Inonotus obliquus polysaccharide with long-acting moisturizing effect, preparation method and application thereof

Abstract

This invention relates to the field of polysaccharide extraction technology and provides a method for preparing Inonotus obliquus polysaccharides with long-lasting moisturizing effects. The method includes: pulverizing dried Inonotus obliquus sclerotia, cold-soaking and defatting to obtain defatted bacterial powder; loading the defatted bacterial powder into a continuous flow fixed-bed reactor, and passing in a composite enzymatic hydrolysate containing immobilized cellulase and immobilized pectinase to obtain a crude polysaccharide extract; pumping the crude polysaccharide extract into an expanded-bed adsorption column to obtain a refined polysaccharide extract; passing the refined polysaccharide extract sequentially through ultrafiltration membranes with molecular weight cutoffs of 300 kDa and 50 kDa to concentrate it, obtaining a polysaccharide concentrate; adding L-serine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to the polysaccharide concentrate, dialysis to remove unreacted reagents, obtaining an L-serine-modified polysaccharide extract; mixing the L-serine-modified polysaccharide extract with sodium polyglutamate, performing supramolecular self-assembly, and then freeze-drying to obtain Inonotus obliquus polysaccharides with long-lasting moisturizing effects; thus achieving long-lasting moisturizing.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide extraction technology, and in particular to Inonotus obliquus polysaccharide with long-lasting moisturizing effects, its preparation method, and its application. Background Technology

[0002] Inonotus obliquus, commonly known as "chaga mushroom," is a rare medicinal fungus that primarily parasitizes birch trees and is naturally distributed in cold regions of Northeast my country, such as the Changbai Mountains and Heilongjiang Province. Inonotus obliquus is rich in various bioactive metabolites, including polysaccharides, triterpenes, steroids, polyphenols, and melanin. Among these, polysaccharides are the most abundant and the most extensively studied core active substance. Inonotus obliquus polysaccharides are mainly composed of monosaccharides such as glucose, galactose, mannose, xylose, and arabinose. Its most characteristic active component is β-D-glucan, which has a β-1,3-glycosidic backbone and β-1,6-branchs. This polysaccharide exhibits structural characteristics such as large molecular weight, high branching degree, and a large number of free hydroxyl groups, giving it natural advantages in hydrogen bonding hydration, film formation, and biocompatibility.

[0003] In the cosmetics field, the moisturizing effects of Inonotus obliquus polysaccharides have received widespread attention. Its moisturizing mechanism is mainly manifested on two levels: firstly, the abundant hydroxyl groups on the surface of the polysaccharide molecules can capture and bind a large number of water molecules through hydrogen bonds, acting as a humectant; secondly, the high molecular weight β-glucan chains can spread on the skin surface to form a breathable film, physically slowing down transdermal water evaporation and achieving a sealing and water-locking effect. Chinese patents CN117398326A and CN117815096A have disclosed the application of Inonotus obliquus extract in enhancing the cohesion of stratum corneum cells, repairing the skin barrier, and moisturizing; these existing technologies indicate that Inonotus obliquus polysaccharides have become a new plant-derived moisturizing raw material.

[0004] The moisturizing properties of natural Inonotus obliquus polysaccharides exhibit a clear "ceiling effect." Regardless of how the molecular skeleton is optimized during extraction, the chemical structure of natural polysaccharides dictates that their moisturizing mechanism primarily relies on the physical hygroscopic effect of hydrogen bonds formed between the inherent hydroxyl groups on the polysaccharide chains and water molecules. Under conditions of high relative humidity, this mechanism can maintain a certain water absorption rate and moisturizing effect. However, when the skin is in a dry environment (such as in air-conditioned rooms, autumn and winter seasons, or in northern low-humidity climates where relative humidity is typically below 50%), the dynamic balance between the hydroxyl groups on the surface of the natural polysaccharides and water molecules is disrupted, the hygroscopic driving force decreases sharply, and it becomes unable to capture enough moisture from the atmosphere to maintain epidermal hydration. At the same time, the physical barrier membrane of natural polysaccharides has limited inhibitory effect on water evaporation, making it difficult to compensate for insufficient hygroscopic absorption under low humidity conditions, resulting in a rapid decline in moisturizing effect after application. This inherent chemical structural limitation means that even after meticulous extraction and purification, the 24-hour continuous moisturizing ability of natural Inonotus obliquus polysaccharides still falls short of the stringent requirements of modern high-end skincare products for "all-day long-lasting hydration," especially in low-humidity, dry environments where effective hydration for more than 24 hours is almost impossible. Therefore, overcoming the natural limitation of Inonotus obliquus polysaccharides at the molecular level—relying on the number of its hydroxyl groups and the humidity ratio of the environment for moisture absorption—and endowing it with strong water-binding capacity and long-lasting moisturizing properties even in low-humidity environments, has become a core technological challenge urgently needing to be overcome in this field. Summary of the Invention

[0005] The purpose of this invention is to provide Inonotus obliquus polysaccharide with long-lasting moisturizing effects, its preparation method, and its application, in order to solve the problems in the background art.

[0006] Specifically, the preparation method of Inonotus obliquus polysaccharide with long-lasting moisturizing effect includes the following steps:

[0007] Step 1: Crush the dried Inonotus obliquus sclerotia and degrease them at least once with a 75% (v / v) ethanol aqueous solution at a temperature of 25-30°C to obtain degreased bacterial powder.

[0008] Step 2: The defatted bacteria powder obtained in Step 1 is loaded into a continuous flow fixed bed reactor, and a composite enzymatic hydrolysate containing immobilized cellulase and immobilized pectinase is introduced. Targeted enzymatic hydrolysis is carried out under the conditions of pH 4.5-5.5 and temperature 45-55℃, while low-frequency weak ultrasound with a frequency of 25 kHz and a power of 120-150 W is applied simultaneously for 1-2 h. After the enzymatic hydrolysis is completed, the bacteria are inactivated, and solid-liquid separation is performed to obtain crude polysaccharide extract.

[0009] Step 3: Pump the crude polysaccharide extract obtained in Step 2 into an expanded bed adsorption column filled with magnetic composite microspheres from bottom to top at a flow rate of 1-3 BV / h. Under the condition of a bed expansion rate of 20%-40%, proteins, pigments and microparticles are removed simultaneously. Collect the effluent to obtain the refined polysaccharide extract.

[0010] Step 4: Pass the purified polysaccharide solution obtained in Step 3 through ultrafiltration membranes with molecular weight cutoffs of 300 kDa and 50 kDa in sequence, operating at a pressure of 0.15-0.25 MPa and a temperature of 25-30℃. Collect and concentrate the polysaccharide fractions with molecular weights between 50 kDa and 200 kDa to obtain a polysaccharide concentrate.

[0011] Step 5: Add 0.5%-2% L-serine and 0.1%-0.5% 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to the polysaccharide concentrate obtained in Step 4. React at pH 5.0-6.0, room temperature and in the dark for 2-4 hours. Dialyze to remove unreacted reagents to obtain L-serine-modified polysaccharide solution.

[0012] Step 6: Mix the L-serine-modified polysaccharide solution obtained in Step 5 with sodium polyglutamate at a mass ratio of 1:0.3-3, adjust the pH to 5.5-7.0, perform supramolecular self-assembly at 30-50℃, and then freeze-dry to obtain Inonotus obliquus polysaccharide with long-lasting moisturizing effect.

[0013] In a further embodiment, in step two, both the immobilized cellulase and the immobilized pectinase are immobilized on an aminoened silica gel carrier using glutaraldehyde cross-linking, and the activity ratio of cellulase to pectinase in the composite enzymatic hydrolysate is 1:2-4.

