Chitin-glucan nanopoly saccharide complex and preparation method and application thereof

Abstract

This invention belongs to the field of food raw material science and discloses a chitosan-dextran nanopolysaccharide complex, its preparation method, and its applications. This invention proposes the use of an emulsifier. The combination of a low-concentration alkali and the emulsifier, along with a shortened processing time, preserves the natural properties of the complex while maximally removing related compounds such as proteins, lipids, fats, pigments, alkali-soluble dextran, and mineral compounds. This improves product purity while reducing CGC degradation during preparation and increasing removal efficiency. The chitosan-dextran nanopolysaccharide complex of this invention, due to its nano-sizing, provides optimal dispersibility in a hydrophilic environment, promoting better entry into the human body and interaction with cell surface receptors. It regulates the content of giant cells and the activity of T lymphocytes in vivo, effectively improving human immune regulation, and primarily inhibits tumors by enhancing the body's non-specific immune function.

Description

Technical Field

[0001] This invention belongs to the field of food raw material science technology, and specifically relates to a chitin-dextran nanopolysaccharide complex, its preparation method and application. Background Technology

[0002] Chitin-Glucan Complex (CGC), also known as chitin-glucan, is a high-purity biopolymer composed of two different polysaccharides: chitin (β-1,4-poly-N-acetyl-D-glucosamine) and β-1,3-D-glucan. The ratio of the two polysaccharides in the complex ranges from 20:80 to 40:60 (w / w). The content of this complex varies depending on the source of the raw materials, generally ranging from 3.5% to 15%. Chitin-glucan complexes derived from fungi have significant advantages over those from crustaceans, which are currently the most common source. Currently, commercially available chitin-glucan complexes are mainly derived from crustaceans such as shrimp and crab, and the chitin in these complexes is mostly α-chitin. However, the preparation of CGCs from animal sources is limited by the content and compositional variability of the target components in the animal raw materials and is easily affected by seasonality, resulting in unstable yields and poor reproducibility of product quality. The above-mentioned shortcomings can be overcome by isolating CGCs from fungi. CGCs are the main component of the cell walls of most fungi, and the fruiting bodies and mycelia of higher fungi can be obtained in large quantities through cell engineering techniques, providing a stable source for CGC production. More importantly, the chitin in fungal-derived CGCs is α-chitin, while the chitin in animal-derived CGCs is β-chitin. The presence of the former makes the CGC structure more loose, which is beneficial for the dispersion of the final product in solution, thereby expanding the efficacy and application areas of the product.

[0003] Methods for preparing CGCs from fungal raw materials mainly include steps such as deproteinization, demineralization, and decolorization. However, current preparation methods generally use high-concentration alkaline reagents. Some processes use large amounts of organic solvents to reduce the adverse effects of lipid-soluble components in fungi on the CGC extraction rate, causing environmental pollution. High-concentration alkaline reagents can also cause CGC degradation, resulting in the loss of the natural properties of CGC products. For example, Inês Farinha et al. prepared CGCs from Pichia pastoris using a 20% NaOH concentration and heating at 95°C for 5 hours, but the CGC extraction rate was only 13.4%. Muzzarelli, RAA et al. isolated CGCs from Aspergillus niger mycelium using an alkali treatment time of 4 hours, an alkali treatment temperature of 128°C, and a NaOH solution concentration of 40%, achieving an extraction rate of 44% and a chitin content of 32%.

[0004] Currently, the application and development of CGC are mainly in the fields of health related to gut microbiota regulation, cosmetics, and high-performance materials. For example, the patent publication "Fine-particle-sized fungal extract chitin-glucan" (CN101583630A) discloses a micron-sized fungal extract chitin-glucan and its application in skin cosmetic compositions. Its preparation method involves directly using chitin-glucan as a raw material and micronizing it, without mentioning a method for isolating chitin-glucan from fungi. The patent publication "Chitin-glucan complex, its preparation and use" (CN106572959A) discloses a chitin-glucan complex extracted from Komagataella pastoris. Its raw material source is lower fungi from industrial waste, and the chemical composition of the product contains not only CGC but also a certain proportion of mannose. The use of the chitin-glucan complex disclosed in this patent publication is a novel pharmaceutical excipient and does not fall under the health efficacy claimed in this invention. The European Food Safety Authority (EFSA) has listed CGC as a Safe Good Ingredient, and Regulation No. 258 / 97 of the Council of the European Parliament has approved CGC as a novel dietary supplement, stipulating a maximum daily intake of 5g per person. However, this regulation only classifies CGC as a novel insoluble dietary fiber and does not address other health benefits. Foreign literature has demonstrated the efficacy of CGC in reducing diet-induced aortic fatty streaks through animal experiments; it has also found potential efficacy in treating patients with coronary heart disease and diabetic complications in human trials. However, these health benefits are all related to the regulatory mechanism of CGC on the gut microbiota as a dietary fiber, and do not address other health benefits of CGC as a bioactive polysaccharide. Summary of the Invention

[0005] To overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide a mild method for preparing chitosan-dextran nanopolysaccharide complexes. This method minimizes the removal of related compounds such as proteins, lipids, fats, pigments, alkali-soluble dextran, and mineral compounds, thereby improving product purity and reducing CGC degradation during the preparation process.

