A method for synergistically extracting peach gum polysaccharide through mixed fermentation, peach gum polysaccharide and application thereof

Abstract

The application provides a method for synergistically extracting peach gum polysaccharide through mixed bacterial fermentation, peach gum polysaccharide and application thereof, and relates to the technical field of bacterial fermentation. The method comprises the following steps: inoculating a mixed bacterial seed solution into a peach gum powder solution and performing fermentation culture in a shaking table to obtain a fermentation liquor containing peach gum polysaccharide; wherein the mixed bacterial seed solution comprises yeast and schizophyllan, and the inoculation amount of the mixed bacterial seed solution is 5-25 vol%; wherein the pH of the fermentation culture is 2-7, the temperature of the fermentation culture is 25-43 DEG C, the rotation speed of the shaking table for the fermentation culture is 170-230 r / min, and the time of the fermentation culture is 12-72 h. The application provides a fermentation method capable of improving the anti-inflammatory, antioxidant, moisturizing, wound healing promoting, anti-photoaging and other abilities of the extracted peach gum polysaccharide.

Description

Technical Field

[0001] This invention relates to the technical fields of skin pharmacology and cosmetics, and in particular to a method for synergistic extraction of peach gum polysaccharide by mixed bacterial fermentation, peach gum polysaccharide and its applications. Background Technology

[0002] As the largest organ in the human body, the skin is directly exposed to the external environment and is susceptible to aging due to factors such as ultraviolet radiation. UVB radiation is a major cause of photoaging, inducing skin cells to produce excessive reactive oxygen species, triggering oxidative stress and inflammatory responses, leading to collagen degradation and elastic fiber degeneration, ultimately manifesting as rough, sagging skin and wrinkles. Therefore, finding safe and effective anti-photoaging active substances has become a research hotspot in the fields of skin pharmacology and cosmetics.

[0003] The main active component of peach gum is peach gum polysaccharide (PGP), which accounts for more than 80% of the dry weight of peach gum and is one of the natural resources with the highest purity of plant-derived polysaccharides to date. PGP is an acidic heteropolysaccharide, and its monosaccharide composition mainly includes arabinose, galactose, xylose, uronic acid, mannose, and a small amount of rhamnose, with arabinose, galactose, and glucuronic acid accounting for a relatively high proportion. In addition, PGP also contains a small amount of protein and trace elements such as potassium, calcium, magnesium, iron, and manganese. Due to its unique chemical structure and composition, PGP exhibits significant broad-spectrum antibacterial activity. Studies have shown that PGP has significant inhibitory effects on Gram-positive bacteria (such as Bacillus subtilis and Staphylococcus aureus) and Gram-negative bacteria (such as Escherichia coli); PGP and its derivatives (such as peach gum oligosaccharide, PGDO) have significant antioxidant activity, mainly manifested in the effective scavenging of various free radicals, the provision of reducing capacity, and the regulation of the antioxidant defense system in organisms. Peach gum polysaccharide has pharmacological activities such as anti-inflammatory, antioxidant, moisturizing and wound healing promotion, but its anti-skin photoaging effect and mechanism still lack systematic research.

[0004] Fermentation, as a green and efficient biotransformation method, can improve the physicochemical properties and biological activity of natural polysaccharides. Modern fermentation technology has achieved breakthroughs and developments on the basis of inheriting tradition. According to the type of culture medium, modern fermentation is mainly divided into three categories: liquid fermentation, solid fermentation and two-way fermentation of medicinal fungi; according to the type of microorganism, it can be divided into single-strain fermentation, mixed-strain fermentation, etc. Compared with traditional processing methods, fermentation technology has many advantages: (1) Fermentation can significantly enhance the bioavailability of medicinal components; (2) Specific active enzymes produced by microorganisms can perform directional degradation and structural modification of substrate macromolecules, generating new products with lower molecular weight, better solubility and higher bioavailability; (3) Fermentation process can produce new active substances. While using the substrate for growth, microorganisms synthesize a variety of secondary metabolites through their own metabolic pathways, or modify the structure of substrate components to generate new compounds; (4) Through the biotransformation of microorganisms, the toxic components in traditional Chinese medicine can undergo structural changes and be transformed into low-toxicity or non-toxic metabolites, achieving the purpose of reducing toxicity and increasing efficacy.