[0014] In a further embodiment, in step three, the magnetic composite microspheres are sodium alginate-coated iron oxide microspheres with a particle size of 50-150 μm, and the inner diameter to height ratio of the expanded bed adsorption column is 1:5-10.

[0015] In a further embodiment, in step four, the ultrafiltration membrane is made of modified polyethersulfone membrane.

[0016] In a further step, a saccharide isomer and / or ectoine are added to step six as synergistic moisturizing active molecules, with the addition amount being 10%-50% of the mass of the L-serine-modified polysaccharide solution.

[0017] Replace step five with:

[0018] S1. First, add L-serine at a mass of 0.5%-2% to the polysaccharide concentrate; use 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride as a condensing agent, which accounts for 0.1%-0.5% of the polysaccharide concentrate mass, and react at pH 5.0-6.0, at room temperature and in the dark for 2 h.

[0019] S2, Second grafting step: Without dialysis, directly add 0.3%-1.5% L-proline (by mass of polysaccharide concentrate) and an additional 0.05%-0.3% 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (by mass of polysaccharide concentrate) to the reaction solution, and continue the reaction at pH 5.5-6.0, room temperature, and in the dark for 1.5-2 hours.

[0020] S3. After the reaction is complete, dialyze the solution for 48 hours using a dialysis bag with a molecular weight cutoff of 3500 Da, and then freeze-dry it.

[0021] Replace step six with:

[0022] S1. Preparation of the coordination solution of epidermal succinate gallate and zinc ions: Dissolve epidermal succinate gallate in deionized water to form a solution with a concentration of 0.5%-1%, and add 0.2% ZnSO4 solution in equal molar ratio dropwise. Stir for 30 min to form a solution of epidermal succinate gallate and zinc coordination oligomers.

[0023] S2. Mix L-serine-modified polysaccharide solution with sodium polyglutamate at a ratio of 1:1, add 5%-10% of the above-mentioned epidermal cadherin gallate and zinc coordination oligomer solution (by mass of L-serine-modified polysaccharide solution), and adjust the pH to 6.0.

[0024] S3, stir and assemble at 40℃ for 3 h. During this period, the epidermal guarcatechin gallate and zinc coordination oligomers are embedded into the network through multi-point hydrogen bonds between galloyl groups and polysaccharide hydroxyl groups. At the same time, metal coordination bonds form strong physical cross-linking points between adjacent chains. Freeze-dry in a light-protected environment and package with nitrogen filling.

[0025] A polysaccharide from Inonotus obliquus with long-lasting moisturizing effects is prepared by the method described above; or, the L-serine-modified polysaccharide solution obtained in step five is emulsified into microdroplets by a pulsed electric field-assisted membrane, and then passed into a supercritical CO2 crystallization vessel and treated at a temperature of 35-40℃ and a pressure of 10-15 MPa for 1-3 h, and the porous core-shell structured nanospheres with a particle size of 100-500 nm are obtained after depressurization.

[0026] Further, the polysaccharide exhibits a moisture absorption rate of no less than 35% and a water evaporation half-life of no less than 48 hours at 24 hours under conditions of 80% relative humidity and 25°C; a moisture retention rate of no less than 42% and a transdermal water loss inhibition rate of no less than 45% at 40% relative humidity and 30°C.

[0027] An application of the above-mentioned Inonotus obliquus polysaccharide with long-lasting moisturizing effect includes its application in cosmetics; the amount added is 3%-10% of the total mass of the cosmetic.

[0028] Compared with the prior art, the present invention can achieve the following:

[0029] 1. In step one, the dried sclerotia are degreased at least once by cold soaking in 75% ethanol at 25-30℃ to obtain a degreased bacterial powder with low pigment and low wax content. In step two, the degreased bacterial powder is loaded into a continuous flow fixed bed reactor and contacted with immobilized cellulase and immobilized pectinase. Targeted enzymatic hydrolysis is performed at pH 4.5-5.5 and 45-55℃, while simultaneously applying 25% ethanol. The extraction system utilizes low-frequency, weak ultrasound at 120-150W (kHz). The entire extraction process is confined to a mild environment, avoiding the use of strong acids, strong alkalis, and high shear forces. The triple linkage of mild pretreatment, immobilized enzyme, and low-frequency, weak ultrasound fundamentally solves the industry-wide problem of random breakage of β-glucan chains caused by traditional hot water extraction and strong ultrasound extraction. Cold ethanol maceration removes waxes and triterpenes that inhibit enzyme activity and cause stickiness in the final product, providing an interference-free substrate interface for the immobilized enzyme. The immobilized enzyme precisely hydrolyzes cell wall cellulose / pectin without touching the related polysaccharide backbone. The low-frequency, weak ultrasound at 25kHz / 120-150W only loosens the cell wall and enhances mass transfer, without generating cavitation effects sufficient to break glycosidic bonds.

[0030] 2. Step 3: The crude polysaccharide extract is pumped from bottom to top into an expanded bed adsorption column (EBA) packed with magnetic composite microspheres at a flow rate of 1-3 BV / h. Under fluidized conditions with a bed expansion rate of 20%-40%, proteins, pigments, and fine particulate matter are simultaneously removed, resulting in a purified polysaccharide extract in one step. This breaks the traditional multi-step tandem purification mode of "centrifugation-Sevag deproteinization-activated carbon / resin decolorization-filtration". The gaps between the fluidized magnetic composite microspheres in the expanded bed adsorption column allow high molecular weight polysaccharides to move freely, while impurities such as proteins and pigments are simultaneously adsorbed and removed, eliminating the need for pre-filtration or removal of suspended solids from the crude extract. The entire process does not use toxic organic solvents such as chloroform or n-butanol. A single device achieves clarification, deproteinization, and decolorization in one step, significantly simplifying the process chain and avoiding the cumulative loss of activity in multi-step operations.

[0031] 3. Step Four: The refined polysaccharide solution is passed sequentially through ultrafiltration membranes with molecular weight cutoffs of 300 kDa and 50 kDa to precisely retain and collect polysaccharide fractions in the 50-200 kDa molecular weight range, followed by concentration. The use of a two-stage ultrafiltration membrane combination of 300 kDa and 50 kDa strictly constructs a molecular weight window of 50-200 kDa, overcoming the limitations of traditional alcohol precipitation or single-stage ultrafiltration in accurately removing ineffective impurities. Large colloidal molecules with molecular weights greater than 300 kDa are retained and discarded, avoiding the problem of a sticky final product and difficulty in skin penetration. Fragmented short chains with molecular weights less than 50 kDa are removed after passing through the membrane pores, avoiding the defect of short moisturizing retention time. The retained 50-200 kDa fraction balances stratum corneum permeability and epidermal film-forming properties, forming a breathable and water-locking film with a thickness of 0.12-0.18 μm on the skin surface, while simultaneously storing water in the shallow layer of the stratum corneum, forming a dual-layer moisturizing structure of "deep water storage - surface water locking."

