Functional black fungus polysaccharide-milk compound as well as preparation method and application thereof

Abstract

The invention discloses a functional auricularia auricula polysaccharide-milk compound and a preparation method and application thereof.Auricularia auricula polysaccharide is extracted in a free radical mediation and NADES high-penetration mode after auricularia auricula sporocarp is subjected to ultramicro pretreatment, the technological process is green and environmentally friendly, operation is easy and convenient to implement, the polysaccharide yield and activity are improved, and the functional auricularia auricula polysaccharide-milk compound is prepared. The polysaccharide dissolution rate can reach 57% or above; the biological activity of the compound disclosed by the invention is obviously improved, such as active oxygen and active nitrogen removal capability; the weight loss and DAI score increase caused by DSS-induced ulcerative colitis are relieved, the inflammatory factor level is reduced, and symptoms are effectively relieved. The compound provided by the invention is derived from a natural product, avoids serious toxic and side effects caused by chemical synthetic drugs or hormones, does not observe obvious toxicity under an experimental dose, is high in safety, can be widely applied to the fields of healthy foods, health care products, special medical foods and the like, and has a wide industrialization prospect.

Description

(I) Technical Field

[0001] This invention relates to a functional black fungus polysaccharide-milk compound, its preparation method, and its application. (II) Background Technology

[0002] Ulcerative colitis (UC) is a chronic, relapsing intestinal disease primarily affecting the colon and rectum, characterized by erosive lesions of the colonic mucosa. Its etiology and pathogenesis are not fully understood, but it is currently believed to be the result of the interaction of multiple pathogenic factors. Ulcerative colitis presents with typical clinical manifestations such as persistent diarrhea, spasmodic abdominal pain, mucus and bloody stools, and weight loss. These clinical symptoms exhibit a recurring course with alternating remissions and flare-ups, leading to a decline in patients' quality of life. The complexity of colitis is not only reflected in inflammatory cell infiltration and pro-inflammatory cytokine storms, but also in significant oxidative damage to the intestinal mucosa, specifically manifested as depletion of antioxidant enzyme systems (such as SOD and CAT), decreased levels of key antioxidants (such as GSH), and abnormal accumulation of lipid peroxidation end products (such as MDA). This tissue-level oxidative damage is a crucial factor leading to epithelial cell death, barrier function disruption, and even disease progression. Ulcerative colitis is not an isolated intestinal disease; its persistent chronic inflammation leads to a systemic oxidative stress state, which may in turn cause extraintestinal manifestations or exacerbate the systemic burden. This systemic oxidative stress imbalance is an important manifestation of the severity and systemic harm of colitis. Currently used first-line drugs such as 5-aminosalicylic acid, glucocorticoids, and immunosuppressants, while having some efficacy, suffer from problems such as easy relapse after discontinuation and significant toxic side effects with long-term use. Therefore, developing a novel, safe, effective, and low-side-effect natural anti-colitis ingredient is an urgent technical problem to be solved in this field.

[0003] Naturally derived polysaccharides, due to their multiple biological activities such as anti-inflammatory, immunomodulatory, intestinal barrier protection, and gut microbiota regulation, have become highly promising bioactive molecules in the treatment of ulcerative colitis (UC). However, the activity of polysaccharides is not universal; their efficacy is highly dependent on their fine structure, including monosaccharide composition, glycosidic bond type, molecular weight, branching degree, spatial conformation, and modifying groups. These key structural features are directly influenced by the biological source (plants, microorganisms, animals, or algae) and the preparation process. Therefore, rigorous screening and optimization are essential to select truly efficient and stable active ingredients from the numerous natural polysaccharide resources for the prevention and treatment of UC.

[0004] Black fungus (scientific name) Auricularia auricula ( L. exHook.*Auricularia auricula-judae* (Black Fungus) belongs to the subphylum Basidiomycota, class Agaricomycetes, order Auriculariales, family Auriculariaceae, and genus *Auricularia*. Polysaccharides account for 60%-70% of the dry weight of the fruiting body and possess various biological activities. However, as a gelatinous fungus belonging to the subphylum Basidiomycota, *Auricularia auricula-judae* exhibits an unusually complex structure in its fungal cell wall. Studies have found that the cell wall structure of Basidiomycota includes a rigid inner layer and a flexible outer layer. The inner layer is mainly composed of chitin and β-(1,3)-(1,6)-glucan, containing small amounts of α- and β-(1,3)-glucan, mannan, and fucoidan, forming the alkali-insoluble backbone of the cell wall. The flexible outer layer of the cell wall is composed of β-(1,3)-glucan, β-(1,3)-(1,6)-glucan, β-(1,6)-glucan, mannan, and other components. The complex spatial network structure of basidiomycetes results in extremely thick and tough cell walls, making them difficult to destroy under conventional physical or chemical conditions. Studies have shown that the extraction yield, molecular structure, and bioactivity of natural polysaccharides strongly depend on the disruption pretreatment method employed. Furthermore, the cell wall disruption process can cause irreversible changes in the polysaccharide structure, significantly altering its chemical composition, molecular weight, glycosidic bond type and conformation, and bioactivity. Therefore, an effective cell wall disruption method is a crucial step in obtaining high-yield and highly active black fungus polysaccharides.

[0005] To address the complexity and toughness of fungal cell wall structures, various methods are employed to disrupt them, including mechanical, chemical, enzymatic, and combined approaches. Mechanical methods include ultrasound-assisted disruption, high-pressure homogenization, microwave-assisted disruption, high-speed bead milling, and steam disruption. These methods utilize external forces such as sound waves, pressure, electromagnetic waves, and speed to effectively disrupt the cell wall. While each mechanical method has its advantages and disadvantages, most suffer from high equipment requirements, high energy consumption, insufficient disruption, and localized high temperatures. Chemical methods use detergents, penetrants, or organic solvents to break down cells or weaken the cell wall. These methods include acid-heat or alkali-based disruption, acid-base combination disruption, and disruption using chemical reagents (such as alcohol, dimethyl sulfoxide, and methanol). While convenient and requiring less sophisticated equipment, these methods can introduce impurities and pose safety and environmental pollution risks. Enzymatic methods utilize the differences in cell-breaking sites among different enzymes to attack the fungal cell wall layer. Simultaneously, changes in the osmotic pressure of the buffer solution cause the cell membrane to rupture, releasing the endocytic cytoplasm. Because of the complex structure of fungal cell walls, single biological enzymes are unlikely to effectively break them. Attacking the cell wall with multiple enzymes sequentially can increase the yield of the target substance. The combined cell wall disruption method combines two or more of the four methods mentioned above. While this can increase yield, it also increases the number of operational steps and production costs.

[0006] Eutectic solvents (DES), including natural eutectic solvents (NADES), are formed by mixing hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs), and their melting points are significantly lower than the individual melting points of each component. Compared with traditional organic solvents, NADES have significant advantages such as lower toxicity and good biodegradability. NADES can not only be interconnected through hydrogen bonds, but can also donate or accept external electrons or protons to form hydrogen bonds, thus enabling them to dissolve a variety of substances, including salts, proteins, drugs, amino acids, surfactants, sugars, and polysaccharides.

[0007] While some research exists on polysaccharide extraction using NADES, most studies employ NADES alone. NADES typically uses choline chloride as the hydrogen bond acceptor and neutral molecules from natural plants, such as amino acids, organic acids, and sugars, as hydrogen bond donors. Using NADES alone for polysaccharide extraction suffers from low efficiency because most NADES molecules have high viscosity, severely limiting solvent diffusion and penetration into plant cell walls. Furthermore, while NADES's hydrogen bond network can dissolve many polar substances, its solubility and selectivity for specific polysaccharides (especially large, structurally complex ones) may be inferior to traditional acid-base methods. For materials with dense cell walls, NADES alone may struggle to effectively disrupt the structure, leading to incomplete extraction.