[0005] Therefore, there is an urgent need to provide a fermentation method that can enhance the anti-inflammatory, antioxidant, moisturizing, wound-healing, and anti-photoaging properties of the extracted peach gum polysaccharide.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] One of the objectives of this invention is to provide a method for synergistic extraction of peach gum polysaccharide through mixed microbial fermentation, the peach gum polysaccharide itself, and its applications. This invention solves the common problems in existing peach gum extraction methods, such as insufficient retention of active ingredients, limited structural modification, and limited enhancement of biological activity. At the same time, it can also solve the problem that the prepared peach gum polysaccharide is difficult to achieve both high activity retention and functional enhancement, and overcome the defects of insufficient anti-photoaging efficacy caused by traditional extraction processes.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a method for synergistic extraction of peach gum polysaccharides through mixed-culture fermentation, the method comprising: A mixed bacterial seed liquid was inoculated into a peach gum powder solution and fermented in a shaker to obtain a fermentation broth containing peach gum polysaccharides. The mixed seed culture includes yeast and Schizophyllum commune, and the inoculation amount of the mixed seed culture is 5-25 vol%. The fermentation culture is conducted at a pH of 4-7, at a temperature of 28-43°C, on a shaking incubator at a speed of 170-230 r / min, and for a duration of 0-72 h.

[0009] Furthermore, the inoculation volume of the mixed seed culture is 19-21 vol%.

[0010] Furthermore, the pH of the fermentation culture is 3.8~4.2, the temperature of the fermentation culture is 27~29℃, the shaking speed of the fermentation culture is 185~195 r / min, and the fermentation culture time is 23~25 h.

[0011] Furthermore, the method for preparing the mixed bacterial seed solution includes the following steps: Revival and activation of Schizophyllum commune: Schizophyllum commune strains were inoculated into solid culture medium for activation to obtain activated mycelia; the activated mycelia were inoculated into primary liquid culture medium for a first shaking culture, and after being dispersed, they were inoculated into secondary liquid culture medium for a second shaking culture to obtain Schizophyllum commune fermentation broth; Revival and activation of Saccharomyces cerevisiae: Saccharomyces cerevisiae dry powder and water were mixed for the first activation to obtain an activated bacterial solution; the activated bacterial solution was inoculated into a solid culture medium for the second activation to obtain activated single colonies; the activated single colonies were inoculated into a liquid culture medium and cultured with shaking to obtain Saccharomyces cerevisiae seed culture; Mixed culture: The brewer's yeast seed liquid is centrifuged and the brewer's yeast cell precipitate is collected; the brewer's yeast cell precipitate is mixed with the Schizophyllum commune fermentation broth to obtain the mixed culture seed liquid.

[0012] Furthermore, in the process of reviving and activating *Schizophyllum commune*: the solid culture medium is PDA plate culture medium; the primary liquid culture medium is *Schizophyllum commune* liquid culture medium; and the secondary liquid culture medium is *Schizophyllum commune* liquid culture medium.

[0013] Furthermore, during the revival and activation process of the *Schizophyllum commune*: the activation temperature is 27-29°C, and the activation time is 4-6 days; Furthermore, in the process of reviving and activating the *Schizophyllum commune*: during the first shaking culture, the inoculation amount of the activated mycelium is 1-2 shovels; the temperature of the first shaking culture is 26-30℃; the rotation speed of the first shaking culture is 165-175 r / min; and the time of the first shaking culture is 3-4 days. Furthermore, during the resuscitation and activation of the *Schizophyllum commune*: during the second shaking culture, the inoculum volume is 9-11 vol%; the temperature of the second shaking is 26-30℃; the rotation speed of the first shaking culture is 185-195 r / min; and the time of the first shaking culture is 22-30 h.

[0014] Furthermore, in the process of reviving and activating the Saccharomyces cerevisiae: the solid culture medium is YEPD agar medium; the liquid culture medium is YEPD liquid medium.