[0032] 4. L-serine was site-specifically grafted onto the polysaccharide backbone of *Inonotus obliquus* via a mild condensation reaction with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, breaking the performance ceiling of natural polysaccharides that rely solely on their own hydroxyl groups for moisture absorption. The additional hydroxyl and carboxyl groups introduced into the L-serine side chain act like miniature water pumps, increasing the 24-hour moisture absorption rate of the modified polysaccharide from less than 15% in natural polysaccharides to over 35% under 80% relative humidity conditions, and increasing the 24-hour moisture retention rate from 18%-19% to over 37% under 40% relative humidity conditions, while extending the water evaporation half-life to over 48 hours. Compared with conventional modifications such as acetylation and carboxymethylation, which require a strongly alkaline environment, resulting in a significant decrease in molecular weight and disintegration of the triple helix conformation, this modification route is carried out under mild, weakly acidic conditions of pH 5.0-6.0, with a molecular weight retention rate of no less than 90% and complete preservation of the higher-order structure of the polysaccharide, truly achieving a qualitative change in moisture absorption capacity while preserving the molecular skeleton.

[0033] 5. By utilizing the intermolecular hydrogen bonds between the numerous hydroxyl groups of the modified polysaccharide backbone and the carboxyl groups of the sodium polyglutamate side chain, as well as the local hydrophobic folding driven by the grafted hydrophobic acetyl / serine residues, a three-dimensional supramolecular network structure is physically constructed without adding any chemical cross-linking agents. When this network is used on the skin surface, it degrades layer by layer, slowly releasing synergistic moisturizing active molecules such as water-locking magnets (saccharide isomers) and ectoine according to the time gradient, achieving 24-48 hours of relay moisturizing.

[0034] 6. The physical level of ultrafine grinding and flash expansion pore-forming, the biological level of biomimetic pre-enzymatic hydrolysis to loosen the outer layer of the cell wall, and the chemical level of gradient / deep solvent degreasing are integrated into a unified pretreatment system. Its synergistic effect enables the subsequent immobilized enzyme and weak ultrasonic extraction to release the whole long-chain polysaccharide efficiently by targeted enzymatic hydrolysis without any high shear force, so as to achieve the process goal of zero damage to long chains and zero impurity residue to the greatest extent.

[0035] 7. Enzymatic biocatalysis was used to enhance the moisturizing properties of Inonotus obliquus polysaccharides, avoiding potential safety controversies related to chemical condensing agents. Simultaneously, hyaluronic acid oligosaccharide-L-serine beads were used as grafting units to preconstruct a three-tiered moisturizing topology of small molecule water absorption, oligosaccharide water storage, and polysaccharide film formation and water locking in a single molecule. This provided enhanced hydrogen bond anchors and bioactivity for subsequent supramolecular self-assembly, achieving a leap from chemical modification to biofunctionalization.

[0036] 8. Natural polyphenol-metal coordination chemistry is introduced into the supramolecular self-assembly system of polysaccharide-peptide to achieve network dual physical cross-linking, namely hydrogen bond cross-linking and metal coordination cross-linking, without the use of any chemical cross-linking agent. At the same time, the spatiotemporal release sequence of active factors is precisely regulated by sequential core-shell assembly, and the resolution behavior of traditional freeze-dried powder is completely changed by creating pores with gas templates. Attached Figure Description

[0037] Figure 1 This is a statistical chart of the results of the in vitro moisturizing rate test.

[0038] Figure 2 This is a statistical chart showing the moisture absorption rate of each sample under RH=80% conditions;

[0039] Figure 3 A statistical chart comparing the moisture absorption rate over 24 hours under different humidity conditions;

[0040] Figure 4 The survival rate statistics of samples A to D at sample concentrations of 0.1 mg / mL, 1.0 mg / mL, and 10.0 mg / mL are presented. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0042] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0043] Example 1

[0044] A method for preparing Inonotus obliquus polysaccharide with long-lasting moisturizing effects includes the following steps:

[0045] Step 1: Crush the dried Inonotus obliquus sclerotia and degrease them at least once with a 75% (v / v) ethanol aqueous solution at a temperature of 25-30°C to obtain degreased bacterial powder.

[0046] Step 2: The defatted bacteria powder obtained in Step 1 is loaded into a continuous flow fixed bed reactor, and a composite enzymatic hydrolysate containing immobilized cellulase and immobilized pectinase is introduced. Targeted enzymatic hydrolysis is carried out under the conditions of pH 4.5-5.5 and temperature 45-55℃, while low-frequency weak ultrasound with a frequency of 25 kHz and a power of 120-150 W is applied simultaneously for 1-2 h. After the enzymatic hydrolysis is completed, the bacteria are inactivated, and solid-liquid separation is performed to obtain crude polysaccharide extract.

[0047] Step one involves cold-soaking the dried sclerotia at 25-30℃ with 75% ethanol at least once to degrease them, obtaining a low-pigment, low-wax degreased bacterial powder. In step two, the degreased bacterial powder is loaded into a continuous flow fixed-bed reactor and contacted with immobilized cellulase and immobilized pectinase. Targeted enzymatic hydrolysis is performed at pH 4.5-5.5 and 45-55℃, with a simultaneous application of 25% ethanol. The extraction system utilizes low-frequency, weak ultrasound at 120-150W (kHz). The entire extraction process is confined to a mild environment, avoiding the use of strong acids, strong alkalis, and high shear forces. The triple linkage of mild pretreatment, immobilized enzyme, and low-frequency, weak ultrasound fundamentally solves the industry-wide problem of random breakage of β-glucan chains caused by traditional hot water extraction and strong ultrasound extraction. Cold ethanol maceration removes waxes and triterpenes that inhibit enzyme activity and cause stickiness in the final product, providing an interference-free substrate interface for the immobilized enzyme. The immobilized enzyme precisely hydrolyzes cell wall cellulose / pectin without touching the related polysaccharide backbone. The low-frequency, weak ultrasound at 25kHz / 120-150W only loosens the cell wall and enhances mass transfer, without generating cavitation effects sufficient to break glycosidic bonds.

[0048] Step 3: Pump the crude polysaccharide extract obtained in Step 2 into an expanded bed adsorption column filled with magnetic composite microspheres from bottom to top at a flow rate of 1-3 BV / h. Under the condition of a bed expansion rate of 20%-40%, proteins, pigments and microparticles are removed simultaneously. Collect the effluent to obtain the refined polysaccharide extract.

[0049] Step 3 involves pumping the crude polysaccharide extract into an expanded bed adsorption column (EBA) packed with magnetic composite microspheres from bottom to top at a flow rate of 1-3 BV / h. Under fluidized conditions with a bed expansion rate of 20%-40%, proteins, pigments, and fine particulate matter are simultaneously removed, yielding a purified polysaccharide extract in one step. This method breaks away from the traditional multi-step purification process of "centrifugation-Sevag protein removal-activated carbon / resin decolorization-filtration." The fluidized magnetic composite microspheres within the expanded bed adsorption column allow high molecular weight polysaccharides to freely pass through, while impurities such as proteins and pigments are simultaneously adsorbed and removed, eliminating the need for pre-filtration or removal of suspended solids from the crude extract. The entire process avoids the use of toxic organic solvents such as chloroform and n-butanol. A single device achieves clarification, protein removal, and decolorization in one step, significantly simplifying the process chain and avoiding the cumulative loss of activity during multi-step operations.

[0050] Step 4: Pass the purified polysaccharide solution obtained in Step 3 through ultrafiltration membranes with molecular weight cutoffs of 300 kDa and 50 kDa in sequence, operating at a pressure of 0.15-0.25 MPa and a temperature of 25-30℃. Collect and concentrate the polysaccharide fractions with molecular weights between 50 kDa and 200 kDa to obtain a polysaccharide concentrate.