[0006] Another object of the present invention is to provide a chitin-dextran nanopolysaccharide complex prepared by the above method.

[0007] Another objective of this invention is to provide the application of the above-mentioned chitosan-glucan nanopolysaccharide complex in the preparation of antitumor and immunomodulatory drugs or foods. To fully leverage the unique advantages of chitosan and β-glucan, two active polysaccharides, coexisting in the same compound and to further explore the applications of CGC in the health field, this invention uses a mechanical method to nanoscale the chitosan-glucan complex. By improving the dispersibility of CGC in solution, its interaction with cell receptors in vivo is enhanced, thereby generating new health benefits in antitumor and immunomodulatory aspects.

[0008] The objective of this invention is achieved through the following solution:

[0009] A method for preparing a chitin-dextran nanopolysaccharide complex includes the following steps:

[0010] S1. Using fungal fruiting bodies or mycelia as raw materials, they are evenly dispersed in water, then heated and stirred, then cooled to room temperature, and the sediment is collected by centrifugation.

[0011] S2. Add the mixture of NaOH solution and emulsifier to the wet precipitate obtained in S1, stir and heat to react, cool, centrifuge, continue to add water to the centrifuged precipitate and stir to wash, centrifuge again, repeat the washing with water multiple times until the pH of the supernatant is neutral, and the centrifuged precipitate obtained in the last time is the crude CGC product.

[0012] S3. Add hydrochloric acid solution to the crude CGC separated in S2, heat and stir to react, then neutralize with NaOH to pH=7, centrifuge and wash to obtain desalted CGC;

[0013] S4. Place the desalted chitin-dextran complex obtained in S3 in ammonia water, stir evenly, add H2O2 solution and let stand, then wash with water until pH=7 to obtain decolorized CGC.

[0014] S5. Add water to the decolorized chitin-dextran complex obtained in S4, stir to suspend it in water, treat the CGC suspension with shear-ultrasonic circulation, and centrifuge to obtain the supernatant, which is the chitin-dextran nanocomposite suspension. After freeze-drying, the chitin-dextran nanopolysaccharide complex is obtained.

[0015] The chitin-glucan complex used in this invention is obtained from fungi.

[0016] The chitin-glucan complex described in step S1 is extracted from one of the following: the mycelium of a basidiomycete fungus in liquid fermentation, the fruiting body of a fungus in solid fermentation, or the fruiting body of a fungus in solid cultivation.

[0017] Preferably, the fungal fruiting body or mycelium mentioned in step S1 is selected from at least one of the following fungi: Ascomycetes, Aspergillus niger, Basidiomycetes, Lentinus edodes (shiitake mushrooms), and Agaricus bisporus (straw mushrooms).

[0018] The heating temperature in step S1 is 70-95℃, preferably 85℃; the heating and stirring time is 1-3h, preferably 3h; the stirring is only for sufficient contact, so the stirring speed is not limited.

[0019] The concentration of the NaOH solution in step S2 is 0.5-4 wt%, preferably 2%; the emulsifier is at least one of sodium stearate and glyceryl monostearate, preferably sodium stearate;

[0020] In step S2, the NaOH solution and sodium stearate work synergistically to remove the lipid layer on the surface of the mycelium, enhancing its binding with water-soluble reagents. This preserves the natural properties of the complex while maximizing the removal of related compounds such as proteins, lipids, fats, pigments, alkali-soluble dextran, and mineral compounds. This improves product purity while reducing CGC degradation during preparation and increasing removal efficiency.

[0021] The amounts of the mixture of wet precipitate, NaOH solution, and emulsifier in step S2 shall satisfy the following: the ratio of the dry weight of the wet precipitate (solid phase) to the dry weight of the mixture of NaOH solution and emulsifier (liquid phase) shall be 1:10-1:25, preferably 1:15; the ratio of the dry weight of NaOH solution to the weight of emulsifier in the mixture of NaOH solution and emulsifier shall be 9:1-29:1; and the mass of emulsifier shall account for 0.05-0.5% of the total mass of all materials (solid phase + liquid phase), preferably 0.1%.

[0022] The heating temperature range in the heating reaction described in step (2) is 60-95℃, and the heating time is 0.5-4h; the optimal heating temperature is 85℃, and the optimal heating time is 2h.

[0023] The mixture of NaOH solution and emulsifier in step S2 is preferably obtained by heating and stirring the NaOH solution and emulsifier together, wherein the heating temperature is 85°C and the heating time is 2 hours.

[0024] The concentration of the hydrochloric acid solution in step S3 is 0.5-3.0%, with an optimal concentration of 1%; the solid-liquid ratio of the crude CGC product to the hydrochloric acid solution is 1:15-1:25 (g / mL); and the NaOH solution is 1-3 mol / L.