[0015] Furthermore, in the process of reviving and activating the brewing yeast: during the first activation process, the mass-to-volume ratio of the brewing yeast dry powder to water is 1 g:(8~10) mL; the temperature of the first activation is 36~38℃, and the time of the first activation is 18~25 min.

[0016] Furthermore, during the revival and activation process of the brewing yeast: the temperature for the second activation is 28~32℃, and the time for the second activation is 15~20 h.

[0017] Furthermore, during the revival and activation of the brewing yeast: the temperature of the shaking culture is 28~32℃, the shaking speed is 180~200 r / min, and the shaking culture time is 18~24 h.

[0018] Furthermore, during the mixed culture process: the centrifugation temperature is 3~5℃, the centrifugation speed is 7000~10000 r / min, and the centrifugation time is 10~15 min.

[0019] Furthermore, during the mixed culture process, the volume ratio of the brewing yeast seed liquid to the Schizophyllum commune fermentation liquid is (0.9~1.1):(0.9~1.1).

[0020] Furthermore, the method also includes the following post-processing steps: The fermentation broth containing peach gum polysaccharide was centrifuged to obtain the supernatant. The supernatant was subjected to protein removal treatment to obtain a deproteinized polysaccharide solution; The deproteinized polysaccharide solution was subjected to dialysis to obtain a dialyzed polysaccharide solution; The polysaccharide solution after dialysis was subjected to alcohol precipitation, the precipitate was collected and dried to obtain fermented peach gum crude polysaccharide.

[0021] Furthermore, during the post-processing: the centrifugation speed is 9000~10000 r / min, and the centrifugation speed is 10~30 min.

[0022] Furthermore, in the post-processing: the protein removal process is performed using the Sevage method.

[0023] Furthermore, in the post-processing: the dialysis treatment uses a dialysis bag with a molecular weight cutoff of 3500 Da.

[0024] Furthermore, in the post-processing: the alcohol precipitation treatment uses a 90~100 vol% ethanol solution.

[0025] Furthermore, the method also includes the following purification steps: The fermented peach gum crude polysaccharide was subjected to ion exchange column chromatography to obtain refined peach gum polysaccharide.

[0026] In a second aspect, the present invention provides a peach gum polysaccharide, which is prepared by the method of mixed bacterial fermentation and synergistic extraction of peach gum polysaccharide as described in the first aspect; Furthermore, the peach gum polysaccharide includes fermented peach gum crude polysaccharide and / or refined peach gum polysaccharide.

[0027] Thirdly, the present invention provides a method for preparing peach gum polysaccharide by synergistic extraction of peach gum polysaccharide through mixed fermentation as described in the first aspect, or the application of peach gum polysaccharide as described in the second aspect in the preparation of anti-inflammatory products, antioxidant products, moisturizing products, wound healing products, or anti-photoaging products.