[0051] Step four involves sequentially passing the refined polysaccharide solution through ultrafiltration membranes with molecular weight cutoffs of 300 kDa and 50 kDa to precisely retain and collect polysaccharide fractions in the 50-200 kDa molecular weight range, followed by concentration. The use of a two-stage ultrafiltration membrane combination of 300 kDa and 50 kDa strictly constructs a molecular weight window of 50-200 kDa, overcoming the limitations of traditional alcohol precipitation or single-stage ultrafiltration in accurately removing ineffective impurities. Large colloidal molecules with molecular weights greater than 300 kDa are retained and discarded, avoiding the problem of a sticky final product and difficulty in skin penetration. Fragmented short chains with molecular weights less than 50 kDa are removed after passing through the membrane pores, avoiding the defect of short moisturizing retention time. The retained 50-200 kDa... The kDa fraction balances stratum corneum permeability and epidermal film-forming properties, forming a breathable and water-locking film with a thickness of 0.12-0.18μm on the skin surface, while simultaneously storing water in the shallow layer of the stratum corneum, forming a dual-layer moisturizing structure of "deep water storage - surface water locking". The 24-hour moisturizing rate of ungraded natural polysaccharides is usually less than 20%, while the moisturizing rate of polysaccharide fractions retained through this window exceeds 25%, verifying the direct enhancement of moisturizing performance by precise grading.

[0052] Step 5: Add 0.5%-2% L-serine and 0.1%-0.5% 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to the polysaccharide concentrate obtained in Step 4. React at pH 5.0-6.0, room temperature and in the dark for 2-4 hours. Dialyze to remove unreacted reagents to obtain L-serine-modified polysaccharide solution.

[0053] L-serine was site-specifically grafted onto the polysaccharide backbone of *Inonotus obliquus* via a mild condensation reaction with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, breaking the performance ceiling of natural polysaccharides that rely solely on their own hydroxyl groups for moisture absorption. The additional hydroxyl and carboxyl groups introduced into the L-serine side chain act like miniature water pumps, increasing the 24-hour moisture absorption rate of the modified polysaccharide from less than 15% in natural polysaccharides to over 35% under 80% relative humidity conditions, and increasing the 24-hour moisture retention rate from 18%-19% to over 37% under 40% relative humidity conditions, while extending the water evaporation half-life to over 48 hours. Compared with conventional modifications such as acetylation and carboxymethylation, which require a strongly alkaline environment, resulting in a significant decrease in molecular weight and disintegration of the triple helix conformation, this modification route is carried out under mild, weakly acidic conditions of pH 5.0-6.0, with a molecular weight retention rate of no less than 90% and complete preservation of the higher-order structure of the polysaccharide, truly achieving a qualitative change in moisture absorption capacity while preserving the molecular skeleton.

[0054] Step 6: Mix the L-serine-modified polysaccharide solution obtained in Step 5 with sodium polyglutamate at a mass ratio of 1:0.3-3, adjust the pH to 5.5-7.0, carry out supramolecular self-assembly at 30-50℃, and then freeze-dry to obtain Inonotus obliquus polysaccharide with long-lasting moisturizing effect.

[0055] By utilizing the intermolecular hydrogen bonds between the numerous hydroxyl groups of the modified polysaccharide backbone and the carboxyl groups of the sodium polyglutamate side chain, as well as the local hydrophobic folding driven by the grafted hydrophobic acetyl / serine residues, a three-dimensional supramolecular network structure is physically constructed without adding any chemical cross-linking agents. When applied to the skin surface, this network degrades layer by layer, slowly releasing synergistic moisturizing active molecules such as water-locking magnets (saccharide isomers) and ectoine according to the time gradient, achieving relay moisturizing for 24-48 hours.

[0056] In a further embodiment, in step two, both the immobilized cellulase and the immobilized pectinase are immobilized on an aminoened silica gel carrier using glutaraldehyde cross-linking, and the activity ratio of cellulase to pectinase in the composite enzymatic hydrolysate is 1:2-4.

[0057] In step three, the magnetic composite microspheres are sodium alginate-coated iron oxide microspheres with a particle size of 50-150 μm, and the inner diameter to height ratio of the expanded bed adsorption column is 1:5-10.

[0058] In step four, the ultrafiltration membrane is made of modified polyethersulfone membrane.

[0059] In step six, saccharide isomers and / or ectoine are added as synergistic moisturizing active molecules, with the amount added being 10%-50% of the mass of the L-serine-modified polysaccharide solution.

[0060] In step six, aseptic filtration and / or filling are performed before freeze-drying.

[0061] Example 2

[0062] The only difference between this embodiment and Embodiment 1 is that step one is replaced with:

[0063] S1. The sclerotium of Inonotus obliquus was cut into 1-2 cm pieces, placed in liquid nitrogen at -196℃, frozen for 10 min, and then ultra-finely pulverized at -20℃ or below until the particle size D was reached. 90 Microbial powder with a particle size not exceeding 50μm is obtained.

[0064] S2. Moisten the ultrafine bacterial powder with a 30% (v / v) ethanol aqueous solution until the water content is 25-30%, and equilibrate in a sealed container for 2 hours.

[0065] S3. The moistened mycelium powder is put into the flash expansion machine and heated to 120-130℃ under a pressure of 0.8-1.2 MPa and held for 30 seconds. The pressure is then released to normal pressure. The volume of the mycelium powder expands by 2-3 times, forming a porous and loose structure, and the flash-expanded mycelium powder is obtained.

[0066] S4. Flash-expanding bacterial powder is dried at 50°C until the moisture content is no higher than 8% to obtain porous activated bacterial powder.

[0067] S5. The porous activated bacterial powder was degreased once by cold soaking in a 75% (v / v) ethanol aqueous solution at a temperature of 25°C for 8 hours, followed by low-temperature drying.

[0068] Before defatting, physical field treatment is introduced. Through low-temperature ultrafine pulverization and instantaneous flash expansion, microcracks are generated in the dense chitin-glucan complex structure of the sclerotia, increasing the specific surface area and porosity, and providing more action sites for subsequent immobilized enzymes. The flash expansion process preactivates some bound polysaccharides, which is beneficial for subsequent release.

[0069] Alternatively, replace step one with:

[0070] S1. The sclerotia of Inonotus obliquus are dried at 40℃ and coarsely crushed and passed through a 40-mesh sieve.

[0071] S2. Preparation of pre-enzymatic hydrolysis buffer: Add 0.02-0.05% w / v free β-glucanase and 0.01-0.03% w / v keratinase to pH 5.2 and 10 mmol / L citrate buffer, and add 0.5% glycerol as enzyme stabilizer;

[0072] S3. Mix the bacterial powder with the pre-enzymatic hydrolysis infusion at a liquid-solid ratio of 4:1, and treat with gentle shaking at 35-38℃ for 1-2 hours.

[0073] S4. Filter the bacterial powder and directly immerse it in a 75% (v / v) ethanol aqueous solution for cold soaking and degreasing without drying. The temperature for cold soaking and degreasing is 25℃, and the cycle is 8-10 h. After filtration, dry at low temperature to obtain pre-activated degreasing bacterial powder.

[0074] In the defatting stage, a trace amount of biomimetic enzyme system is introduced in advance to loosen the outer layer of the cell wall under mild conditions, allowing the defatting solvent to penetrate deeper, while opening up a green channel when the immobilized enzyme enters.

[0075] Alternatively, replace step one with:

[0076] S1. The sclerotium of Inonotus obliquus was cut into 1-2 cm pieces, placed in liquid nitrogen at -196℃, frozen for 10 min, and then ultra-finely pulverized at -20℃ or below until the particle size D was reached. 90 Microbial powder with a particle size not exceeding 50μm is obtained.

[0077] S2. Moisten the ultrafine bacterial powder with a 30% (v / v) ethanol aqueous solution until the water content is 25-30%, and equilibrate in a sealed container for 2 hours.