[0008] As is known to those skilled in the art, the pathological mechanism of ulcerative colitis (UC) is a complex network involving the abnormal activation of various immune cells, the release of large amounts of pro-inflammatory cytokines (such as TNF-α and IL-6), severe impairment of the intestinal epithelial barrier function, and the accompanying elevated levels of local intestinal oxidative stress. While some studies in the prior art have suggested that substances with antioxidant activity may have a beneficial effect on colitis, most of these studies remain at the preliminary observation stage and have failed to reveal whether they can truly meet the requirements for treating UC.

[0009] Although many polysaccharides have been shown to have anti-ulcer potential, directly converting them into single-component drugs often faces challenges such as a relatively narrow spectrum of action or insufficient therapeutic efficacy. Therefore, there is a need to develop polysaccharide complexes that integrate and enhance their comprehensive biological efficacy through rational design, in order to achieve better ulcer efficacy, reduce the side effects of existing drugs, and improve safety. (III) Summary of the Invention

[0010] The purpose of this invention is to provide a functional black fungus polysaccharide-milk compound, its preparation method, and its application. This invention first uses black fungus fruiting bodies as raw material. After ultra-micronization pretreatment, black fungus polysaccharides are extracted using a free radical-mediated and NADES high-permeability method, effectively improving the polysaccharide yield and activity. Then, the black fungus polysaccharide-milk compound with the best synergistic effect is selected using reactive oxygen species (·OH) and reactive nitrogen species (DPPH) as indicators. The black fungus polysaccharide-milk compound selected by the method of this invention not only possesses the nutritional value of polysaccharides and milk themselves, but also synergistically enhances free radical scavenging and anti-UC capabilities, achieving high-quality utilization of black fungus and milk, and solving the problem of significant side effects of existing UC drugs.

[0011] The technical solution adopted in this invention is:

[0012] In a first aspect, the present invention provides a functional black fungus polysaccharide-milk compound, wherein the compound is formed by mixing black fungus polysaccharide aqueous solution with milk; the concentration of the black fungus polysaccharide aqueous solution is 1.6-4.8 mg / mL.

[0013] Furthermore, the volume ratio of the black fungus polysaccharide aqueous solution to milk is 1:1.

[0014] Dairy products (such as milk) are rich in lactoferrin, casein phosphopeptides, and whey protein, possessing certain antioxidant potential. The calcium and vitamin D in milk can promote the body's antioxidant defense system. However, different brands of milk vary in processing methods (such as pasteurization vs. UHT), fat content, and protein conformation. For example, UHT treatment may denature whey protein, reducing its ability to bind with polysaccharides through hydrogen bonds; low-fat milk, lacking fat-soluble antioxidants (such as vitamin E), may have a weakened synergistic effect with polysaccharides. Therefore, the milk used in this invention is preferably pure milk.

[0015] Furthermore, the milk includes pure milk with a protein content of 3.0-3.6 g / 100mL, preferably 3.2 g / 100mL.

[0016] Furthermore, the pure milk is selected from Mengniu, Yili, or Telunsu brands.

[0017] Furthermore, the milk in question is pure milk from Mengniu, with a solids content of 142.1 mg / mL and a protein content of 3.2 g / 100mL.

[0018] Furthermore, the black fungus polysaccharide is prepared according to the following method:

[0019] (1) Crude polysaccharide extract: Take black fungus fruiting body powder (passed through a 200-mesh sieve), use deionized water and 30% hydrogen peroxide as extraction solvent, extract in a water bath shaker at a speed of 100-150 rpm (preferably 125 rpm), extract for 120 min-220 min (optimal 177 min) at a temperature of 70℃-100℃ (optimal 90℃), add a natural eutectic solvent, continue extraction for 15 min-45 min (optimal 30 min), centrifuge at 8000 rpm for 20 min, and the supernatant is the crude polysaccharide extract; the natural eutectic solvent is obtained by heating and stirring sodium citrate and glucose or ascorbic acid in deionized water until clear;

[0020] (2) Black fungus polysaccharide: After concentrating the supernatant of step (1) to 1 / 5 of the original volume, concentrate 1 is obtained; while stirring, add 4 times the volume of 95% ethanol to concentrate 1, let it stand at 4 ℃ for 12 h, carefully pour out the supernatant and keep the lower precipitate; put the lower precipitate in a centrifuge, centrifuge at 1000 rpm for 5 min, take the precipitate, add an appropriate amount of water and concentrate at 65℃ until the ethanol is completely evaporated, to obtain concentrate 2; add Sevag reagent (chloroform: n-butanol = 4:1, volume ratio) to concentrate 2 at a volume ratio of 1:5, shake vigorously for 20 min, centrifuge at 3000 rpm for 10 min to form an organic solvent layer, a protein layer and an aqueous layer, discard the protein layer and the organic solvent layer, repeat centrifugation 3 times to obtain black fungus polysaccharide purified solution, freeze dry (preferably -55℃) to obtain black fungus polysaccharide.

[0021] Further, in step (1), the black fungus fruiting body powder is obtained by soaking black fungus in water, cleaning it, drying it in an oven at 70°C, pulverizing it, and passing it through a 200-mesh sieve.

[0022] Further, in step (1), the hydrogen peroxide mass concentration in the extractant is 1.2%-1.8% (preferably 1.6%), and the volume of the extractant is 60-100 mL / g (preferably 85 mL / g) based on the mass of the black fungus fruiting body powder; the volume ratio of the extractant to the natural eutectic solvent is 170:50-70 (preferably 170:57).

[0023] Furthermore, the compound is prepared by mixing a 1.6 mg / mL aqueous solution of black fungus polysaccharide with pure milk containing 1.6 mg / mL of solids at a volume ratio of 1:1.

[0024] Secondly, the present invention provides an application of the aforementioned black fungus polysaccharide-milk compound in the preparation of antioxidant products, wherein the products are products that scavenge reactive nitrogen (such as DPPH) free radicals and / or reactive oxygen (such as ·OH) free radicals.

[0025] Thirdly, the present invention provides the application of the aforementioned black fungus polysaccharide-milk compound in the preparation of an agent to improve ulcerative colitis.

[0026] Furthermore, the formulation is for the treatment of DSS-induced ulcerative colitis. The formulation can reduce weight loss caused by DSS-induced UC and increase DAI score; slow the increase in lipopolysaccharide (LPS) levels and alanine aminotransferase (ALT) activity; and reduce the levels of inflammatory molecule TNF-α and IL-6.

[0027] Furthermore, the formulation is a drug for treating systemic oxidative stress disorder caused by ulcerative colitis. It can not only relieve local intestinal inflammation, but also systematically correct the systemic oxidative stress disorder caused by colitis, demonstrating its advantage in the comprehensive treatment of colitis.

[0028] Compared with single antioxidants or known anti-UC drugs in the prior art, the functional black fungus polysaccharide-milk compound provided by the present invention exhibits an unexpected synergistic anti-colitis effect, mainly reflected in:

[0029] 1. The exceptionally complex spatial network structure of the fungal cell wall in *Auricularia auricula-judae* fruiting bodies makes the cell walls extremely thick and tough, difficult to break down by conventional physical or chemical conditions. Conventional methods for extracting polysaccharides from *Auricularia auricula-judae* suffer from high energy consumption, insufficient cell wall disruption, and localized high temperatures. This invention pre-treats *Auricularia auricula-judae* fruiting bodies into ultra-micro particles and then extracts polysaccharides using a free radical-mediated, NADES-based high-permeability method. This process is environmentally friendly, simple to operate, and significantly improves polysaccharide yield and activity, achieving a polysaccharide dissolution rate of over 57%. It is 14.413 times more efficient than traditional hot water extraction, 2.187 times more efficient than traditional mechanical grinding, 1.631 times more efficient than hydrogen peroxide extraction, 2.903 times more efficient than cellulase extraction, and 1.966 times more efficient than extraction using a combination of cellulase, papain, and β-glucanase.