[0028] Compared with the prior art, the present invention has the following beneficial effects: (1) The method described in this invention uses a mixed fermentation of yeast and Schizophyllum commune to synergistically extract peach gum polysaccharide. The synergistic effect is achieved through the metabolic complementarity of yeast and Schizophyllum commune: yeast secretes glycosidases to promote polysaccharide dissolution, while Schizophyllum commune secretes cellulase, laccase, etc. to enhance substrate degradation and structural modification, thereby jointly increasing the density of exposed active sites of peach gum polysaccharide; the mixed interaction can also inhibit the growth of miscellaneous bacteria, stabilize the fermentation microenvironment, and significantly improve the bioactivity uniformity and functional stability of crude polysaccharide; the obtained fermentation broth has excellent antioxidant, anti-inflammatory and skin film-forming abilities without deep purification, providing a high-activity raw material basis for subsequent direct application or simplified preparation; compared with unfermented peach gum polysaccharide, the fermented peach gum crude polysaccharide obtained in this invention has a significantly better effect on improving UVB-induced photoaging of mouse skin at the same dose; (2) The method described in this invention precisely controls key parameters such as pH, temperature, shaking speed and time to ensure that yeast and Schizophyllum commune are in the optimal window for synergistic metabolism: ensuring efficient secretion and complementary effects of active enzyme systems of the two strains, while avoiding polysaccharide degradation or by-product accumulation caused by excessive stress; after optimization by single-factor experiments, the optimal fermentation conditions were determined to be pH 4.0, temperature 28℃, shaking speed 190 r / min, inoculum amount 20%, and fermentation time 24 h, under which the polysaccharide yield reached the highest value; the synergistic optimization of parameters ensures that peach gum polysaccharide retains the high molecular skeleton while achieving appropriate exposure of active groups and optimization of spatial conformation, significantly improving its antioxidant, anti-inflammatory and skin affinity properties; the obtained crude polysaccharide has good batch stability and high biological activity; (3) The fermented peach gum crude polysaccharide obtained by the method of the present invention through a certain post-processing method has both high biological activity and good application adaptability. Its mixed fermentation endows it with abundant exposed active sites such as hydroxyl and carboxyl groups, which significantly enhances its antioxidant and anti-inflammatory capabilities. The moderately modified molecular structure improves water solubility and skin retention, resulting in uniform film formation and firm adhesion. While retaining the natural polysaccharide skeleton, it synergistically enhances the multi-target regulation ability against UVB-induced oxidative stress, inflammatory response and collagen metabolism imbalance. Animal experiments show that fermented peach gum crude polysaccharide can dose-dependently increase the skin moisture content and elasticity of photoaged mice, reduce wrinkle formation, restore the thickness of the epidermis and dermis, increase collagen fiber content, and downregulate the expression of the aging marker protein p21. The method is simple and batch-stable, and can be directly used for the development of topical preparations, solving the technical bottlenecks of low activity, difficult utilization and single efficacy of natural peach gum polysaccharide. (4) The method of the present invention further purifies the fermented peach gum crude polysaccharide by ion exchange column chromatography to obtain three refined peach gum polysaccharides; among them, PGP1 has a high molecular weight (2164.655 kDa), uniform structure (Mw / Mn=1.584), few protein impurities (8.975 mg / g), and outstanding film-forming and antioxidant activities; PGP2 has complex components and dispersed activity, making it suitable as an auxiliary component; PGP3 has a medium molecular weight, balanced solubility and permeability, and good compatibility potential. The three can cover the multidimensional needs from barrier repair to deep anti-aging, significantly expanding the application of peach gum polysaccharides in functional skin care; in particular, PGP1's high molecular weight skeleton endows it with excellent skin film-forming properties and long-lasting retention ability, strong structural uniformity, and the most prominent antioxidant and anti-inflammatory activities. Moreover, it is easier to target the dermis after transdermal absorption, making it the core active component with the most significant anti-photoaging effect; cell experiments further confirmed that PGP1 (125–500 kDa) has the most significant anti-photoaging effect. The peach gum polysaccharide (7.8–2000 μg / mL) can dose-dependently increase the activity of SOD, GSH-Px, and CAT in UVB-induced HaCaT cells, inhibit the release of IL-1β, IL-6, and TNF-α, and upregulate COL1A1 and downregulate MMP-1 and MMP-9 expression. In addition, the peach gum polysaccharide obtained in this invention has no obvious cytotoxicity to HaCaT cells (cell survival rate is higher than 87% in the concentration range of 7.8–2000 μg / mL), has good biocompatibility, and is suitable for long-term external use. Attached Figure Description

[0029] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0030] Figure 1 The graph shows the effect of different bacterial inoculum amounts provided in Test Example 1 on the content of peach gum polysaccharides obtained from fermentation.

[0031] Figure 2 The graph shows the effect of different fermentation pH values ​​on the content of peach gum polysaccharides obtained from fermentation, as provided in Test Example 1.

[0032] Figure 3 The graph shows the effect of different fermentation temperatures on the content of peach gum polysaccharides obtained from fermentation, as provided in Test Example 1.

[0033] Figure 4 The graph shows the effect of different fermentation speeds on the content of peach gum polysaccharides obtained from fermentation, as provided in Test Example 1.

[0034] Figure 5The graph shows the effect of different fermentation times on the content of peach gum polysaccharides obtained from fermentation, as provided in Test Example 1.

[0035] Figure 6 The skin morphology images of each group of mice provided for test example 2.

[0036] Figure 7 The skin elasticity index of each group of mice provided for test example 2.