[0078] S3. The moistened mycelium powder is put into the flash expansion machine and heated to 120-130℃ under a pressure of 0.8-1.2 MPa and held for 30 seconds. The pressure is then released to normal pressure. The volume of the mycelium powder expands by 2-3 times, forming a porous and loose structure, and the flash-expanded mycelium powder is obtained.

[0079] S4. The porous activated flash-expanding bacteria powder was added to a pre-enzymatic hydrolysate containing 0.03% w / v free β-glucanase and 0.02% w / v cutinase and treated at 35°C for 1.5 h.

[0080] S5. After filtration, the bacterial powder is directly added to a 75% (v / v) ethanol aqueous solution and cold-soaked at 25°C for 8 hours to remove fat; then dried at low temperature to obtain activated-pre-enzymatic hydrolysis-degreased bacterial powder.

[0081] The system integrates the physical aspects of ultrafine grinding and flash expansion to create pores, the biological aspects of biomimetic pre-enzymatic hydrolysis to loosen the outer cell wall, and the chemical aspects of gradient / deep solvent degreasing into a unified pretreatment system. Its synergistic effect allows subsequent immobilized enzymes and weak ultrasonic extraction to efficiently release whole long-chain polysaccharides through targeted enzymatic hydrolysis without any high shear force, maximizing the process goal of zero damage to long chains and zero impurity residue.

[0082] Example 3

[0083] The only difference between this embodiment and Embodiment 1 is that step five is replaced with:

[0084] S1. First, add L-serine at a mass of 0.5%-2% to the polysaccharide concentrate; use 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride as a condensing agent, which accounts for 0.1%-0.5% of the polysaccharide concentrate mass, and react at pH 5.0-6.0, at room temperature and in the dark for 2 h.

[0085] S2, Second grafting step: Without dialysis, directly add 0.3%-1.5% L-proline (by mass of polysaccharide concentrate) and an additional 0.05%-0.3% 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (by mass of polysaccharide concentrate) to the reaction solution, and continue the reaction at pH 5.5-6.0, room temperature, and in the dark for 1.5-2 hours.

[0086] S3. After the reaction is complete, dialyze the solution for 48 hours using a dialysis bag with a molecular weight cutoff of 3500 Da, and then freeze-dry it.

[0087] Based on L-serine, L-proline is sequentially introduced as the second graft monomer. L-proline is one of the most abundant amino acids in natural moisturizing factors. Its unique pyrrolidine ring structure can stabilize the hydration layer through preferential exclusion effect in dry environments, preventing polysaccharides from losing water rapidly under extremely low humidity.

[0088] Alternatively, step five can be replaced with:

[0089] S1. Amino introduction pretreatment: Add 0.2%-0.5% of food-grade small molecule chitosan oligosaccharide (Mw=1-3kDa) or lysine short peptide to the polysaccharide concentrate and stir and premix for 30 min at pH 6.0 to provide sufficient free amino anchors for subsequent enzymatic grafting.

[0090] S2. Add L-serine to the system at a concentration of 0.5%-2% of the polysaccharide concentrate.

[0091] S3. Add transglutaminase (enzyme activity ≥1000U / g polysaccharide) and react gently for 4-6 hours at pH 6.0-6.5 and temperature 45-50℃.

[0092] S4. Inactivate enzymes at 80℃ for 10 minutes, then remove small molecule impurities by ultrafiltration or dialysis.

[0093] The bioenzymatic method of transglutaminase replaces the chemical condensation of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, catalyzing the amide bond of L-serine to connect to existing amino residues on the polysaccharide chain or the lysine side chain of the polysaccharide-protein complex, achieving green modification without chemical cross-linking agents, with stronger reaction specificity and fewer side reactions.

[0094] Alternatively, step five can be replaced with:

[0095] S1. Pre-synthesis of beaded unit: Hyaluronic acid oligosaccharide (Mw=5kDa) and L-serine were mixed at a molar ratio of 1:5, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was used as a condensing agent, accounting for 0.5% of the mass of the polysaccharide concentrate. The mixture was condensed for 1 hour to obtain hyaluronic acid oligosaccharide-L-serine conjugate.

[0096] S2. Add the conjugate from step S1 to the polysaccharide concentrate at a ratio of 1%-4% of the total mass of the polysaccharide, and then add a trace amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride for overall grafting, which accounts for 0.05%-0.15% of the polysaccharide concentrate mass. React at pH 5.0-6.0 at room temperature in the dark for 2-3 hours.

[0097] S3. Dialyze to remove impurities, then freeze dry or proceed directly to step six.

[0098] Instead of grafting L-serine small molecules separately, hyaluronic acid oligosaccharides (Mw=3-10 kDa) and L-serine are first linked by ester bonds to form amphiphilic group beads, and then grafted onto the polysaccharide backbone as a whole. This structure of large and small water droplets not only has stronger moisture absorption capacity, but also pre-constructs recognition anchors for subsequent assembly with sodium polyglutamate supramolecularly. The hyaluronic acid oligosaccharide segments can form a stronger complementary hydrogen bond network with sodium polyglutamate.

[0099] Alternatively, step five can be replaced with:

[0100] S1. First, add 0.3% by mass of small molecule chitosan oligosaccharide (introducing amino anchor points) to the polysaccharide concentrate and premix for 30 min; at the same time, pre-condense hyaluronic acid oligosaccharide (Mw=6kDa) and L-serine in a molar ratio of 1:3 with a trace amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride for 30 min to form hyaluronic acid oligosaccharide-L-serine beaded conjugate.

[0101] S2. Add the hyaluronic acid oligosaccharide-L-serine bead conjugate to the polysaccharide concentrate at a ratio of 2% of the total mass of the polysaccharide.

[0102] S3. Add transglutaminase (1200 U / g polysaccharide) and react at pH 6.2 and 48℃ for 5 h;

[0103] S4, enzyme inactivation at 80℃ for 10 min, dialyzed in a 3500Da dialysis bag for 48 h, to obtain enzyme-modified polysaccharide solution by beading.

[0104] Enzymatic biocatalysis was used to enhance the moisturizing properties of Inonotus obliquus polysaccharides, avoiding potential safety controversies surrounding chemical condensing agents. Simultaneously, hyaluronic acid oligosaccharide-L-serine beads were used as grafting units to preconstruct a three-tiered moisturizing topology of small molecule water absorption, oligosaccharide water storage, and polysaccharide film formation and water locking in a single molecule. This provided enhanced hydrogen bond anchors and bioactivity for subsequent supramolecular self-assembly, achieving a leap from chemical modification to biofunctionalization.

[0105] Example 4

[0106] The only difference between this embodiment and Embodiment 1 is that step six is ​​replaced with:

[0107] S1. Preparation of the coordination solution of epidermal succinate gallate and zinc ions: Dissolve epidermal succinate gallate in deionized water to form a solution with a concentration of 0.5%-1%, and add 0.2% ZnSO4 solution in equal molar ratio dropwise. Stir for 30 min to form a solution of epidermal succinate gallate and zinc coordination oligomers.

[0108] S2. Mix L-serine-modified polysaccharide solution with sodium polyglutamate at a ratio of 1:1, add 5%-10% of the above-mentioned epidermal cadherin gallate and zinc coordination oligomer solution (by mass of L-serine-modified polysaccharide solution), and adjust the pH to 6.0.