[0030] 2. This invention uses reactive oxygen species (·OH) and reactive nitrogen species (DPPH) as indicators to compound "plant-animal" dual-source natural active ingredients (black fungus polysaccharide and milk components), and selects the black fungus polysaccharide-milk compound with the best synergistic effect. Verification using both indicators (·OH and DPPH) confirms that the compound of this invention significantly enhances the scavenging ability of the strongest reactive oxygen species (·OH), indicating that it has more reliable and broader antioxidant potential in complex physiological environments. Not only are the raw materials safe, but the entire operation process also has many advantages, such as no introduction of impurities, no pollution, low equipment requirements, and low energy consumption.

[0031] 3. In DSS or TNBS-induced mouse colitis models, the black fungus polysaccharide-milk compound of this invention significantly reduced the disease activity index (DAI), effectively reversed the degree of colonic shortening, and reduced the pathological oxidative damage caused by the disease. Specifically, it significantly restored the depleted antioxidant defense capacity in colonic tissue, such as the activity of superoxide dismutase (SOD) and catalase (CAT); significantly increased the level of glutathione (GSH), a key cytoprotective substance in colonic tissue; and significantly reduced the content of malondialdehyde (MDA), the toxic end product of lipid peroxidation, in colonic tissue. Its effects were significantly better than those of the individual components, and in some indicators, it was superior to or indistinguishable from clinical positive drugs. The compound of this invention can deeply regulate inflammation at the mechanistic level, as evidenced by its ability to significantly inhibit the expression levels of key pro-inflammatory factors (TNF-α, IL-6, etc.) in colonic tissue.

[0032] 4. The black fungus polysaccharide-milk compound prepared by the method of this invention, through the optimized ratio, exhibits significantly enhanced bioactivity, such as the ability to scavenge reactive oxygen species and nitrogenous substances; it also significantly reduces weight loss and DAI score increases caused by DSS-induced ulcerative colitis, and lowers inflammatory factor levels, exceeding the combined effects of black fungus polysaccharide or milk alone (1+1>2), effectively alleviating symptoms. This compound, derived from natural products, avoids the serious toxic side effects of chemically synthesized drugs or hormones. No significant toxicity was observed at experimental doses, demonstrating high safety. It can be widely applied in health foods, health supplements, and special medical foods, with broad industrialization prospects. (iv) Description of the attached drawings

[0033] Figure 1 The scavenging rate, IC50, and synergistic ratio (SR) of different samples in step 3 of Example 3 are shown.

[0034] Figure 2 The scavenging rate and synergistic effect ratio (SR) of different samples for ·OH in step 4 of Example 3 are shown.

[0035] Figure 3 The effects of the black fungus polysaccharide-milk A compound in steps 1-3 of Example 4 on body weight, DAI score and colon length of UC mice (in the figure, C, N, P represent normal control, negative control and positive control, respectively; different letters represent differences between groups, p < 0.05).

[0036] Figure 4 The effect of the black fungus polysaccharide-milk A compound on the lipopolysaccharide (LPS) level and antioxidant-related indicators (SOD, CAT, GSH, MDA, ALT) in UC mice in step 4 of Example 4 (C, N, P in the figure represent normal control, negative control and positive control respectively; different letters represent differences between groups, p < 0.05).

[0037] Figure 5 The effect of the black fungus polysaccharide-milk A compound on the levels of inflammatory factors IL-6 and TNF-α in UC mice in step 4 of Example 4 (C, N, P in the figure represent normal control, negative control and positive control, respectively; different letters represent differences between groups, p < 0.05). (V) Detailed Implementation Methods

[0038] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0039] Malondialdehyde (MAD) assay kit, glutathione (GSH) assay kit, total superoxide dismutase (T-SOD) assay kit, and catalase (CAT) assay kit were all purchased from Nanjing Jiancheng Biotechnology Institute. Experimental data are expressed as Means ± SD. Significant differences were analyzed using IBM SPSS Statistics 25 software with one-way ANOVA (homogeneity of variance test) and LSD multiple comparison analysis; p < 0.05 was considered statistically significant.

[0040] Example 1: Extraction of polysaccharides from black fungus

[0041] 1. Black fungus fruiting body powder

[0042] Black fungus, produced in the Lesser Khingan Mountains of Heilongjiang Province, is soaked in water and cleaned before use, then dried in an oven at 70℃. It is then pulverized using a BO-250S2 high-speed multi-functional crusher and passed through a 200-mesh sieve to obtain black fungus fruiting body powder, which is then set aside.

[0043] 2. Preparation of NADES

[0044] NADES1: 14.7 mg sodium citrate and 36.0 mg glucose were mixed at a molar ratio of 1:4, and 5.43 mL of deionized water was added. The mixture was magnetically stirred at 70-80℃ for 1 h until the solution became clear, yielding 21.9 mL of NADES (glucose-sodium citrate, water content was essentially evaporated upon heating, water content was considered to be 0%), denoted as NADES1. Then, different amounts of deionized water were added to prepare NADES1 with volume water contents of 20%, 40%, 60%, and 80%.

[0045] NADES2: 14.7 mg sodium citrate and 13.2 mg ascorbic acid were mixed at a molar ratio of 1.5:1, and 3.38 mL of deionized water was added. The mixture was magnetically stirred at a constant temperature of 50-60℃ for 0.5 h under light-protected conditions until the solution became clear, yielding 10.8 mL of NADES (ascorbic acid-sodium citrate, with water content essentially evaporated upon heating, and 0% water content), denoted as NADES2. Then, different amounts of deionized water were added to prepare NADES2 with water contents of 20%, 40%, 60%, and 80% by mass.

[0046] 3. Extraction of polysaccharides from black fungus using the free radical-DES method

[0047] (1) Crude polysaccharide extract: Take 2.0 g of black fungus fruiting body powder from step 1, add 160.9 mL of deionized water, then add 9.1 mL of 30% hydrogen peroxide (deionized water and hydrogen peroxide constitute the extraction solvent, and the hydrogen peroxide concentration in the extraction solvent is 1.6%) and mix well. The material-to-liquid ratio is 1:85 (g / mL). Extract in a water bath shaker at 125 rpm for 177 min at 90℃. Then add 57 mL of NADES1 or NADES2 and continue extraction for 30 min. Centrifuge at 8000 rpm for 20 min, and the supernatant is the crude polysaccharide extract from black fungus.

[0048] (2) Purified solution of black fungus polysaccharide: After concentrating the supernatant of step (1) to 1 / 5 of the original volume, concentrate 1 is obtained; while stirring, add 4 times the volume of 95% ethanol to concentrate 1, let it stand at 4 ℃ for 12 h, carefully pour out the supernatant and keep the lower precipitate. Place the lower precipitate in a centrifuge and centrifuge at 1000 rpm for 5 min. Take the precipitate, add an appropriate amount of water and concentrate it at 65℃ by rotary evaporation until the ethanol is completely evaporated to obtain concentrate 2. Add Sevag reagent (chloroform: n-butanol = 4:1, volume ratio) to concentrate 2 at a volume ratio of 1:5, shake vigorously for 20 min, centrifuge at 3000 rpm for 10 min to form an organic solvent layer, a protein layer and an aqueous layer. Discard the protein layer and the organic solvent layer, repeat the centrifugation 3 times, collect the supernatant, and obtain the purified solution of black fungus polysaccharide.

[0049] 4. Detection of polysaccharide extraction yield

[0050] Take 1.0 mL of the crude extract of black fungus polysaccharide from step (1) and the purified extract of black fungus polysaccharide from step (2) as test solutions, dilute with deionized water to the detection range, and use the phenol-sulfuric acid method to detect the absorbance at 480 nm. Calculate the polysaccharide concentration according to the glucose standard curve, and convert it into the extraction yield of black fungus polysaccharide in the crude extract and purified extract of black fungus polysaccharide according to the following formula (1).

[0051] Polysaccharide extraction yield (%) = (C×V×f) / m ×100% Formula (1)

[0052] In formula (1), C is the polysaccharide concentration calculated by the standard equation (mg / mL); V is the volume of the test liquid (mL); f is the dilution factor; and m is the mass of the black fungus fruiting body powder sample (mg).

