngii immunoreactive peptide solution and incubated in a 37°C water bath at 105 rpm for 2 h to simulate gastric digestion. Gastric samples were adjusted to pH 7.0 with 0.1 mol / L sodium hydroxide solution and dissolved in artificial intestinal fluid (AIJ, sterile, Beijing Solaibao Technology Co., Ltd.). The mixture was incubated in a 37°C water bath at 105 rpm for 2 h to simulate intestinal digestion to obtain the digested material after in vitro simulated digestion.

[0066] To prepare cell culture medium: Pour 79 mL of DMEM high-glucose medium into a sterile container, add 20 mL of fetal bovine serum, and mix slowly (avoiding air bubbles). Add 1 mL of 100× penicillin-streptomycin solution and shake gently. Mix thoroughly with a sterile pipette (or gently invert the container) to ensure complete homogenization. All reagents were obtained from Beijing Solaibao Technology Co., Ltd.

[0067] Caco-2 cell monolayer transport: Caco-2 cells were plated at an appropriate density (e.g. 1×10 5 cells / cm2) were seeded on a Transwell polycarbonate membrane chamber (pore size 0.4 μm), and cell culture medium was added to the lower chamber; culture was continued for about 21 days, and the cell culture medium was replaced regularly. When the daily cell transmembrane resistance value was greater than 300 Ω·cm 2 , indicating that the Caco-2 cell monolayer has been formed. Before the experiment, the cell monolayer was washed several times with Hank's balanced salt solution (preheated to 37°C). The chyme after in vitro simulated digestion was added to the top chamber of the Transwell chamber, and fresh 1× Hank's balanced salt solution (Beijing Solebold Technology Co., Ltd.) was added to the lower chamber and incubated at 37°C and 5% CO2 for 4 hours. Samples were taken from the lower chamber and supplemented with an equal volume of fresh buffer. After the experiment, the cell monolayer was washed several times with 1× Hank's balanced salt solution (Beijing Solebold Technology Co., Ltd.) precooled to 4°C. The cells were ultrasonically pulverized in a 4°C low-temperature constant temperature water bath (120W, pulse 1s / 3s, 10 times in total), the lysate was collected, and the intracellular target content was determined by high performance liquid chromatography. The bioavailability was calculated according to Formula 1.

[0068]

[0069] like Figure 6 As shown in the data, compared with free King Oyster Mushroom immune active peptide, the bioavailability of the three nanoparticles was increased by at least 50% after the digestive chyme of simulated gastric fluid and simulated small intestinal fluid was transported through the Caco-2 cell monolayer. Among them, the King Oyster Mushroom immune active peptide in the lysozyme-xanthan gum conjugate nanoparticles had the highest bioavailability, reaching 59.11%±2.67%.

[0070] 4. Immunomodulatory activity of lysozyme nanoparticles, lysozyme-xanthan gum mixture nanoparticles, and lysozyme-xanthan gum conjugate nanoparticles

[0071] Lysozyme nanoparticles (Comparative Example 1), lysozyme-xanthan gum mixture nanoparticles (Comparative Example 2), lysozyme-xanthan gum conjugate nanoparticles (Example 2), and free Pleurotus eryngii immune active peptide were prepared using 1× Hanks' balanced salt solution to prepare a nanoparticle solution or a free Pleurotus eryngii immune active peptide solution with a concentration of 0.5 mg / mL.

[0072] According to "2. Loading rate and encapsulation rate of Pleurotus eryngii active peptides in lysozyme nanoparticles, lysozyme-xanthan gum mixture nanoparticles and lysozyme-xanthan gum conjugate nanoparticles", lysozyme nanoparticles, lysozyme-xanthan gum mixture nanoparticles and lysozyme-xanthan gum conjugate nanoparticles were prepared respectively.

[0073] Caco-2 cells were grown at an appropriate density (e.g. 2 × 10 4 cells / cm 2 ) were seeded on a Transwell polycarbonate membrane chamber (pore size 0.4 μm), and cell culture medium was added to the lower chamber. Caco-2 cells were cultured in the upper chamber of the Transwell for 21 days, and the transmembrane resistance was monitored daily. When the transmembrane resistance value reached a stable value of 400 Ω·cm 2 The above confirms that the monolayer barrier is intact. Aspirate the cell culture medium and gently wash the cell monolayer 1-2 times with pre-warmed phosphate buffer. 5 A 1.5 mL RAW264.7 cell suspension was seeded into the lower chamber of a Transwell plate. Fresh culture medium was added to the upper chamber and cocultured with the Caco-2 monolayer for 24 hours. After 24 hours of coculture, the cells were treated with a positive control (1 μg / mL lipopolysaccharide), free Pleurotus eryngii immunoreactive peptide, or a nanoparticle solution. An equal volume of cell culture medium without any test sample was added to the negative control and cultured for another 24 hours. The culture medium from the lower chamber was collected and centrifuged at 3000 × g at 4°C for 10 minutes to remove cell debris. The supernatant was collected and aliquoted. Nitric oxide concentration in the supernatant was determined using the Griess method. The cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were also measured by ELISA.