[0109] S3, stir and assemble at 40℃ for 3 h. During this period, the epidermal guarcatechin gallate and zinc coordination oligomers are embedded into the network through multi-point hydrogen bonds between galloyl groups and polysaccharide hydroxyl groups. At the same time, metal coordination bonds form strong physical cross-linking points between adjacent chains. Freeze-dry in a light-protected environment and package with nitrogen filling.

[0110] By introducing epidermal cadherin gallate and trace zinc ions into the self-assembly system, a second physical cross-linking point is formed in the supramolecular network through polyphenol-metal coordination, which at the same time endows the composition with strong antioxidant activity, thus protecting the skin barrier from damage caused by environmental oxidative stress while moisturizing.

[0111] Alternatively, step six can be replaced with:

[0112] S1 and L-serine modified polysaccharide solutions were first pre-assembled at 35℃ and pH 5.5 for 1 h to form core prepolymers;

[0113] S2, add sugar isomers, ectoine, pre-prepared epidermal catechin gallate and zinc ion coordination oligomer (accounting for 6% of the total mass of polysaccharides), stir for 30 min to penetrate into the core;

[0114] S3. Slowly add sodium polyglutamate (L-serine modified polysaccharide solution: sodium polyglutamate = 1:0.6), adjust pH to 6.3, and stir and assemble at 42℃ for 3.5 h. The epidermal echinocatechin gallate and zinc coordination bonds form enhanced physical cross-links between the shell layers.

[0115] S4. Cool the assembly solution to 2°C and stir with N2 at 50 mL / min for 4 min to form a microbubble suspension.

[0116] S5, -40℃ rapid pre-freezing, vacuum freeze-drying for 36 h, N2 filling and packaging.

[0117] By introducing natural polyphenol-metal coordination chemistry into the supramolecular self-assembly system of polysaccharides and peptides, dual physical cross-linking of the network, namely hydrogen bond cross-linking and metal coordination cross-linking, is achieved without the use of any chemical cross-linking agent. At the same time, the spatiotemporal release sequence of active factors is precisely regulated by sequential core-shell assembly, and the resolution behavior of traditional freeze-dried powder is completely changed by creating pores with gas templates.

[0118] Example 5

[0119] A polysaccharide from Inonotus obliquus with long-lasting moisturizing effects is prepared by the preparation method described in Example 1 above; or, the L-serine-modified polysaccharide solution obtained in step five of Example 1 is emulsified into microdroplets by a pulsed electric field-assisted membrane, and then passed into a supercritical CO2 crystallization vessel and treated at a temperature of 35-40℃ and a pressure of 10-15 MPa for 1-3 h, and the porous core-shell structured nanospheres with a particle size of 100-500 nm are obtained after depressurization.

[0120] Further, the polysaccharide exhibits a moisture absorption rate of no less than 35% and a water evaporation half-life of no less than 48 hours at 24 hours under conditions of 80% relative humidity and 25°C; a moisture retention rate of no less than 42% and a transdermal water loss inhibition rate of no less than 45% at 40% relative humidity and 30°C.

[0121] Example 6

[0122] An application of the Inonotus obliquus polysaccharide with long-lasting moisturizing effect described in Example 5 above includes its application in cosmetics; the amount added is 3%-10% of the total mass of the cosmetic.

[0123] I. Testing Basis and Standards

[0124] This experimental procedure strictly follows the following currently effective national / industry standards, as detailed in Table 1:

[0125] Table 1

[0126] Test Project According to the standard Hygroscopicity test of chemical products GB / T 26374-2010 In vitro cytotoxicity test ISO 10993-5:2009

[0127] II. Experimental Objective

[0128] This experiment aims to systematically verify the innovative progressive path of the six-step process of this invention, which consists of "preservation of the entire long-chain framework - breakthrough of the moisture absorption ceiling - extension of the moisturizing time window". By setting up a stratified control group, the specific contribution of each step of the technical feature combination to the long-lasting moisturizing performance is revealed step by step, and a comprehensive comparison is made with existing technical benchmarks.

[0129] III. Experimental Samples and Group Design

[0130] 3.1 Sample preparation and numbering (see Table 2 for details)

[0131] Table 2

[0132] Sample number Process coverage Corresponding technical aspects Purpose of setting Sample A Hot water extraction after defatting Degreasing only in step one Simulating traditional processes as a benchmark for natural ungraded crude polysaccharides Sample B Steps one through four Degreasing + immobilized enzyme weak ultrasonic extraction + EBA purification + two-stage ultrafiltration fractionation Verify the contribution of gentle extraction and precise grading to basic moisturizing. Sample C Steps one through five L-serine grafting modification was performed on sample B. Verifying the core effect of chemical modification in breaking through the limits of natural moisture absorption. Sample D Steps one through six (i.e., Example 1) Based on sample C, supramolecular self-assembly was performed, and a cooperation factor was loaded. Verify the ability of supramolecular self-assembly sustained release to extend the moisturizing time window. glycerin Analytical Pure - General Cosmetic Moisturizer Reference Standard Sodium hyaluronate Mw=1000-1500kDa - The industry's current benchmark for high-end moisturizing raw materials blank Deionized water / blank matrix emulsion - Baseline control

[0133] Solution preparation: All solid samples were prepared into 1% (w / v) solutions (or suspensions) with deionized water.

[0134] 3.2 Test Indicators and Group Comparison (See Table 3 for details)

[0135] Table 3

[0136] Experiment number Test Project Main comparison group Validating creativity Experiment 1 In vitro moisturizing rate (gravimetric method) Comparison of Sample A-Sample D with Glycerin / Sodium Hyaluronate The overall effect of the six-step process and the progressive contribution of each link Experiment 2 Moisture absorption rate Comparison of Sample B with Sample C with Sample D; Comparison of Glycerin / Sodium Hyaluronate; Low Humidity Comparison Step 5: Breaking Through the Natural Moisture Absorption Ceiling Experiment 3 Cytotoxicity Sample A - Sample D Safety assurance through green processes throughout the entire process

[0137] IV. Experiment 1: In vitro moisturizing rate test (gravimetric method)

[0138] 4.1 Test Basis

[0139] QB / T 4256-2011; refer to GB / T 26374-2010, the method for determining the hygroscopicity of humectants.

[0140] 4.2 Instruments and Conditions

[0141] Constant temperature and humidity chamber (30±1℃, RH=40±2%)

[0142] Analytical balance (accuracy 0.0001 g)

[0143] 3M breathable tape (3M) TM Transpore TM Surgical Tape)

[0144] Micropipette (100μL)

[0145] 4.3 Operating Procedures

[0146] Cut the breathable tape into 5 cm × 5 cm pieces, equilibrate it in a constant temperature and humidity chamber for 2 h, and weigh it to obtain M0.

[0147] Accurately transfer 100 μL of sample solution and spread it evenly (coating amount 3.0 mg / cm).2 Immediately weigh to obtain M 0h (0h mass);

[0148] The coated tape was placed in a constant temperature and humidity chamber and weighed at 2 h, 4 h, 8 h, and 24 h to obtain M. t ;

[0149] calculate:

[0150]

[0151] Each sample was replicated in six parts, and the results are expressed as Mean ± SD.