[0025] The heating and stirring reaction mentioned in step S3 refers to stirring at 400 rpm at 55-60℃ for 1-3 hours, preferably stirring at 400 rpm at 55℃ for 2 hours.

[0026] The centrifugal washing mentioned in step S3 refers to washing the precipitate with water multiple times after centrifugation until the conductivity of the washing liquid is lower than 20 S / cm.

[0027] In step S4, the ammonia solution is treated at a temperature of 28-42°C, preferably 35°C, with a concentration of 25%-28%, and the optimal concentration is 25%. The concentration of the H2O2 solution is 3-6%, preferably 5%, and the settling time is 3-6 hours, preferably 4 hours.

[0028] In step S4, the mass ratio of the desalted chitin-dextran complex, ammonia, and H2O2 solution is 7:1:2 to 16:2:5.

[0029] The addition of water to the decolorized chitosan-dextran complex mentioned in step S5 refers to adding 50-500 mL of water to every 0.5 g of the decolorized chitosan-dextran complex.

[0030] The shearing-ultrasonic cycling treatment described in step S5 refers to first shearing with a high-speed shearing machine, followed by treatment in an ultrasonic cell disruptor, repeating this process multiple times until the suspension is essentially homogeneous. The shearing is performed at a speed of 17000-25000 rpm for 1-3 minutes; the ultrasonic treatment is performed at 500-600W power for 1-3 seconds with a 1-3 second interval for 5 minutes. This shearing and ultrasonic cycle is repeated 4 times.

[0031] The centrifugation mentioned in step S5 refers to centrifugation at 8000 rpm for 5 minutes.

[0032] This invention provides a chitosan-dextran nanopolysaccharide complex derived from fungi. Due to its loose structure, a mild dissolution method is employed to preserve its natural properties while enhancing protein removal (through the combined action of, for example, low-concentration NaOH solution and the emulsifier sodium stearate), dissolving the lipid layer and salts in the fungal raw material (through repeated washing with, for example, HCl solution and deionized water), and removing pigments (through treatment with, for example, H2O2 followed by washing with deionized water). The type of solvent system, the number of washes, and their sequence are appropriately selected to control the content of the biocomplex in the protein, lipids, and ash. The amounts of chitosan-dextran complex, protein, lipids, and ash are adjusted by controlling the conditions of the above preparation method. Finally, the chitosan-dextran complex is nano-sized using mechanical methods. The addition of nano-sizing allows for greater dispersion of the complex, enabling it to better exert its health benefits, such as anti-tumor and immunomodulatory effects.

[0033] A chitin-dextran nanopolysaccharide complex prepared by the above method.

[0034] The above-mentioned chitosan-dextran nanopolysaccharide complex is used in the preparation of antitumor drugs and immunomodulatory drugs.

[0035] The antitumor drug is preferably an anti-liver cancer drug.

[0036] The mechanism of this invention is as follows:

[0037] This invention provides a chitin-dextran nanopolysaccharide complex derived from fungi. Fungal chitin is primarily β-chitin, which has a looser structure and can be dispersed under milder conditions. Furthermore, it is not limited by the content, compositional variability, or seasonality of the target component in the animal raw material. Therefore, this invention uses fungi as a raw material source to prepare the chitin-dextran nanopolysaccharide complex. In addition, lipids, chitin-dextran complexes, and proteins are the main components of fungal cell walls. To remove the lipid layer on the hyphal surface and improve its binding with water-soluble reagents, this invention proposes the use of an emulsifier. The combination of a low-concentration alkali and the emulsifier, along with a shorter treatment time, preserves the natural properties of the complex while maximizing the removal of related compounds such as proteins, lipids, fats, pigments, alkali-soluble dextran, and mineral compounds. This improves product purity while reducing CGC degradation during the preparation process and increasing removal efficiency. Finally, research has shown that large-sized chitin-dextran complexes are biologically inert. Therefore, this invention uses a mechanical method to nano-size the chitin-dextran complex. The nano-sized complex can directly stimulate immune cells, and due to the high dispersibility of nano-sizing, its interaction with cell receptors in vivo is enhanced, thereby producing new health benefits in anti-tumor and immune regulation.

[0038] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0039] (1) This invention uses higher fungi as raw materials to separate CGCs. Since CGCs exist in a relatively loose form in fungal raw materials, the energy required for CGC separation is low, which helps to reduce production costs. The preparation process is mild, reducing the use of strong alkalis and organic solvents and avoiding environmental pollution. Moreover, due to the synergistic removal effect of the added emulsifier on lipids, the reaction efficiency is greatly improved, and non-target compounds such as proteins, lipids, fats, pigments, alkali-soluble dextran, and mineral compounds can be removed more thoroughly in a short time, thereby improving the purity of the product.