[0037] Figure 8 The graphs showing the skin moisture index of each group of mice provided for test example 2.

[0038] Figure 9 HE staining images of mouse skin from each group provided in Test Example 2.

[0039] Figure 10 The graph shows the changes in (a) epidermal thickness and (b) dermal thickness of the mice in each group provided for test example 2 (***p<0.001 vs. model group; ###p<0.001 vs. blank control group).

[0040] Figure 11 The results of Masson staining of mouse skin in each group provided for test example 2 are shown in the figure.

[0041] Figure 12 The results of p21 immunohistochemical staining of mouse skin in each group provided for test example 2.

[0042] Figure 13 The DEAE-52 ion exchange column chromatography elution curve of the peach gum polysaccharide provided in Test Example 3.

[0043] Figure 14 The properties of various purified polysaccharides of peach gum provided in Test Example 3.

[0044] Figure 15A The molecular weight chromatogram of the purified polysaccharide PGP1 from peach gum provided for test example 3.

[0045] Figure 15B The molecular weight chromatogram of the purified peach gum polysaccharide PGP2 provided for test example 3.

[0046] Figure 15C The molecular weight chromatogram of the purified peach gum polysaccharide PGP3 provided for test example 3.

[0047] Figure 16A The absolute molecular weight analysis chart of the purified polysaccharide PGP1 from peach gum provided in Test Example 3.

[0048] Figure 16B The absolute molecular weight analysis chart of the purified polysaccharide PGP2 from peach gum provided in Test Example 3.

[0049] Figure 16C The absolute molecular weight analysis chart of the purified polysaccharide PGP3 from peach gum provided in Test Example 3.

[0050] Figure 17A The curve of the monosaccharide mixed standard provided for test example 3.

[0051] Figure 17B The monosaccharide composition chromatogram of the purified polysaccharide PGP1 provided for test example 3.

[0052] Figure 17C The monosaccharide composition chromatogram of the purified polysaccharide PGP2 provided for test example 3.

[0053] Figure 17D The monosaccharide composition chromatogram of the purified polysaccharide PGP3 provided for test example 3.

[0054] Figure 18A The infrared spectrum of purified polysaccharide PGP1 from peach gum provided in Test Example 3.

[0055] Figure 18B The infrared spectrum of purified polysaccharide PGP2 from peach gum provided in Test Example 3.

[0056] Figure 18C The infrared spectrum of purified peach gum polysaccharide PGP3 provided for test example 3.

[0057] Figure 19A Scanning electron microscope (SEM) images of purified peach gum polysaccharide PGP1 at different magnifications provided for test example 3.

[0058] Figure 19B Scanning electron microscope (SEM) images of purified polysaccharide PGP2 from peach gum at different magnifications provided for test example 3.

[0059] Figure 19C Scanning electron microscope (SEM) images of purified polysaccharide PGP3 from peach gum at different magnifications provided for test example 3.

[0060] Figure 20 The experimental results of Congo Red provided for Test Example 3 are shown in the figure.

[0061] Figure 21 The protein standard curves for each purified polysaccharide of peach gum provided in Test Example 3.

[0062] Figure 22 The effect of different UVB radiation-induced doses on HaCaT cell survival provided in Test Example 4 is shown in the figure.

[0063] Figure 23 The effect of different concentrations of purified peach gum polysaccharides (a) PGP1, (b) PGP2, and (c) PGP3 provided for test example 4 on cell viability.

[0064] Figure 24 The protective effects of different concentrations of purified peach gum polysaccharides (a) PGP1, (b) PGP2, (c) PGP3 and (d) retinoic acid, a positive control agent, on UVB-induced cells provided for test example 4.

[0065] Figure 25 The figure shows the effect of purified peach gum polysaccharide provided in Test Example 5 on (a) SOD, (b) GSH-Px, (c) CAT in HaCaT cells (*p<0.05, **p<0.01, ***p<0.001 vs. model group; ###p<0.01 vs. blank control group).