[0152] 4.4 Experimental Results

[0153] Table 4: Results of in vitro moisturizing rate test (Mean±SD, n=6)

[0154] Test sample 2-hour hydration rate (%) 4-hour hydration rate (%) 8-hour hydration rate (%) 24-hour hydration rate (%) Blank (deionized water) 45.8 18.2 8.7 3.1 Glycerin (1%) 68.5 42.6 28.3 15.2 Sodium hyaluronate (1%) 72.3 50.8 35.4 22.6 Sample A 58.2 36.4 22.5 14.8 Sample B 70.5 56.8 38.5 18.9 Sample C 82.6 68.7 52.4 37.2 Sample D 89.3 78.4 65.1 46.5

[0155] Table 5: Stepwise Progressive Effect and Relative Improvement of 24-hour Moisturizing Rate

[0156] control group 24-hour moisture retention rate difference relative improvement Revealed technical contributions Sample B vs. Sample A 18.9% vs. 14.8% +27.7% Gentle extraction of bone structure preservation and precise graded removal of ineffective fragments Sample C vs. Sample B 37.2% vs. 18.9% +96.8% L-serine grafting breaks through the limitations of natural moisture absorption. Sample D vs. Sample C 46.5% vs. 37.2% +24.7% Supramolecular self-assembled sustained-release system extends the moisturizing time window Sample D for glycerol 46.5% vs. 15.2% +205.9% The overall technology of this invention surpasses that of general moisturizers. Sample D for sodium hyaluronate 46.5% vs. 22.6% +105.9% Significantly surpassing the current benchmark in the high-end moisturizing raw material industry

[0157] pass Figure 1 As can be seen from Tables 4 and 5:

[0158] 1. The obvious effect of preserving the framework: Compared with the traditional hot water extraction of sample A, sample B has a 27.7% higher 24-hour moisture retention rate, which confirms that the gentle extraction strategy of "immobilized enzyme and weak ultrasound" can effectively preserve the film-forming framework of polysaccharide long chains. After the precise removal of macromolecular colloids and small molecule fragments by the combination of 300kDa and 50kDa ultrafiltration, the 50-200kDa fractions can indeed take into account both penetration and film formation, forming a double-layer moisturizing structure of "deep water storage and surface water locking".

[0159] 2. A qualitative breakthrough through chemical modification: Compared to the unmodified sample B, sample C exhibited a 96.8% increase in moisture retention, almost doubling. Even in a dry environment (RH=40%), it maintained a high moisture retention rate of 37.2%, completely breaking through the performance bottleneck of natural polysaccharides relying solely on passive moisture absorption through surface hydroxyl groups. The additional -OH and -COOH groups introduced by L-serine fundamentally transformed the mechanism of polysaccharide from passive moisture absorption via hydrogen bonding to active chemical capture of water molecules.

[0160] 3. Synergistic extension of physical modeling: Based on sample C, sample D further increased the 24-hour moisturizing rate to 46.5% through supramolecular self-assembly, proving that the physical cross-linking network can effectively delay the release of moisturizing active factors and water evaporation, forming a relay-style long-lasting moisturizing effect.

[0161] V. Experiment 2: Moisture Absorption Rate Test

[0162] 5.1 Test Basis

[0163] QB / T 4256-2011 Gravimetric method and GB / T 26374-2010.

[0164] 5.2 Instruments and Conditions

[0165] Weighing bottle (φ40 mm);

[0166] Dryer (containing a saturated ammonium sulfate solution to maintain RH of approximately 80%, or a saturated potassium carbonate solution to maintain RH of approximately 43%).

[0167] Analytical balance (accuracy 0.0001 g);

[0168] Constant temperature environment (25±1℃);

[0169] 5.3 Operating Procedures

[0170] The sample was dried to constant weight in a vacuum oven at 60℃, cooled in a desiccator, and weighed to obtain W0. The sample weighing bottle (with the cap off) was placed in a desiccator with RH=80% or RH=43% and kept at a constant temperature of 25℃. The bottle was removed at 2 h, 4 h, 8 h, and 24 h, capped, and weighed quickly to obtain W0. t ;calculate:

[0171]

[0172] Each sample was replicated in six parts.

[0173] 5.4 Experimental Results (See Table 6 for details)

[0174] Table 6: Moisture absorption rate of each sample under RH=80% environment (Mean±SD, n=6)

[0175] Test sample 2-hour moisture absorption rate (%) 4-hour moisture absorption rate (%) 8-hour moisture absorption rate (%) 24-hour moisture absorption rate (%) glycerin 4.5 8.2 14.6 27.8 Sodium hyaluronate 6.8 12.4 20.3 35.2 Sample B 2.8 5.3 9.6 15.3 Sample C 7.2 15.8 26.5 38.1 Sample D 6.8 14.5 24.3 35.8

[0176] Table 7: Comparison of 24-hour moisture absorption rate under different humidity conditions (Mean±SD, n=6)

[0177] Test sample 24-hour moisture absorption rate (%) RH=80% 24-hour moisture absorption rate (%) RH = 43% glycerin 27.8 12.5 Sample B 15.3 4.8 Sample C 38.1 18.6

[0178] pass Figure 2 , Figure 3 From Tables 6 and 7, we can see that:

[0179] 1. The clear limitations of natural polysaccharides: The moisture absorption rate of unmodified polysaccharides (sample B) was only 15.3% after 24 hours at RH=80%, which is much lower than that of sodium hyaluronate (35.2%); when the humidity dropped to RH=43%, the moisture absorption rate plummeted to 4.8% - which reveals the physical nature of natural polysaccharides passively absorbing moisture by relying on the ambient humidity.

[0180] 2. The transformative effect of L-serine modification: Sample C exhibited a moisture absorption rate of 38.1% at RH=80%, a 149% increase compared to sample B. Even at a dry environment with RH=43%, its moisture absorption rate of 18.6% was still higher than the overall moisture absorption curve of sample B under high humidity (80%) (15.3%). More importantly, at RH=43%, the moisture absorption rate of sample C (18.6%) exceeded that of glycerol (12.5%), demonstrating that after L-serine grafting, the polysaccharide transformed from passively driven moisture absorption to actively chemically capturing water molecules. This is a fundamental breakthrough, distinct from any process optimization relying solely on physical extraction.

[0181] 3. Post-assembly coordination and balance: The moisture absorption rate of sample D is slightly lower than that of sample C (35.8%-38.1%), which confirms the structural fact that some hydrophilic groups participate in the supramolecular hydrogen bond network. This is a functional regulation that sacrifices a small amount of moisture absorption to achieve a significant extension of the moisturizing time window.

[0182] VI. Experiment 3: Safety Verification—In vitro cytotoxicity test

[0183] 6.1 Test Basis

[0184] ISO 10993-5:2009 Medical devices - Biological evaluation - Part 5: In vitro cytotoxicity tests.

[0185] 6.2 Methods

[0186] Cell line: L929 mouse fibroblasts;

[0187] Method: MTT assay;

[0188] Sample concentration gradient: 0.1 mg / mL, 1.0 mg / mL, 10.0 mg / mL;

[0189] Incubation time: 24 hours;

[0190] Judgment criteria: Cell viability ≥70% indicates no potential cytotoxicity;

[0191] 6.3 Experimental Results (See Table 8 for details)

[0192] Table 8

[0193] Sample concentration (mg / mL) Survival rate of sample A (%) Survival rate of sample B (%) Survival rate of sample C (%) Survival rate of sample D (%) 0.1 98.5 98.6 98.8 99.1 1.0 96.8 96.9 97.3 97.8 10.0 94.1 94.2 95.1 94.8

[0194] Through Table 8 and Figure 4 It can be seen that at the highest test concentration of 10 mg / mL, the cell survival rate of all samples is ≥94%, which is far higher than the threshold of no potential cytotoxicity (70%) specified in ISO 10993-5. This indicates that the entire series of samples prepared by the green process of this invention (no chloroform, no strong acids or bases, no EDC residues and fully removed by dialysis) are all within the scope of non-cytotoxic safety and can be used in sensitive skin care products.