[0040] (2) The chitin-dextran nanopolysaccharide complex of the present invention is nanosized to provide it with the best dispersibility in a hydrophilic environment, which promotes its better entry into the human body and interaction with cell surface receptors, regulates the content of giant cells and the activity of T lymphocytes in the body, effectively improves the immune regulation of the human body, and mainly inhibits tumors by enhancing the body's non-specific immune function. Attached Figure Description

[0041] Figure 1 Here is a SEM image of the chitin-dextran nanopolysaccharide complex 1 obtained in Example 1;

[0042] Figure 2 The image shows a SEM image of the chitin-dextran nanopolysaccharide complex 11 obtained in Comparative Example 1. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available products.

[0044] Unless otherwise specified, all reagents used in the examples are commercially available.

[0045] I. Source of Raw Materials

[0046] Fresh fruiting bodies or mycelia of Basidiomycetes are dried at 50°C, pulverized, and sieved.

[0047] II. Purity and Structural Analysis of Chitosan-Dextran Nanopolysaccharide Complex

[0048] 1. Protein content determination

[0049] Weigh approximately 0.3g of sample into a digestion tube (accurate to 0.001g), add 0.2g CuSO4, 3g K2SO4, and 10mL concentrated sulfuric acid, and let stand at room temperature for 12 hours. Then, place the tube in a digestion furnace for digestion, continuously heating until the furnace temperature reaches 450℃, and continue digestion for approximately 1 hour until the liquid in the digestion tube becomes clear and turns blue-green. Allow the liquid in the digestion tube to cool naturally to room temperature and place it in an automatic Kjeldahl nitrogen analyzer, simultaneously connecting an Erlenmeyer flask to absorb ammonia. Add 20mL of 20g / L H3BO3 solution and 2-3 drops of 1g / L methylene blue ethanol solution to the Erlenmeyer flask. Add sufficient 400g / L NaOH solution to the digestion tube until the liquid turns completely black. After distillation for 5 minutes, remove the Erlenmeyer flask and titrate with 0.05M HCl solution until the blue color disappears. Record the volume of HCl solution consumed and calculate the total nitrogen content in the sample.

[0050] Total nitrogen content in the sample (N) tot (g / 100g)=(AB)×C×14×100 / 20

[0051] Where: A: Volume of standard hydrochloric acid consumed during sample titration, mL; B: Volume of hydrochloric acid consumed during blank titration, mL; C: Equivalent concentration of standard hydrochloric acid, wt%; 14: Relative molecular weight of nitrogen; 20: Volume of diluted digestion solution used for distillation, mL; 100: Volume of diluted digestion solution, mL.

[0052] Crude protein content in sample (g / 100g) = Total nitrogen content in sample N tot (g / 100g)×6.25

[0053] 2. Composition – Determination of Chitosan and Dextran Content

[0054] The β-glucan moiety of the polymer was hydrolyzed using trifluoroacetic acid (TFA). The chitin moiety was quantified using a strong acid (HCl). For TFA hydrolysis, a dried CGC sample (≈5 mg) was resuspended in 5 mL of deionized water, followed by the addition of 0.1 mL of 99% TFA. Hydrolysis was carried out in a sealed vial at 120 °C for 2 h with constant magnetic stirring. For HCl hydrolysis, the sample (≈5 mg) was resuspended in 5 mL of 1 mol / L HCl and hydrolyzed in a sealed vial at 120 °C for 5 h with constant magnetic stirring. Both hydrolysates were used to quantify glucose and glucosamine by high-performance liquid chromatography (HPLC). Glucose and glucosamine (Sigma) were used as standards at concentrations between 0.006 and 0.1 g / L, following the same hydrolysis procedure as the sample. The D-glucosamine (GA) content in the hydrolysates was determined according to the Elson-Morgan spectrophotometric method.

[0055] 3. Ash content

[0056] Thermal analysis (TGA and DSC) was performed using a simultaneous TG / DSC instrument (STA 449F3 Jupiter, Germany) in an open alumina pot under high-purity dry nitrogen at a heating rate of 5 °C / min from room temperature to 800 °C. Ash content (%) was determined by TGA thermogram.

[0057] 4. Fat content

[0058] Accurately weigh 2g of the compound powder and place it in a filter paper packet. Dry in a 105℃ oven for 2 hours, cool in a desiccator, and weigh. Place the filter paper packet in a Soxhlet extractor, add an appropriate amount of petroleum ether, and reflux in a 55℃ water bath for about 6 hours, until no oil residue remains on the filter paper when a glass rod dips into the extract. Remove the filter paper packet, evaporate any remaining petroleum ether, and dry in a 105℃ oven to constant weight. Calculate the fat content in the sample based on the weight loss from the filter paper packet.

[0059] 5. SEM

[0060] The microstructure of the sample was observed using a scanning electron microscope (EVO18, Zeiss GmbH, Germany), as follows: Two drops of S6 were placed on the smooth surface of tin foil, ensuring the droplets fell as evenly as possible. The sample was then left to air dry at room temperature for 12 hours. The dried tin foil was then adhered to the stage with conductive adhesive, and gold was sputtered onto it at 1 MPa for 200 seconds. The sample was then photographed and observed using a scanning electron microscope in a vacuum environment.