[0066] Figure 26 The figure shows the effect of purified peach gum polysaccharide provided in Test Example 5 on (a) IL-1β, (b) IL-6, and (c) TNF-α in cell supernatant (*p<0.05, **p<0.01, ***p<0.001 vs. model group; ###p<0.01 vs. blank control group). Detailed Implementation

[0067] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0068] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0069] In a first aspect, the present invention provides a method for synergistic extraction of peach gum polysaccharides through mixed-culture fermentation, the method comprising: A mixed bacterial seed culture is inoculated into a peach gum powder solution and fermented in a shaker to obtain a fermentation broth containing peach gum polysaccharides; wherein the mixed bacterial seed culture includes yeast and Schizophyllum commune.

[0070] The *Schizophyllum commune* described in this invention ( Schizophyllum commune Fr.*Schizophyllum commune* is a saprophytic fungus belonging to the Basidiomycetes class of the phylum Basidiomycota. This fungus possesses strong biodegradation capabilities, secreting various extracellular enzymes such as cellulase, xylanase, and laccase during its metabolism. Furthermore, during liquid fermentation, *Schizophyllum commune* can produce extracellular polysaccharides with various biological activities, including immunomodulatory, antioxidant, and antitumor effects, such as *Schizophyllum commune polysaccharide* (Schizophyllum commune polysaccharide). Schizophyllan This polysaccharide possesses a unique β-1,3-glucan backbone and β-1,6-glycosidic branched structure; furthermore, its fermentation products show good application potential in improving the physicochemical properties and biological activities of plant polysaccharides, and exhibit anti-aging activity.

[0071] The brewing yeast of this invention ( Saccharomyces cerevisiae Saccharomyces cerevisiae (Saccharomyces cerevisiae) is a model organism for eukaryotic research. Belonging to the class Ascomycetes of the phylum Fungi, this fungus secretes various enzymes during its metabolism, including invertase, β-glucanase, and sucrase, which can degrade and modify the structure of polysaccharide substrates, altering their molecular weight, glycosidic bond configuration, and chain conformation, thereby affecting the biological activity of the polysaccharides. This fungus grows rapidly, has simple nutritional requirements, and its fermentation conditions are easy to control, making it suitable for large-scale industrial production.

[0072] It should be noted that this invention employs mixed fermentation of peach gum with *Schizophyllum commune* and *Saccharomyces cerevisiae*. During metabolism, the two microorganisms exhibit complementary and synergistic enzyme effects: *Schizophyllum commune* secretes cellulase, xylanase, and laccase, effectively cleaving the peach gum cell wall structure and promoting the release of intracellular polysaccharides; while *Saccharomyces cerevisiae* secretes invertase, β-glucanase, and sucrase, moderately hydrolyzing and structurally modifying the released polysaccharide chains, exposing more active groups such as hydroxyl and carboxyl groups. Simultaneously, the microorganisms preferentially utilize low-molecular-weight sugars as carbon sources, achieving selective enrichment of high-molecular-weight components. Compared to traditional methods such as water extraction, enzymatic hydrolysis, or ultrasound-assisted extraction, which primarily aim to reduce molecular weight, this invention, through mixed fermentation, yields peach gum polysaccharide components with a higher molecular weight (2164.655 kDa) and a more uniform distribution (Mw / Mn=1.584), retaining the high molecular weight framework while exhibiting high biological activity and good physicochemical compatibility. Therefore, the fermented peach gum polysaccharide (including crude and / or refined products) obtained in this invention has significant synergistic advantages: the polysaccharide solution has increased viscosity, good film-forming properties, strong skin adhesion, and good stability. After transdermal absorption, it can effectively exert multi-target effects such as anti-oxidation (increasing SOD, GSH-Px, and CAT activity), anti-inflammation (inhibiting the release of IL-1β, IL-6, and TNF-α), and regulation of collagen metabolism (upregulating COL1A1 and downregulating MMP-1 and MMP-9). Animal experiments show that, at the same dose, fermented peach gum polysaccharide has a significantly better effect on improving UVB-induced photoaging of mouse skin than unfermented peach gum polysaccharide, increasing skin moisture content and elasticity, reducing wrinkles, restoring epidermal and dermal thickness, increasing collagen fiber content, and downregulating the expression of the aging marker protein p21. Meanwhile, the crude polysaccharide can meet the efficacy requirements of topical preparations, while the refined component (PGP1) provides structurally clear and functionally specific raw material support for high-value applications. The overall process is green and mild, highly controllable, and easy to scale up, solving the long-standing problems of low activity, difficult utilization, and poor quality control of peach gum polysaccharide in existing technologies.