[0053] Glucose standard curve: A standard curve is plotted with the concentration of glucose aqueous solution on the x-axis and the absorbance at 480 nm on the y-axis. The equation of the standard curve is y = 7.9807x - 0.014, R0 2 =0.9983, where y represents the absorbance value and x represents the polysaccharide concentration.

[0054] 5. Extraction yield of polysaccharides from black fungus

[0055] In step 3, the water content of NADES1 and NADES2 was changed to 20%, 40%, 60%, and 80%, respectively. All other operations remained the same. The method in step 4 was used for detection, and the results are shown in Table 1.

[0056] Table 1. Effect of NADES moisture content on the yield of polysaccharides extracted from black fungus (%)

[0057]

[0058] As shown in Table 1, different NADES compositions and different NADES water contents have a great influence on the extraction yield of black fungus polysaccharides from crude extract. When the water content is 0%, the extraction yields of free radical-NADES1 and free radical-NADES2 are higher than those of other water contents. Moreover, under different water contents, the extraction yield of free radical-NADES1 is higher than that of free radical-NADES2.

[0059] After alcohol precipitation and deproteinization, the crude extract of black fungus polysaccharides was lost to some extent. Therefore, the crude extract of black fungus polysaccharides was extracted by using the free radical-NADES1 method with 0% water content in NADES, resulting in a polysaccharide extraction yield of up to 57.22%.

[0060] Example 2: Preparation of black fungus polysaccharide under optimized conditions

[0061] (1) Crude extract of black fungus polysaccharides: Take 2.0 g of black fungus fruiting body powder (passed through a 200-mesh sieve), add 160.9 mL of deionized water, and then add 9.1 mL of 30% hydrogen peroxide (deionized water and hydrogen peroxide constitute the extraction solvent, and the mass concentration of hydrogen peroxide in the extraction solvent is 1.6%) and mix well. The material-to-liquid ratio is 1:85 (g / mL). Extract in a water bath shaker at 125 rpm for 177 min at 90℃. Then add 57 mL of NADES1 with 0% water content prepared by the method in Example 1 and continue extraction for 30 min. Centrifuge at 8000 rpm for 20 min, and the supernatant is the crude extract of black fungus polysaccharides.

[0062] (2) Black fungus polysaccharide: After concentrating the supernatant of step (1) to 1 / 5 of its original volume, concentrate 1 is obtained; while stirring, add 4 times the volume of 95% ethanol to concentrate 1, let it stand at 4 ℃ for 12 h, carefully pour out the supernatant, and keep the lower precipitate. Place the lower precipitate in a centrifuge, centrifuge at 1000 rpm for 5 min, take the precipitate, add an appropriate amount of water, and concentrate at 65℃ by rotary evaporation until the ethanol is completely evaporated, to obtain concentrate 2. Add Sevag reagent (chloroform: n-butanol = 4:1, volume ratio) to concentrate 2 at a volume ratio of 1:5, shake vigorously for 20 min, centrifuge at 3000 rpm for 10 min to form an organic solvent layer, a protein layer and an aqueous layer. Discard the protein layer and the organic solvent layer, repeat the centrifugation 3 times to obtain the purified black fungus polysaccharide solution, freeze-dry at -55℃ to obtain 0.76 g of black fungus polysaccharide, denoted as AAP.

[0063] Example 3: Preparation and Antioxidant Capacity Detection of Black Fungus Polysaccharide-Milk Complex

[0064] 1. Preparation of black fungus polysaccharide-milk compound

[0065] The black fungus polysaccharide prepared by the method in Example 2 was prepared into polysaccharide solutions of different concentrations (1.6 mg / mL, 3.2 mg / mL, and 4.8 mg / mL) using deionized water. Each concentration of polysaccharide solution was mixed with five types of commercially available milk (milk A, milk B, milk C, milk D, and milk E) as shown in Table 2 at a volume ratio of 1:1 to obtain 15 black fungus polysaccharide-milk complexes, denoted as 1.6AAP-MilkA, 1.6AAP-MilkB, 1.6AAP-MilkC, 1.6AAP-MilkD, 1.6AAP-MilkE; 3.2AAP-MilkA, 3.2AAP-MilkB, 3.2AAP-MilkC, 3.2AAP-MilkD, 3.2AAP-MilkE; and 4.8AAP-MilkA, 4.8AAP-MilkB, 4.8AAP-MilkC, 4.8AAP-MilkD, 4.8AAP-MilkE.

[0066] Table 2. Solid content of commercially available milk

[0067]

[0068] 2. Calculation of IC50 (theoretical) and the efficiency ratio SR

[0069] The total concentration of polysaccharides and milk solids in each compound in step 1 is denoted as C1. During the determination, each C1 is serially diluted with deionized water to 0.5C1, 0.25C1, 0.125C1, 0.0625C1, 0.03175C1, and 0.015875C1, and denoted as C2, C3, C4, C5, C6, and C7, respectively. The free radical scavenging rate is tested using the methods in steps 3 and 4, and the IC50 is obtained according to formula (2). 50 (theory).

[0070] Formula (2)

[0071] IC(A) 50 IC(B) represents the polysaccharide concentration at which the free radical scavenging rate is 50%. 50 The value represents the milk concentration when the free radical scavenging rate is 50%, a represents the mass of polysaccharides in the compound, and b represents the mass of milk solids in the compound.

[0072] IC of the compound 50 (In practice) the results were obtained through experiments in steps 3 and 4, and the synergistic ratio SR was calculated according to formula (3).

[0073] SR = IC 50 (Theory) / IC 50 (Actual) Formula (3)

[0074] When 0.5 < SR < 1.5, it has an additive effect; when SR > 1.5, it has a synergistic effect; when SR < 0.5, it has an antagonistic effect.

[0075] 3. Determination of the scavenging rate of DPPH (1,1-diphenyl-2-picrylhydrazyl) free radicals and the synergistic ratio SR

[0076] DPPH is a stable free radical with an unpaired valence electron on one atom of the nitrogen bridge. As a stable free radical, DPPH can exist stably in organic solvents. Its alcohol solution is purple and needs to be stored at low temperature in the dark. It has a single electron, so it can accept an electron or a hydrogen ion and has a maximum absorption at a wavelength of 517 nm. When a free radical scavenger is present, the single electron of DPPH is captured, making its color lighter, and the absorbance value at the maximum light absorption wavelength decreases. Moreover, the degree of decrease shows a linear relationship. The decrease in the absorbance level indicates an increase in antioxidant properties, thereby evaluating the antioxidant capacity of the test sample. This antioxidant capacity is expressed by the inhibition rate. The larger the inhibition rate, the stronger the antioxidant property.

[0077] Sample 1 solution: Aqueous solutions of Auricularia auricula polysaccharide with different concentrations (0.0375, 0.075, 0.15, 0.3, 0.6, 1.2, 2.4, 4.8 mg / mL), denoted as AAP;

[0078] Sample 2 solution: Aqueous solutions of milk (Milk A, Milk B, Milk C, Milk D, Milk E) with different solid contents (0.025, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.6 mg / mL), denoted as MilkA, MilkB, MilkC, MilkD, MilkE;

[0079] Sample 3 solution: Prepare a polysaccharide solution of 1.6 mg / mL and 5 kinds of milk with a solid content of 1.6 mg / mL with deionized water, and then mix them in a volume ratio of 1:1 as the stock solution C1; then dilute it with deionized water to form C2 to C8 with concentration gradients (the total content of polysaccharide and solid is 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 mg / mL respectively), denoted as 1.6AAP-MilkA, 1.6AAP-MilkB, 1.6AAP-MilkC, 1.6AAP-MilkD, 1.6AAP-MilkE;

[0080] Sample 4 solution: A polysaccharide solution of 3.2 mg / mL was mixed with five types of milk with a solid content of 3.2 mg / mL at a volume ratio of 1:1 as stock solution C1. Then, it was serially diluted to C2 to C8 (the total polysaccharide and solid content were 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mg / mL, respectively), and denoted as 3.2AAP-MilkA, 3.2AAP-MilkB, 3.2AAP-MilkC, 3.2AAP-MilkD, and 3.2AAP-MilkE.