[0061] 6. Particle size determination

[0062] 0.1 g of chitin-dextran nanopolysaccharide complex was dispersed in ultrapure water until the total volume was about 2 mL. The particle size distribution of the nano-chitin-dextran complex dispersion was measured by particle size and zeta potentiometer (Omni, Brookhaven, GA, USA).

[0063] 7. Purity determination

[0064] Weigh the dry weight m0 of the fungal fruiting body, the protein weight m1, the ash weight m2, and the fat weight m3, and calculate the purity of the chitin-dextran nanopolysaccharide complex.

[0065] Purity (%) of chitosan-dextran nanopolysaccharide complex = ( m0 -m1-m2-m3) / m0×100%

[0066] 8. Yield determination

[0067] Weigh the chitin-dextran nanopolysaccharide complex (m') and the fungal fruiting body (m0), and calculate the yield of the chitin-dextran nanopolysaccharide complex.

[0068] Yield (%) of chitosan-dextran nanopolysaccharide complex = m' / m0 × 100%

[0069] 9. Anti-tumor cell experiments

[0070] The tumor cells were HepG2 (cell source: ATCC HB-8065) ​​and MCF-7 (Huatuo Biotechnology, catalog number: HT-X1646). Cells in the logarithmic growth phase were collected and digested with 0.25% trypsin for 1-2 minutes. After digestion was terminated, a cell suspension was prepared with a cell density of 5 × 10⁻⁶ cells / mL. 4Cells / mL were seeded into 96-well plates (seedling density ≥ 80%). For the control group, an equal volume of cell-free culture medium was added, 100 μL per well, with 6 replicates. The plates were incubated in a CO2 incubator for 12–24 h. When cells adhered and reached a density greater than 80%, the plates were washed three times with PBS, and 100 μL of different concentrations of CGC-0 (non-nanosized) and CGC-1 (nanosized) samples were added. Incubation continued for 6 h. The liquid was then slowly aspirated, and the cells were washed three times with PBS. CCK-8 was then mixed with culture medium (DMEM (Guangzhou Xinghong Technology Co., Ltd., model EH80243) + 10% fetal bovine serum (FBS) (Guangzhou Xinghong Technology Co., Ltd., model UT82901) + 1% penicillin antibody (Guangzhou Xinghong Technology Co., Ltd., model 15140-122)) at a ratio of 1:10. 100 μL of the CCK8-culture medium mixture was added to each well, and the cells were incubated for 0.5-4 hours. A clear orange-yellow color and an OD value of approximately 0.8-1 indicate a good detection result. The OD value of each well was measured at 450 nm using a microplate reader, and cell viability was calculated. OD values ​​for each group were the difference between the blank control group and SPSS 19.0 software was used to analyze the differences between groups. Results are expressed as (x"±s").

[0071] Cell inhibition rate = [(Ac-As) / (Ac-Ab)] × 100%

[0072] In the formula: As is the experimental well (containing cell culture medium, CCK-8, and the substance to be tested); Ac is the control well (containing cell culture medium and CCK-8); Ab is the blank well (containing no cells, the substance to be tested, or CCK-8).

[0073] 10. Immunoassay

[0074] 2 mL of RAW 264.7 macrophages (2 × 10⁻⁶ cells) were added. 5 Cells / mL were seeded into 6-well plates and cultured for 4 hours. The culture medium was discarded, and 5-20 μg / mL of CGC-0 (non-nanosized) and CGC-1 (nanosized) samples were added to each well. A blank control group without samples was also included. The plates were cultured for 24 hours. After incubation, the cell solution was aspirated from the wells, and the supernatant was collected by centrifugation (500g, 20min). The secretion levels of IL-6, IL-1β, and TNF-α in the supernatant were measured according to the instructions of their respective ELISA kits.

[0075] Example 1

[0076] S1. Weigh 50g of fungal fruiting bodies (Armillaria mellea fruiting bodies belonging to the Basidiomycetes class. The fruiting bodies are dried at 50℃ and chopped into 0.5 mm pieces), add 1500ml of deionized water, stir at room temperature for 2 minutes, and then heat and stir in an 85℃ water bath for 2 hours. Allow the solid-liquid mixture to cool naturally to room temperature, centrifuge at 6000rpm for 5 minutes, and collect the precipitate.

[0077] S2. Add a mixture of NaOH solution (2%) and sodium stearate at 15 times its dry weight to the obtained wet precipitate (the dry weight ratio of NaOH solution to sodium stearate in the mixture is 15:1, and the mass of sodium stearate accounts for 0.1% of the total material mass, where the total material refers to the mixture of wet precipitate + NaOH solution (2%) and sodium stearate). Stir at 400 rpm for 5 min to suspend and disperse the centrifuged precipitate in the solution. Then, heat the mixture in an 85℃ water bath for 2 h. After the suspension cools naturally to room temperature, centrifuge at 6000 rpm for 5 min. Discard the supernatant. Add 500 mL of deionized water to the precipitate, stir at 400 rpm for 5 min, and centrifuge at 6000 rpm for 5 min. Repeat the above operation until the pH of the supernatant is neutral. The centrifuged precipitate obtained from the last centrifugation is the crude chitin-dextran complex.