[0195] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0196] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing Inonotus obliquus polysaccharide with long-lasting moisturizing effects, characterized in that, Includes the following steps: Step 1: Crush the dried Inonotus obliquus sclerotia and degrease them at least once with a 75% ethanol aqueous solution at a temperature of 25-30℃ to obtain degreased bacterial powder. Step 2: The defatted bacteria powder obtained in Step 1 is loaded into a continuous flow fixed bed reactor, and a composite enzymatic hydrolysate containing immobilized cellulase and immobilized pectinase is introduced. Targeted enzymatic hydrolysis is carried out under the conditions of pH 4.5-5.5 and temperature 45-55℃, while low-frequency weak ultrasound with a frequency of 25 kHz and a power of 120-150 W is applied simultaneously for 1-2 h. After the enzymatic hydrolysis is completed, the bacteria are inactivated, and solid-liquid separation is performed to obtain crude polysaccharide extract. Step 3: Pump the crude polysaccharide extract obtained in Step 2 into an expanded bed adsorption column filled with magnetic composite microspheres from bottom to top at a flow rate of 1-3 BV / h. Under the condition of a bed expansion rate of 20%-40%, proteins, pigments and microparticles are removed simultaneously. Collect the effluent to obtain the refined polysaccharide extract. Step 4: Pass the purified polysaccharide solution obtained in Step 3 through ultrafiltration membranes with molecular weight cutoffs of 300 kDa and 50 kDa in sequence, operating at a pressure of 0.15-0.25 MPa and a temperature of 25-30℃. Collect and concentrate the polysaccharide fractions with molecular weights between 50 kDa and 200 kDa to obtain a polysaccharide concentrate. Step 5: Add 0.5%-2% L-serine and 0.1%-0.5% 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to the polysaccharide concentrate obtained in Step 4. React at pH 5.0-6.0, room temperature and in the dark for 2-4 hours. Dialyze to remove unreacted reagents to obtain L-serine-modified polysaccharide solution. Step 6: Mix the L-serine-modified polysaccharide solution obtained in Step 5 with sodium polyglutamate at a mass ratio of 1:0.3-3, adjust the pH to 5.5-7.0, perform supramolecular self-assembly at 30-50℃, and then freeze-dry to obtain Inonotus obliquus polysaccharide with long-lasting moisturizing effect.

2. The method for preparing the Inonotus obliquus polysaccharide with long-lasting moisturizing effect according to claim 1, characterized in that, In step two, both the immobilized cellulase and the immobilized pectinase are immobilized on an aminoened silica gel carrier using glutaraldehyde cross-linking, and the activity ratio of cellulase to pectinase in the composite enzymatic hydrolysate is 1:2-4.

3. The method for preparing the Inonotus obliquus polysaccharide with long-lasting moisturizing effect according to claim 1, characterized in that, In step three, the magnetic composite microspheres are sodium alginate-coated iron oxide microspheres with a particle size of 50-150 μm, and the inner diameter to height ratio of the expanded bed adsorption column is 1:5-10.

4. The method for preparing the Inonotus obliquus polysaccharide with long-lasting moisturizing effect according to claim 1, characterized in that, In step four, the ultrafiltration membrane is made of modified polyethersulfone membrane.

5. The method for preparing the Inonotus obliquus polysaccharide with long-lasting moisturizing effect according to claim 1, characterized in that, In step six, saccharide isomers and / or ectoine are added as synergistic moisturizing active molecules, with the amount added being 10%-50% of the mass of the L-serine-modified polysaccharide solution.

6. The method for preparing the Inonotus obliquus polysaccharide with long-lasting moisturizing effect according to claim 1, characterized in that, Replace step one with: S1. The sclerotium of Inonotus obliquus was cut into 1-2 cm pieces, placed in liquid nitrogen at -196℃, frozen for 10 min, and then ultra-finely pulverized at -20℃ or below until the particle size D was reached. 90 Microbial powder with a particle size not exceeding 50μm is obtained. S2. Wet the ultrafine bacterial powder with a 30% (v / v) ethanol aqueous solution until the water content is 25-30%, and equilibrate in a sealed container for 2 hours. S3. The moistened mycelium powder is put into the flash expansion machine and heated to 120-130℃ under a pressure of 0.8-1.2 MPa and held for 30 seconds. The pressure is then released to normal pressure. The volume of the mycelium powder expands by 2-3 times, forming a porous and loose structure, and the flash-expanded mycelium powder is obtained. S4. The porous activated flash-expanding bacteria powder was added to a pre-enzymatic hydrolysate containing 0.03% w / v free β-glucanase and 0.02% w / v cutinase and treated at 35°C for 1.5 h. S5. After filtration, the bacterial powder is directly added to a 75% (v / v) ethanol aqueous solution and cold-soaked at 25°C for 8 hours to remove fat; then dried at low temperature to obtain activated-pre-enzymatic hydrolysis-degreased bacterial powder.

7. The method for preparing the Inonotus obliquus polysaccharide with long-lasting moisturizing effect according to claim 1, characterized in that, Replace step five with: S1. Add 0.3% (by mass) of small molecule chitosan oligosaccharide to the polysaccharide concentrate and premix for 30 min. At the same time, pre-condense hyaluronic acid oligosaccharide and L-serine in a molar ratio of 1:3 with a trace amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride for 30 min to form hyaluronic acid oligosaccharide-L-serine beaded conjugate. S2. Add the hyaluronic acid oligosaccharide-L-serine bead conjugate to the polysaccharide concentrate at a ratio of 2% of the total mass of the polysaccharide. S3. Add transglutaminase and react at pH 6.2 and 48℃ for 5 h; S4, enzyme inactivation at 80℃ for 10 min, dialyzed in a 3500Da dialysis bag for 48 h, to obtain enzyme-modified polysaccharide solution.

8. The method for preparing the Inonotus obliquus polysaccharide with long-lasting moisturizing effect according to claim 1, characterized in that, Replace step six with: S1 and L-serine modified polysaccharide solutions were first pre-assembled at 35℃ and pH 5.5 for 1 h to form core prepolymers; S2, add sugar isomers, ectoine, pre-prepared epidermal galactoate gallate and zinc ion coordination oligomer, stir for 30 min to penetrate into the core; S3. Slowly add sodium polyglutamate (L-serine modified polysaccharide solution: sodium polyglutamate = 1:0.6), adjust pH to 6.3, and stir and assemble at 42℃ for 3.5 h. The epidermal echinocatechin gallate and zinc coordination bonds form enhanced physical cross-links between the shell layers. S4. Cool the assembly solution to 2°C and stir with N2 at 50 mL / min for 4 min to form a microbubble suspension. S5, -40℃ rapid pre-freezing, vacuum freeze-drying for 36 h, N2 filling and packaging.

9. A polysaccharide from Inonotus obliquus with long-lasting moisturizing effects, characterized in that, It is prepared by any one of the preparation methods of claims 1-5; or, the L-serine-modified polysaccharide solution obtained in step five of claim 1 is emulsified by a pulsed electric field-assisted membrane to form microdroplets, and then introduced into a supercritical CO2 crystallization vessel and treated at a temperature of 35-40℃ and a pressure of 10-15 MPa for 1-3 h, and the porous core-shell structured nanospheres with a particle size of 100-500 nm are obtained by depressurization.

10. An application of a polysaccharide from Inonotus obliquus with long-lasting moisturizing effects, characterized in that, This includes the application of the long-lasting moisturizing effect of Inonotus obliquus polysaccharide as described in claim 9 in cosmetics; the amount added is 3%-10% of the total mass of the cosmetic.

Images