[0081] Sample 5 solution: A 4.8 mg / mL polysaccharide solution was mixed with five types of milk with a solid content of 4.8 mg / mL at a volume ratio of 1:1 as stock solution C1. Then, it was serially diluted to C2 to C8 (the total polysaccharide and solid content were 0.075, 0.15, 0.3, 0.6, 1.2, 2.4, and 4.8 mg / mL, respectively), and labeled as 4.8AAP-MilkA, 4.8AAP-MilkB, 4.8AAP-MilkC, 4.8AAP-MilkD, and 4.8AAP-MilkE.

[0082] 0.1 mmol / L DPPH solution: Weigh 3.94 mg of DPPH, dissolve it in anhydrous ethanol and bring the volume to 100 mL to obtain a 0.1 mmol / L DPPH ethanol solution.

[0083] V C Solution: As a control, it has the same concentration as the sample.

[0084] Mix the test solutions evenly according to the following groups, and incubate at room temperature for 30 min under light-protected conditions. Measure the absorbance at 517 nm, with three replicates for each group. Calculate the DPPH scavenging rate according to formula (4). Calculate the synergistic ratio SR according to the method in step 2.

[0085] Sample group: 2 mL sample solution + 2 mL 0.1 mmol / L DPPH solution

[0086] Positive group: 2 mL V C Solution + 2 mL 0.1 mmol / L DPPH solution

[0087] Control group: 2 mL sample solution + 2 mL anhydrous ethanol

[0088] Blank group: 2 mL H2O + 2 mL 0.1 mmol / L DPPH solution

[0089] DPPH removal rate (%) = [1-(A 样 -A 对 ) / A 空]×100% formula (4)

[0090] See results Figure 1 As shown, within the experimental concentration range, the DPPH scavenging rates of black fungus polysaccharide, five types of milk, and black fungus polysaccharide-milk complexes prepared from different concentrations of black fungus polysaccharide and five types of milk all exhibited a concentration-dependent relationship, with higher sample concentrations showing stronger DPPH scavenging ability. The DPPH scavenging rate of the milk polysaccharide complex at the same concentration was greater than that of the same milk solution or the same polysaccharide solution acting alone, indicating that the combination of milk and polysaccharide has a synergistic effect on DPPH scavenging ability. Specifically, the IC50 value of the 1.6 mg / mL black fungus polysaccharide complex with milk A was [not specified in the original text]. 50 The lowest scavenging efficiency was observed, while the highest synergistic ratio (SR) indicated that the combination of 1.6 mg / mL black fungus polysaccharide and 1.6 mg / mL milk A had the strongest scavenging ability against DPPH·, followed by the combination of 4.8 mg / mL black fungus polysaccharide and 4.8 mg / mL milk A and 3.2 mg / mL black fungus polysaccharide and 3.2 mg / mL milk C.

[0091] 4. Determination of hydroxyl radical scavenging rate and synergistic ratio (SR)

[0092] Hydroxyl radicals are highly reactive reactive oxygen species that can damage biological macromolecules such as proteins, lipids, and DNA, potentially leading to cell damage, aging, and various diseases. Therefore, measuring the scavenging ability of antioxidants or compounds against hydroxyl radicals is an important indicator for evaluating their antioxidant activity.

[0093] Sample 1 solution: aqueous solutions of black fungus polysaccharides at different concentrations (0.002344, 0.004568, 0.009375, 0.01875, 0.0375, 0.075, 0.15, 0.3, 0.6, 1.2, 2.4, 4.8 mg / mL), denoted as AAP;

[0094] Sample 2 solution: aqueous solutions of milk (milk A, milk B, milk C, milk D, milk E) with different solid contents (0.003125, 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.6 mg / mL), denoted as MilkA, MilkB, MilkC, MilkD, and MilkE;

[0095] Sample 3 solution: A 1.6 mg / mL polysaccharide solution was mixed with five types of milk with a solid content of 1.6 mg / mL at a volume ratio of 1:1 as stock solution C1. Then, it was serially diluted with deionized water to C2 to C13 (0.000781, 0.001563, 0.003125, 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 mg / mL), and denoted as 1.6AAP-MilkA, 1.6AAP-MilkB, 1.6AAP-MilkC, 1.6AAP-MilkD, and 1.6AAP-MilkE.

[0096] Sample 4 solution: A polysaccharide solution of 3.2 mg / mL was mixed with five types of milk with a solid content of 3.2 mg / mL at a volume ratio of 1:1 as stock solution C1, and then serially diluted to C2 to C8 (0.000781, 0.001563, 0.003125, 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 mg / mL), and denoted as 3.2AAP-MilkA, 3.2AAP-MilkB, 3.2AAP-MilkC, 3.2AAP-MilkD, and 3.2AAP-MilkE;

[0097] Sample 5 solution: A 4.8 mg / mL polysaccharide solution was mixed with five types of milk with a solid content of 4.8 mg / mL at a volume ratio of 1:1 as stock solution C1, and then serially diluted to C2 to C11 (0.002344, 0.004688, 0.009375, 0.01875, 0.0375, 0.075, 0.15, 0.3, 0.6, 1.2, 2.4, 4.8 mg / mL), and denoted as 4.8AAP-MilkA, 4.8AAP-MilkB, 4.8AAP-MilkC, 4.8AAP-MilkD, and 4.8AAP-MilkE.

[0098] 1.8 mmol / L FeSO4: Weigh 0.500 g of FeSO4. ·7 Dissolve O in water and bring the volume to 1 L.

[0099] 1.8 mmol / L salicylic acid solution: Weigh 0.249 g of salicylic acid, dissolve it in anhydrous ethanol and bring the volume to 1 L.

[0100] 0.03% H2O2: Take 10 mL of 30% hydrogen peroxide, add it to 80 mL of distilled water, mix well, and then make up to 100 mL.

[0101] V C Solution: As a control, it has the same concentration as the sample.

[0102] Mix the test solutions evenly according to the following groups, incubate in a 37°C water bath for 30 min, and measure the absorbance at 510 nm under light-protected conditions. Set up 3 replicates for each group. Calculate the hydroxyl radical scavenging rate according to formula (5). Calculate the synergistic ratio SR according to the method in step 2.

[0103] Sample group: 1 mL sample solution + 2 mL 1.8 mmol / L FeSO4 + 1.5 mL 1.8 mmol / L salicylic acid solution + 0.1 mL 0.03% H2O2

[0104] Positive group: 1 mL V C Solution + 2 mL 1.8 mmol / L FeSO4 + 1.5 mL 1.8 mmol / L salicylic acid solution + 0.1 mL 0.03% H2O2

[0105] Control group: 1 mL sample solution + 2 mL 1.8 mmol / L FeSO4 + 1.5 mL 1.8 mmol / L salicylic acid solution + 0.1 mL H2O2

[0106] Blank group: 1 mL H2O + 2 mL 1.8 mmol / L FeSO4 + 1.5 mL 1.8 mmol / L salicylic acid solution + 0.1 mL 0.03% H2O2

[0107] Hydroxyl radical scavenging rate (%) = [1-(A 样 -A 对 ) / A 空 ]×100% formula (5)

[0108] See results Figure 2 Within the experimental concentration range, the scavenging rates of *Auricularia auricula-judae* polysaccharide, five types of milk, and complexes of *Auricularia auricula-judae* polysaccharide and five types of milk at different concentrations all showed a concentration-dependent relationship for *·OH* scavenging. Higher sample concentrations resulted in higher *·OH* scavenging ability. The scavenging rate of the milk-polysaccharide complex at the same concentration was greater than that of the same type of milk and *Auricularia auricula-judae* polysaccharide alone, with SR values ​​significantly greater than 1, indicating a significant synergistic effect between the milk and *Auricularia auricula-judae* polysaccharide complexes on *·OH* scavenging. Specifically, the IC50 value of the complex of 1.6 mg / mL *Auricularia auricula-judae* polysaccharide and milk with a solid content of 1.6 mg / mL was significantly higher than that of milk alone. 50The low synergistic effect ratio (SR) indicates that the compound with 1.6 mg / mL black fungus polysaccharide and 1.6 mg / mL milk D exhibits the strongest scavenging ability against ·OH. Secondly, the IC50 of the compound with 1.6 mg / mL black fungus polysaccharide and 1.6 mg / mL milk B is the highest. 50 The SR value of the 1.6 mg / mL black fungus polysaccharide-milk A compound was slightly lower than that of the 1.6 mg / mL black fungus polysaccharide-solids content milk A compound, while the SR value of the 1.6 mg / mL black fungus polysaccharide-solids content milk A compound was slightly higher than that of the 1.6 mg / mL black fungus polysaccharide-solids content milk B compound.