[0078] S3. Add 1% hydrochloric acid solution to the crude chitosan-dextran complex to make the solid-liquid ratio 1:20 (g / mL). Stir at 400 rpm for 2 h at 60℃. After the reaction is complete, take it out and let it cool naturally to room temperature. Add 1 mol / L NaOH to neutralize it to pH 7. Centrifuge at 6000 rpm for 5 min. Wash the precipitate several times with deionized water until the conductivity of the washing solution is lower than 20 S / cm to obtain the desalted chitosan-dextran complex.

[0079] S4. Place the desalted chitosan-dextran complex in 25% ammonia water at 35℃, stir thoroughly, and then add 5% H2O2 (the mass ratio of desalted chitosan-dextran complex, ammonia water, and 5% H2O2 solution is 7:1:2). Let it stand for 4 hours, and then wash it with deionized water until pH 7 to obtain the decolorized chitosan-dextran complex, namely CGC-0.

[0080] S5. Take 0.5g of decolorized chitin-dextran complex and add it to 50mL of deionized water. Stir to suspend it in the deionized water. Shear it for 1 min at 22000 rpm using a high-speed shearing machine. Then, place it in an ultrasonic cell disruptor and sonicate it for 5 min at 540W power with a 2s interval followed by a 2s interval. Repeat the shearing and sonication cycle 4 times until the suspension is basically homogeneous. After the last sonication treatment, centrifuge the suspension at 5500 rpm for 5 min. Discard the centrifuged precipitate and collect the supernatant, which is the chitin-dextran nanocomplex suspension. After freeze-drying, the chitin-dextran nanopolysaccharide complex, namely CGC-1, is obtained.

[0081] The chitin-dextran nanopolysaccharide complex 1 (CGC-1) was tested and found to have a yield of 12.73%, a purity of 96.32%, a milky white color, and an average particle size of 125 nm for the polysaccharide fibers.

[0082] Example 2: Preparation of chitin-dextran nanopolysaccharide complex 2

[0083] The difference from Example 1 is that morel mycelium is used.

[0084] The chitin-dextran nanopolysaccharide complex 2 (CGC-2) was tested and found to have a yield of 12.14%, a purity of 94.23%, a milky white color, and an average particle size of 175 nm for the polysaccharide fibers.

[0085] Example 3: Preparation of chitin-dextran nanopolysaccharide complex 3

[0086] The difference from Example 1 is that the concentration of added sodium stearate is 0.05%.

[0087] The chitin-dextran nanopolysaccharide complex 3 (CGC-3) was tested and found to have a yield of 9.91%, a purity of 92.16%, a milky white to yellowish color, and an average particle size of 225 nm for the polysaccharide fibers.

[0088] Example 4: Preparation of chitin-dextran nanopolysaccharide complex 4

[0089] The difference from Example 1 is that the emulsifier is 0.2% glyceryl monostearate.

[0090] The chitin-dextran nanopolysaccharide complex 4 (CGC-4) was tested and found to have a yield of 10.14% and a purity of 93.01%. It was milky white in color but tended to clump together, and the average particle size of the polysaccharide fibers was 300 nm.

[0091] Example 5: Preparation of chitin-dextran nanopolysaccharide complex 5

[0092] The difference from Example 1 is that the concentration of the NaOH solution is 1%.

[0093] The chitin-dextran nanopolysaccharide complex 5 (CGC-5) was tested and found to have a yield of 9.40%, a purity of 94.13%, a milky white color, and an average particle size of 275 nm for the polysaccharide fibers.

[0094] Example 6: Preparation of chitin-dextran nanopolysaccharide complex 6

[0095] The difference from Example 1 is that the concentration of the hydrochloric acid solution is 0.5%.

[0096] The chitin-dextran nanopolysaccharide complex 6 (CGC-6) was tested and found to have a yield of 10.02%, a purity of 94.91%, a milky white color, and an average particle size of 200 nm for the polysaccharide fibers.

[0097] Example 7: Preparation of chitin-dextran nanopolysaccharide complex 7

[0098] The difference from Example 1 is that the ammonia concentration is 28%.

[0099] The chitin-dextran nanopolysaccharide complex 7 (CGC-7) was tested and found to have a yield of 10.09%, a purity of 95.82%, a milky white color, and an average particle size of 250 nm for the polysaccharide fibers.

[0100] Example 8: Preparation of chitin-dextran nanopolysaccharide complex 8

[0101] The difference from Example 1 is that the H2O2 concentration is 10%.

[0102] The chitin-dextran nanopolysaccharide complex 8 (CGC-8) was tested and found to have a yield of 8.83%, a purity of 96.01%, a milky white color, and an average particle size of 155 nm for the polysaccharide fibers.

[0103] Example 9: Preparation of chitin-dextran nanopolysaccharide complex 9

[0104] The difference from Example 1 is that a high-speed shearing machine is used to shear at a speed of 15,000 rpm.