[0074] See the results Figure 7 , showing that: compared with the blank group, free King Oyster Mushroom immune active peptide and three types of King Oyster Mushroom immune active peptide-loaded nanoparticles can increase the concentrations of nitric oxide, TNF-α, IL-6 and IL-1β in RAW264.7 cells, among which lysozyme-xanthan gum conjugate nanoparticles loaded with King Oyster Mushroom immune active peptide have higher immunomodulatory activity than the other two nanoparticles.

Claims

1. A lysozyme-xanthan gum conjugate nanoparticle for improving the bioavailability of an immune-active peptide from King Oyster Mushroom, characterized by: The nanoparticles are prepared from lysozyme, xanthan gum and King Oyster Mushroom immune active peptide as raw materials through a Maillard reaction controlled wet heat method. The amino acid sequence of the King Oyster Mushroom immune active peptide is LLGVD.

2. The lysozyme-xanthan gum conjugate nanoparticles according to claim 1, wherein: The lysozyme-xanthan gum conjugate nanoparticles are prepared by the following method: dissolving lysozyme and xanthan gum in ultrapure water, sequentially performing hydration, wet-heat reaction, and centrifuging using an ultrafiltration centrifuge tube to obtain a lysozyme-xanthan gum conjugate solution; mixing the Pleurotus eryngii immune active peptide with the lysozyme-xanthan gum conjugate solution, stirring at room temperature, and freeze-drying to obtain the Pleurotus eryngii immune active peptide-loaded lysozyme-xanthan gum conjugate nanoparticles.

3. The lysozyme-xanthan gum conjugate nanoparticles according to claim 1 or 2, characterized in that: The mass ratio of lysozyme to xanthan gum is 1:2 to 1:4, preferably 1:2.5 to 1:4, and more preferably 1:

3.

4. The lysozyme-xanthan gum conjugate nanoparticles according to claim 2, characterized in that: The lysozyme and xanthan gum are dissolved in ultrapure water, and the total concentration of the lysozyme and xanthan gum is 3%.

5. The lysozyme-xanthan gum conjugate nanoparticles according to claim 2, characterized in that: The mass ratio of King Oyster Mushroom immune active peptide to lysozyme is 1:1 to 5:1, preferably 1:

3.

6. A method for preparing the lysozyme-xanthan gum conjugate nanoparticles according to claim 1, characterized in that: The following steps are involved: Step (1), dissolving lysozyme and xanthan gum in ultrapure water, stirring at room temperature until completely dissolved, hydrating, and then performing a wet heat reaction. After the reaction is completed, centrifuging using an ultrafiltration centrifuge tube, collecting a sample in the inner tube, and obtaining a lysozyme-xanthan gum conjugate solution; Step (2): adding the Pleurotus eryngii immune active peptide to the lysozyme-xanthan gum conjugate solution, stirring continuously at room temperature, and freeze-drying to obtain the Pleurotus eryngii immune active peptide-loaded lysozyme-xanthan gum conjugate nanoparticles.

7. The method for preparing lysozyme-xanthan gum conjugate nanoparticles according to claim 6, wherein: In step (1), the hydration temperature is 4° C. and the hydration time is 12 h.

8. The method for preparing lysozyme-xanthan gum conjugate nanoparticles according to claim 6, wherein: In step (1), the temperature of the wet heat reaction is 70 to 90° C., preferably 80° C.; the time of the wet heat reaction is 1 to 9 hours, preferably 6 to 8 hours, more preferably 7 hours; The ultrafiltration membrane of the ultrafiltration centrifuge tube has a molecular weight cutoff of 1 kDa to 10 kDa; the centrifugation time is 10 to 30 minutes, and the centrifugation speed is 4000 to 8000 rpm.

9. The method for preparing lysozyme-xanthan gum conjugate nanoparticles according to claim 6, wherein: In step (2), the stirring time is 1 to 5 hours; the stirring speed is 1000 to 2000 rpm.

10. Use of the lysozyme-xanthan gum conjugate nanoparticles according to claim 1 in preparing health products or medicines that help enhance immunity.

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