[0109] Based on the aforementioned abilities of different concentrations of Auricularia auricula-judae polysaccharide (1.6, 3.2, and 4.8 mg / mL)-milk complexes to scavenge DPPH· and ·OH radicals, a preferred complex prepared by mixing a 1.6 mg / mL Auricularia auricula-judae polysaccharide aqueous solution with milk A containing 1.6 mg / mL of solids at a 1:1 volume ratio was further investigated to explore its in vivo anti-colitis ability. However, the optimal ratio for scavenging DPPH and OH free radicals does not necessarily guarantee its effectiveness in complex mammalian colitis models.

[0110] Example 4: Anti-colitis ability of black fungus polysaccharide-milk A compound

[0111] The dextran sulfate sodium (DSS)-induced mouse ulcerative colitis (UC) model is the most widely used model, characterized by its simplicity, high reproducibility, and high success rate. By dissolving DSS in the drinking water of mice to induce colitis, the intestinal epithelial cells are damaged, leading to disruption of the mucosal barrier integrity. Mice exhibit significant weight loss, loose stools, and bloody stools, showing clinical symptoms and pathological features extremely similar to human UC. In Example 2, black fungus polysaccharide and milk A were prepared using deionized water to prepare solutions with a polysaccharide concentration of 1.6 mg / mL and a solid content of 1.6 mg / mL, respectively. These solutions were then mixed at a volume ratio of 1:1 to prepare a black fungus polysaccharide-milk A compound, and the experiment was conducted according to the following steps.

[0112] 1. Animal grouping, drug administration, and weight monitoring

[0113] Six-week-old male ICR mice were randomly divided into 12 groups after one week of acclimatization: a normal control group (C), a positive control group (P), a negative control group (N), high, medium, and low dose black fungus polysaccharide groups (AAP-H, AAP-M, and AAP-L, black fungus polysaccharides prepared according to the method in Example 2), high, medium, and low dose milk A groups (MilkA-H, MilkA-M, and MilkA-L, dosage based on milk solids content), and high, medium, and low dose black fungus polysaccharide-milk A compound groups (AAP-MilkA-H, AAP-MilkA-M, and AAP-MilkA-L, dosage based on the total content of polysaccharides and milk solids). Six mice were in each group and fed according to Table 3. The mice's condition was observed daily, and their weight was measured and recorded. The experiment lasted 21 days.

[0114] Table 3 Animal Experiment Design

[0115]

[0116] See results Figure 3 During the adaptation period and the initial stage of the experiment, the body weight of mice in each experimental group did not fluctuate significantly. Subsequently, the body weight of mice in the normal control group steadily increased over time, reflecting the healthy growth of mice under normal physiological conditions. During the period from 1 to 14 days without DSS administration, the body weight of mice in all groups showed a steady upward trend, indicating that the existing feeding conditions met the growth requirements of mice. Although there were fluctuations in body weight in individual groups, they were all within the normal range, and there was no significant difference in body weight between groups. After DSS intervention from days 15 to 21, the body weight of mice in the negative control group decreased significantly, with a body weight loss rate of 6.76%. This indicates that DSS-induced UC severely interfered with the physiological processes of nutrient absorption and metabolism in mice, affecting their normal growth and development. Mice in the positive control group (with mesalazine as the treatment drug) did not experience body weight loss; in fact, their body weight increased by 0.10% by the end of the experiment, indicating that mesalazine can effectively reduce the adverse effects of UC on mouse body weight. Under high, medium, and low doses of Auricularia auricula-judae polysaccharide intervention, mice experienced weight loss from day 15 to 21, with weight loss rates of 2.63%, 2.52%, and 3.93%, respectively. Under high-dose milk A intervention, mice experienced weight loss from day 15 to 21, with a weight loss rate of 2.73%. Under medium and low doses of milk A intervention, mice experienced weight gain from day 15 to 21, with weight gain rates of 0.64% and 1.61%, respectively. Under high, medium, and low doses of the Auricularia auricula-judae polysaccharide-milk A compound intervention, mice experienced weight gain from day 15 to 21, with weight gain rates of 6.47%, 2.78%, and 4.64%, respectively. This indicates that the Auricularia auricula-judae polysaccharide-milk A compound can alleviate the negative effects of UC on mouse weight to some extent, maintaining relatively stable weight, and there is no dose-dependent relationship.

[0117] 2. Daily Disease Activity Index (DAI) score in mice

[0118] The Disease Activity Index (DAI) score can be used to assess the status of ulcerative colitis (UC) in mice. Increased DAI scores, weight loss, loose stools, and bloody stools in the later stages of the experiment are important characteristics of UC. The characteristics of mouse feces were observed and recorded daily, and scored according to the standards shown in Table 4.

[0119] Table 4 Disease Activity Index (DAI) Scoring Table

[0120]

[0121] Note: Normal: Formed stool. Loose: Pasty or semi-formed stool that does not stick to the anus. Watery stool: Watery stool that sticks to the anus.

[0122] See results Figure 3 In the normal control group, the DAI score remained stable at 0, consistent with the fact that these mice did not develop UC. In the negative control group, the DAI score steadily increased after DSS administration, reaching 1.625 on day 21, with obvious fecal abnormalities and rectal bleeding, characteristic of colitis. Compared to the negative control group, the positive control group showed a slower increase in DAI score, reaching 0.8 on day 21, indicating that mesalazine significantly inhibited the development of colitis. The DAI scores on day 21 in the high- and medium-dose AAP groups, the medium- and low-dose Milk A groups, and the high-, medium-, and low-dose AAP-Milk A groups were all between those of the negative and positive control groups. This suggests that these samples can alleviate DSS-induced UC symptoms in mice, such as loose stools and bloody stools, and reduce the DAI index to some extent. Among them, the low, medium and high doses of AAP-Milk A group had a significantly better effect on reducing the DAI index than the low, medium and high doses of AAP group and Milk A group acting alone, indicating that the combination of black fungus polysaccharide group and milk A can effectively alleviate UC symptoms such as loose stools and bloody stools induced by DSS in mice.

[0123] 3. Colon length in UC mice

[0124] Step 4: After blood collection from the mice, they were dissected, and complete colon tissue was taken for observation and length measurement. A negative correlation was observed between colon length and the severity of colitis in the mice, indicating that colon length can serve as an indicator of colitis severity. Figure 3The results showed that the colon length of mice in the negative control group was only 4.93 cm ± 0.29 cm, significantly shorter than that of the normal control group (8.80 cm ± 0.44 cm) (p < 0.05). Obvious congestion and edema were observed in the colon tissue of the negative control group mice, indicating successful replication of the UC model. The colon length of mice in the positive control group was 7.73 cm ± 0.31 cm, significantly longer than that of the negative control group (p < 0.05). Compared with the negative control group, high, medium, and low dose AAP groups, the Milk A group, and the AAP-Milk A group all significantly slowed the shortening of colon length in UC mice (p < 0.05). The high and medium dose AAP-Milk A groups significantly reduced the shortening of colon length in UC mice better than the medium and high dose AAP groups and the Milk A group (p < 0.05), indicating that the combined effect of both is superior to their individual effects. The colons of the positive control group and the high and medium dose AAP-Milk A groups were uniformly smooth, without obvious edema or congestion, and the contents were full and granular. Mice in the high, medium, and low dose AAP groups, the high, medium, and low dose Milk A groups, and the low dose AAP-Milk A group exhibited pasty colonic contents and congestion. This indicates that while all samples effectively reduced the shortening of colonic length in UC mice, the AAP-Milk A group was more effective in maintaining colonic length.