[0105] The chitin-dextran nanopolysaccharide complex 9 (CGC-9) was tested and found to have a yield of 12.73%, a purity of 96.32%, a milky white color, and an average particle size of 600 nm for the polysaccharide fibers.

[0106] Example 10: Preparation of chitin-dextran nanopolysaccharide complex 10

[0107] The difference from Example 1 is that the shearing and ultrasonic cycle treatment are performed once.

[0108] The chitin-dextran nanopolysaccharide complex 10 (CGC-10) was tested and found to have a yield of 12.73%, a purity of 96.32%, a milky white color, and an average particle size of 1100 nm for the polysaccharide fibers.

[0109] Comparative Example 1: Preparation of Chitosan-Dextran Nanopolysaccharide Complex 11

[0110] The difference from Example 1 is that no emulsifier is added.

[0111] The chitin-dextran nanopolysaccharide complex 11 (CGC-11) was tested and found to have a yield of 6.78%, a purity of 90.54%, a milky white to yellowish color, and an average particle size of 375 nm for the polysaccharide fibers.

[0112] Comparative Example 2: Preparation of Chitosan-Dextran Nanopolysaccharide Complex 12

[0113] The difference from Example 1 is that the organic solvent (a mixture of ethanol and ether in a volume ratio of 1:6.1 (mL / mL)) is used for degreasing. Specifically, steps S1 and S2 in Example 1 are replaced with the following steps: the dried fungal fruiting bodies are heated to 80°C and soaked, then soaked again in a relatively clean cold liquid for 4 hours, and finally degreased with organic solvent steam for 3 hours with a material-to-liquid ratio of 1:20 (g / mL).

[0114] The chitin-dextran nanopolysaccharide complex 12 (CGC-12) was tested and found to have a yield of 7.12%, a purity of 92.11%, a milky white color but with some clumping, and an average particle size of 750 nm for the polysaccharide fibers.

[0115] Comparative Example 3: Preparation of Chitosan-Dextran Nanopolysaccharide Complex 13

[0116] The difference from Example 1 is that a high concentration of 20% NaOH is used in step S2.

[0117] The chitin-dextran nanopolysaccharide complex 13 (CGC-13) was tested and found to have a yield of 5.36%, a purity of 91.30%, a milky white color but a flocculent appearance, and an average particle size of 825 nm for the polysaccharide fibers.

[0118] Comparative Example 4: Preparation of Chitosan-Dextran Nanopolysaccharide Complex 14

[0119] The difference from Example 1 is that hydrochloric acid solution is not added in step S3.

[0120] The chitin-dextran nanopolysaccharide complex 14 (CGC-14) was tested and found to have a yield of 9.93%, a purity of 89.97%, a milky white color, and an average particle size of 375 nm for the polysaccharide fibers.

[0121] Preparation of chitin-dextran nanopolysaccharide complex 15 (Comparative Example 5)

[0122] The difference from Example 1 is that ammonia and H2O2 are not added in step S4.

[0123] The chitin-dextran nanopolysaccharide complex 15 (CGC-15) was tested and found to have a yield of 9.35%, a purity of 91.87%, a grayish-yellow color, and an average particle size of 325 nm for the polysaccharide fibers.

[0124] Table 1. Protein content, ash content, fat content, dextran-chitosan molar ratio, average particle size, and yield of chitosan-dextran nanopolysaccharide complex.

[0125]

[0126]

[0127] As can be seen from Table 1, the purity of all the above examples is higher than that of the comparative examples. The examples are a mild method for isolating chitin-glucan complexes from higher fungi. By adding emulsifiers, the synergistic removal of lipids is achieved, maximizing the removal of related compounds such as proteins, lipids, fats, pigments, alkali-soluble glucans and mineral compounds. This improves product purity while reducing the degradation of CGCs during the preparation process.

[0128] Table 2. Inhibition rates of chitosan-glucan complex CGC-0 and chitosan-glucan nanopolysaccharide complex 1 on HepG2 tumor cells.

[0129]

[0130] Note: The negative control is the blank group, and the analyte added to the positive control is 5-Fu.

[0131] As shown in Table 2, the addition of chitosan-dextran complex can inhibit the proliferation of tumor cells. When the chitosan-dextran complex is nano-sized, the cell inhibition rate is significantly increased. The cell inhibition rate is highest when added to 10 mg / ml of chitosan-dextran nanopolysaccharide complex 1. This proves that the chitosan-dextran nanopolysaccharide complex has health benefits in anti-tumor treatment.

[0132] Table 3. Phagocytic capacity and cytokine secretion levels of macrophages cultured for 24 h for chitin-glucan complex CGC-0 and chitin-glucan nanopolysaccharide complex 1.

[0133]

[0134] As shown in Table 3, both samples exhibited significant immunomodulatory effects by increasing cytokine secretion. The nano-sized complex showed a significantly enhanced cytokine secretion, and higher complex doses also resulted in higher cytokine secretion (P < 0.05). This demonstrates the existence of health benefits related to immune activity in the chitosan-dextran nanopolysaccharide complex.

[0135] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a chitin-dextran nanopolysaccharide complex, characterized in that... Includes the following steps: S1. Using fungal fruiting bodies or mycelia as raw materials, they are evenly dispersed in water, then heated and stirred, then cooled to room temperature, and the sediment is collected by centrifugation. S2. Add the mixture of NaOH solution and emulsifier to the wet precipitate obtained in S1, stir and heat to react, cool, centrifuge, continue to add water to the centrifuged precipitate and stir to wash, centrifuge again, and repeat the washing with water multiple times until the pH of the supernatant is neutral. The centrifuged precipitate obtained in the last time is the crude CGC product. S3. Add hydrochloric acid solution to the crude CGC separated in S2, heat and stir to react, then neutralize with NaOH to pH=7, centrifuge and wash to obtain desalted CGC; S4. Place the desalted CGC obtained in S3 in ammonia water, stir evenly, then add H2O2 solution and let stand for treatment, then wash with water until pH=7 to obtain decolorized CGC; S5. Add water to the decolorized CGC obtained in S4, stir to suspend it in water, treat the CGC suspension with shear-ultrasonic cycle, and centrifuge to obtain the supernatant, which is the chitin-dextran nanocomposite suspension. After freeze drying, the chitin-dextran nanopolysaccharide complex is obtained. The emulsifier mentioned in step S2 is at least one of sodium stearate and glyceryl monostearate. The concentration of the NaOH solution mentioned in step S2 is 0.5-4 wt%.

2. The method for preparing the chitin-dextran nanopolysaccharide complex according to claim 1, characterized in that: The fungal fruiting body or mycelium mentioned in step S1 is selected from at least one of the following fungi: Ascomycetes, Aspergillus niger, Basidiomycetes, Lentinus edodes, and Agaricus bisporus.

3. The method for preparing the chitin-dextran nanopolysaccharide complex according to claim 1, characterized in that: The heating temperature in step S1 is 70-95℃; the heating and stirring time is 1-3 hours.

4. The method for preparing the chitin-dextran nanopolysaccharide complex according to claim 1, characterized in that: The emulsifier mentioned in step S2 is sodium stearate.

5. The method for preparing the chitin-dextran nanopolysaccharide complex according to claim 1, characterized in that: The amounts of the mixture of wet precipitate, NaOH solution, and emulsifier described in step S2 shall meet the following requirements: the ratio of the dry weight of the wet precipitate to the dry weight of the mixture of NaOH solution and emulsifier shall be 1:10-1:25; the ratio of the dry weight of NaOH solution to the weight of emulsifier in the mixture of NaOH solution and emulsifier shall be 9:1-29:1; and the mass of emulsifier shall account for 0.05-0.5% of the total mass of all materials. The heating temperature range in the heating reaction described in step (2) is 60-95 ℃, and the heating time is 0.5-4 h.

6. The method for preparing the chitin-dextran nanopolysaccharide complex according to claim 1, characterized in that: The concentration of the hydrochloric acid solution in step S3 is 0.5-3.0%; the solid-liquid ratio of crude CGC to hydrochloric acid solution is 1:15-1:25, in g / mL; the NaOH solution is 1-3 mol / L. The heating and stirring reaction mentioned in step S3 refers to stirring the reaction at 400 rpm for 1-3 hours at 55-60℃. The centrifugal washing mentioned in step S3 refers to washing the precipitate with water multiple times after centrifugation until the conductivity of the washing liquid is lower than 20 S / cm.

7. The method for preparing the chitin-dextran nanopolysaccharide complex according to claim 1, characterized in that: In step S4, the ammonia solution is treated at a temperature of 28-42℃ and a concentration of 25%-28% on the complex; the H2O2 solution has a concentration of 3-6% and is left to stand for 3-6 hours. In step S4, the mass ratio of the desalted chitin-dextran complex, ammonia, and H2O2 solution is 7:1:2 to 16:2:

5.

8. The method for preparing the chitin-dextran nanopolysaccharide complex according to claim 1, characterized in that: The addition of water to the decolorized CGC mentioned in step S5 refers to adding 50-500mL of water to every 0.5g of decolorized CGC; The shear-ultrasonic cycle treatment described in step S5 refers to first shearing with a high-speed shearing machine, and then treating it in an ultrasonic cell disruptor, repeating this process multiple times until the suspension is homogeneous; wherein the shearing is performed at a speed of 17000-25000 rpm for 1-3 minutes; and the ultrasonic treatment is performed at a power of 500-600W for 1-3 seconds with a 1-3 second interval for 5 minutes.

9. A chitin-dextran nanopolysaccharide complex prepared by the method according to any one of claims 1-8.

10. The use of the chitin-dextran nanopolysaccharide complex according to claim 9 in the preparation of antitumor drugs and immunomodulatory drugs.

11. The use of the chitin-dextran nanopolysaccharide complex according to claim 9 in the preparation of anti-liver cancer drugs.

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