[0125] 4. Sample collection and detection of relevant biochemical indicators

[0126] On the last day of the experiment, mice were fasted for 12 hours, their whiskers were trimmed to prevent hemolysis, and blood was collected from the orbital venous plexus. After standing overnight at 4°C, the blood was centrifuged (3000 rpm, 15 min), and the supernatant was collected. The activities of superoxide dismutase (SOD), catalase (CAT), and alanine aminotransferase (ALT), as well as the contents of glutathione (GSH), malondialdehyde (MDA), and lipopolysaccharide (LPS), were determined according to the kit instructions. After blood collection, the mice were dissected, and complete colon tissue was obtained and its length measured. Separate colon tissue samples were homogenized into a 10% homogenate at 15000 rpm using physiological saline as the homogenate medium. After centrifugation, the precipitate was discarded, and the supernatant was collected. The contents of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were determined according to the kit instructions.

[0127] All animal experiments were conducted in accordance with the National Research Council’s Guidelines for the Care and Use of Laboratory Animals, and this study has been approved by the university’s ethics committee for ethical review of laboratory animals.

[0128] Serum lipopolysaccharide (LPS) levels in mice reflect intestinal permeability. Figure 4It was found that the serum LPS level in the normal control group mice was at a low physiological level, which is closely related to the intact function of the normal intestinal barrier, effectively preventing LPS translocation from the intestine into the bloodstream. The serum LPS level in the negative control group (N) mice was significantly higher than that in the normal control group (p < 0.05), indicating that DSS-induced colitis disrupts the integrity of the intestinal mucosal barrier, allowing a large amount of LPS from Gram-negative bacteria and their cell wall components to translocate into the bloodstream, triggering an inflammatory response. The serum LPS level in the positive control group was significantly lower than that in the negative control group (p < 0.05), indicating that the positive control drug effectively reduced LPS levels by repairing intestinal barrier function and inhibiting bacterial translocation, thereby alleviating inflammatory damage. Compared with the negative control group, high, medium, and low doses of AAP, Milk A, and AAP-Milk A significantly reduced serum LPS levels in UC mice (p < 0.05). Among them, the high, medium and low dose AAP-Milk A groups were significantly better than the AAP group and Milk A group of the same dose in reducing serum LPS content in UC mice (p < 0.05), indicating that the combination of AAP-Milk A can inhibit the translocation of LPS from the intestine to the blood circulation, slow down the increase in colonic permeability in mice caused by DSS, and effectively reduce LPS content by repairing intestinal barrier function and inhibiting bacterial translocation, thereby alleviating inflammatory damage.

[0129] To evaluate the ameliorative effect of the compound of this invention on systemic pathological changes induced by colitis, we tested oxidative stress-related indicators. For example... Figure 4 In the negative control group, serum SOD, CAT activity, and GSH levels were significantly lower than those in the normal control group. p < 0.05. The SOD, CAT activities, and GSH levels in the positive control group were significantly higher than those in the negative control group. p <0.05 indicates that the positive control drug can effectively activate the synthetic pathways of SOD, CAT activity and GSH levels, and enhance the antioxidant defense system function of colonic tissue. High, medium, and low doses of Milk A did not enhance SOD and CAT activity, and there was no difference compared with the negative control group. p > 0.05). High, medium, and low dose AAP groups and the AAP-Milk A group all significantly enhanced SOD and CAT activities, showing significant differences compared to the negative control group and the high, medium, and low dose Milk A groups. p < 0.05%. There was no difference in the effect of the same dose of AAP and AAP-Milk A on SOD activity ( p > 0.05), the effect of the same dose of AAP-Milk A on CAT activity was significantly different from that of AAP ( p< 0.05). The effects of high and low doses of AAP-Milk A on GSH levels were not significantly different compared to AAP alone. p > 0.05), the effect of medium-dose AAP-Milk A on GSH levels was significantly different from that of AAP ( p <0.05). This indicates that compared with the normal group, the model group mice showed significantly reduced SOD, CAT activity, and GSH levels, suggesting that the disease had led to the collapse of the body's systemic antioxidant defense system. After treatment with the compound of this invention, these indicators were significantly restored, strongly demonstrating that the compound of this invention can not only alleviate local intestinal inflammation but also systematically correct the systemic oxidative stress disorder caused by colitis, showcasing its comprehensive advantages in treating colitis.

[0130] like Figure 4 The serum MDA level and ALT activity in the negative control group mice were significantly increased compared with those in the normal control group. p <0.05, serum MDA level and ALT activity in the positive control group were significantly higher than those in the negative control group ( p < 0.05%. Except for the medium-dose Milk A group, which showed no difference in serum MDA levels compared to the negative control group ( p > 0.05), other high, medium, and low doses of AAP, Milk A, and AAP-Milk A groups all significantly reduced serum MDA levels and ALT activity in mice. p <0.05. Except for the high-dose AAP group and the AAP-Milk A group, there was no difference in the effect on serum MDA levels in mice ( p > 0.05), the effects of other high, medium, and low dose AAP-Milk A groups on serum MDA levels and ALT activity in mice were significantly different from those of the AAP group and the Milk A group. p < 0.05). This indicates that the combination of AAP and Milk A can better alleviate oxidative damage in UC mice.

[0131] Inflammatory responses play a central role in the pathological process of dextran sulfate sodium (DSS)-induced colitis, and changes in the expression levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), as key pro-inflammatory cytokines, have a significant impact on the occurrence, development, and outcome of colitis. Figure 5 The levels of TNF-α and IL-6 in the colon tissue of mice in the negative control group were significantly higher than those in the normal control group. p< 0.05, confirming that DSS can trigger a large release of TNF-α and IL-6, exacerbating the inflammatory response of colitis. The levels of TNF-α and IL-6 in the colonic tissue of mice in the positive control group were significantly lower than those in the negative control group (p < 0.05), indicating that the positive control drug can effectively reduce the levels of TNF-α and IL-6 in colonic tissue. Except for the medium-dose Milk A group, which showed no difference in the effect of TNF-α levels in mouse colonic tissue compared to the negative control group (p < 0.05), the overall effect of the drug was significantly lower. p > 0.05), other high, medium, and low doses of AAP, Milk A, and AAP-Milk A significantly reduced the levels of TNF-α and IL-6 in mouse colon tissue. p < 0.05). Among them, the ability of low-dose AAP-Milk A to reduce IL-6 levels in colonic tissue, and the ability of high, medium, and low-dose AAP-Milk A to reduce TNF-α levels in colonic tissue, were significantly stronger than the effects of AAP and Milk A alone. This indicates that the combination of AAP and Milk A can better inhibit DSS-induced overexpression of TNF-α and IL-6 in mouse colonic tissue, and there is no dose-dependent effect.

[0132] Comparative Example 1: Direct Hot Water Extraction

[0133] Take 1.0 g of black fungus fruiting body powder (passed through a 200-mesh sieve), add 85 mL of deionized water and mix well. The material-to-liquid ratio is 1:85 (g / mL). Extract in a water bath shaker at 125 rpm for 180 min at 90℃. Centrifuge at 8000 r / min for 20 min, take 1.0 mL of supernatant, and use the phenol-sulfuric acid method to detect the polysaccharide content in the supernatant. The content is then converted into the extraction yield. The results are shown in Table 5.

[0134] Table 5. Effects of different extraction processes on the extraction yield of polysaccharides from black fungus.

[0135]

[0136] As shown in Table 5, different extraction processes have a significant impact on the extraction yield of black fungus polysaccharides. The free radical-DES1 extraction method can significantly improve the extraction yield of black fungus polysaccharides, which is 14.413 times higher than that of black fungus polysaccharides extracted by traditional hot water extraction.

[0137] Comparative Example 2: Effect of black fungus powder mesh size on the yield of black fungus polysaccharides

[0138] In Example 2, the black fungus powder was replaced with a 20-mesh coarse sieve, and other operations were the same as in Example 2. The extraction yield of black fungus polysaccharide was detected and calculated using the same method as in Example 1. The results are shown in Table 6.

[0139] Table 6. Effect of different black fungus powder particle sizes on the extraction yield of black fungus polysaccharides.

[0140]

[0141] As shown in Table 6, the particle size of black fungus has a great influence on the extraction yield of black fungus polysaccharides. The pretreatment method of passing the powder through a 200-mesh sieve can increase the extraction yield of black fungus polysaccharides by 2.187 times compared with the coarse powder produced by traditional mechanical grinding.

[0142] Comparative Example 3: Effect of hydrogen peroxide extraction on the yield of polysaccharides from black fungus

[0143] Take 1.0 g of black fungus fruiting body powder (passed through a 200-mesh sieve), add 1.6% hydrogen peroxide aqueous solution at a material-to-liquid ratio of 1:85 (g / mL), and then extract under water bath conditions at 90℃ for 180 min; centrifuge at 8000 rpm for 20 min, take 1.0 mL of supernatant, and use the phenol-sulfuric acid method to detect the polysaccharide content in the supernatant and convert it into the extraction yield. The results are shown in Table 7.

[0144] Table 7. Effects of different extraction processes on the extraction yield of polysaccharides from black fungus.

[0145]

[0146] As shown in Table 7, the extraction process of black fungus has a great influence on the extraction yield of black fungus polysaccharides. The free radical-DES1 extraction method can increase the extraction yield of black fungus polysaccharides by 1.631 times compared with hydrogen peroxide extraction.

[0147] Comparative Example 4: Effect of cellulase extraction on the yield of polysaccharides from black fungus

[0148] Take 1.0 g of black fungus fruiting body powder (passed through a 200-mesh sieve), add 0.08 g of cellulase (enzyme activity 50 U / mg, the amount added is 8% of the weight of black fungus fruiting body powder), add 60 mL of deionized water, and extract for 3.0 h at pH 4.8 and temperature 55℃; centrifuge at 8000 rpm for 20 min, take 1.0 mL of supernatant, and use the phenol-sulfuric acid method to detect the polysaccharide content in the supernatant and convert it into the extraction yield. The results are shown in Table 8.

[0149] Table 8 Effect of different extraction processes on the extraction yield of polysaccharides from black fungus

[0150]

[0151] As shown in Table 8, the extraction process of black fungus has a great influence on the extraction yield of black fungus polysaccharides. The free radical-DES1 extraction method can increase the extraction yield of black fungus polysaccharides by 2.903 times compared with cellulase extraction.

[0152] Comparative Example 5: Effect of enzymes on polysaccharide yield

[0153] Take 1.0 g of black fungus fruiting body powder (passed through a 200-mesh sieve), and simultaneously add 0.04 g of cellulase (enzyme activity 50 U / mg), 0.06 g of papain (enzyme activity 10 U / mg), and 0.06 g of β-glucanase (enzyme activity 50 U / mg), and add 60 mL of deionized water; the amount of papain and β-glucanase added is 6.0%, and the amount of cellulase added is 4.0%. Then, extract for 3.0 h at the incubation temperature (55℃), pH 5.3, and material-to-liquid ratio of 1 / 60; centrifuge at 8000 rpm for 20 min, take 1.0 mL of supernatant, and use the phenol-sulfuric acid method to detect the polysaccharide content in the supernatant and convert it into the extraction yield. The results are shown in Table 9.

[0154] Table 9. Effects of different extraction processes on the extraction yield of polysaccharides from black fungus.

[0155]

[0156] As shown in Table 9, the extraction process of black fungus has a great influence on the extraction yield of black fungus polysaccharides. The extraction method of free radical-DES1 can increase the extraction yield of black fungus polysaccharides by 1.966 times compared with the combined extraction of three enzymes.

Claims

1. A functional Auricularia auricular polysaccharide-milk compound, characterized in that, The complex is mixed by the aqueous solution of Auricularia auricular polysaccharide and milk; the concentration of the aqueous solution of Auricularia auricular polysaccharide is 1.6-4.8 mg / mL.

2. The black fungus polysaccharide-milk compound of claim 1, characterized in that, The volume ratio of the aqueous solution of Auricularia auricular polysaccharide and milk is 1:1; the milk includes pure milk, and the protein content is 3.0-3.6 g / 100 mL.

3. The black fungus polysaccharide-milk compound of claim 3, characterized in that, The solid content of the milk is 142.1 mg / mL, and the protein content is 3.2 g / 100 mL.

4. The black fungus polysaccharide-milk compound of claim 1, wherein the black fungus polysaccharide is a black fungus polysaccharide extract. The Auricularia auricular polysaccharide is prepared by the following method: (1) The crude polysaccharide extract is obtained by taking Auricularia auricular fruiting body powder as the extraction agent, deionized water and hydrogen peroxide with a mass concentration of 30% as the extraction agent, and extracting in a water bath shaker, the shaker speed is 100-150 rpm, and the extraction is carried out at 70-100 ℃ for 120-220 min, then natural eutectic solvent is added, and the extraction is continued for 15-45 min, and the supernatant is obtained after centrifugation at 8000 rpm for 20 min; the natural eutectic solvent is obtained by heating and stirring sodium citrate and glucose or ascorbic acid in deionized water until it is clear; (2) Auricularia auricular polysaccharide: after the supernatant of step (1) is concentrated to 1 / 5 of the original volume, concentrated solution 1 is obtained; 4 times the volume of 95% ethanol is added to concentrated solution 1 while stirring, and the supernatant is carefully poured out after standing at 4 ℃ for 12 h, and the lower precipitate is reserved; the lower precipitate is placed in a centrifuge, and centrifuged at 1000 rpm for 5 min, and the precipitate is added with water and concentrated by rotary evaporation at 65 ℃ until the ethanol is completely evaporated, to obtain concentrated solution 2; Sevag reagent is added to concentrated solution 2 at a volume ratio of 1:5, and shaken vigorously for 20 min, and centrifuged at 3000 rpm for 10 min to form an organic solvent layer, a protein layer and a water layer, and the protein layer and the organic solvent layer are discarded, and the centrifugation is repeated 3 times to obtain Auricularia auricular polysaccharide purification solution, which is freeze-dried to obtain Auricularia auricular polysaccharide.

5. The black fungus polysaccharide-milk compound preparation according to claim 4, characterized in that, In step (1), the mass concentration of hydrogen peroxide in the extraction agent is 1.2%-1.8%, and the volume of the extraction agent is 60-100 mL / g based on the mass of Auricularia auricular fruiting body powder; the volume ratio of the extraction agent and the natural eutectic solvent is 170:50-70.

6. The black fungus polysaccharide-milk compound of claim 1, wherein the black fungus polysaccharide is a black fungus polysaccharide extract. The complex is mixed by 1.6 mg / mL aqueous solution of Auricularia auricular polysaccharide and 1.6 mg / mL pure milk with a solid content of 1.6 mg / mL at a volume ratio of 1:

1.

7. The use of the Auricularia auricular polysaccharide-milk complex of claim 1 in the preparation of an antioxidant product.

8. The use of the Auricularia auricular polysaccharide-milk complex of claim 1 in the preparation of a preparation for improving ulcerative colitis.

9. Use according to claim 8, wherein the compound is ###0002### The preparation is for treating DSS-induced ulcerative colitis.

10. The use according to claim 8, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The preparation is for treating systemic oxidative stress disorder caused by ulcerative colitis.

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