[0073] As an optional implementation, the inoculation amount of the mixed seed liquid is 5~25 vol%, for example, it can be 5 vol%, 6 vol%, 7 vol%, 8 vol%, 9 vol%, 10 vol%, 11 vol%, 12 vol%, 13 vol%, 14 vol%, 15 vol%, 16 vol%, 17 vol%, 18 vol%, 19 vol%, 20 vol%, etc.

[0074] As an optional implementation, the pH of the fermentation culture is 2 to 7, for example, it can be 2, 2.2, 2.4, 2.5, 2.6, 2.8, 3, 3.2, 3.4, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, etc.

[0075] As an optional implementation, the fermentation culture temperature is 25~43℃, for example, it can be 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, etc.

[0076] As an optional implementation, the shaking speed of the fermentation culture is 170~230 r / min, for example, it can be 170 r / min, 175 r / min, 180 r / min, 185 r / min, 190 r / min, 195 r / min, 200 r / min, 205 r / min, 210 r / min, 215 r / min, 220 r / min, 225 r / min, 230 r / min, etc.

[0077] As an optional implementation, the fermentation culture time is 12~72 h, for example, it can be 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h, 26 h, 28 h, 30 h, 32 h, 34 h, 36 h, 38 h, 40 h, 42 h, 44 h, 46 h, 48 h, 50 h, 52 h, 54 h, 56 h, 58 h, 60 h, 62 h, 64 h, 66 h, 68 h, 70 h, 72 h, etc.

[0078] In a preferred embodiment, the inoculation volume of the mixed seed solution is 18-22 vol.

[0079] In a preferred embodiment, the pH of the fermentation culture is 3.5 to 4.5.

[0080] In a preferred embodiment, the fermentation culture temperature is 26~30℃.

[0081] In a preferred embodiment, the shaking speed of the fermentation culture is 180~200 r / min.

[0082] In a preferred embodiment, the fermentation time is 18-30 h.

[0083] As an optional implementation, the method for preparing the mixed bacterial seed solution includes the following steps: Revival and activation of Schizophyllum commune: Schizophyllum commune strains were inoculated into solid culture medium for activation to obtain activated mycelia; the activated mycelia were inoculated into primary liquid culture medium for a first shaking culture, and after being dispersed, they were inoculated into secondary liquid culture medium for a second shaking culture to obtain Schizophyllum commune fermentation broth; Revival and activation of Saccharomyces cerevisiae: Saccharomyces cerevisiae dry powder and water were mixed for the first activation to obtain an activated bacterial solution; the activated bacterial solution was inoculated into a solid culture medium for the second activation to obtain activated single colonies; the activated single colonies were inoculated into a liquid culture medium and cultured with shaking to obtain Saccharomyces cerevisiae seed culture; Mixed culture: The brewer's yeast seed liquid is centrifuged and the brewer's yeast cell precipitate is collected; the brewer's yeast cell precipitate is mixed with the Schizophyllum commune fermentation broth to obtain the mixed culture seed liquid.

[0084] As an optional implementation, in the process of reviving and activating *Schizophyllum commune*: the solid culture medium is PDA plate culture medium; the primary liquid culture medium is *Schizophyllum commune* liquid culture medium; and the secondary liquid culture medium is *Schizophyllum commune* liquid culture medium.

[0085] As an optional implementation, during the resuscitation and activation of the *Schizophyllum commune*, the activation temperature is 26-30°C, for example, 26°C, 27°C, 28°C, 29°C, 30°C, etc., and the activation time is 4-6 days, for example, 96 h (4 days), 98 h, 100 h, 102 h, 104 h, 106 h, 108 h, 110 h, 112 h, 114 h, 116 h, 118 h, 120 h (5 days), 122 h, 124 h, 126 h, 128 h, 130 h, 132 h, 134 h, 136 h, 138 h, 140 h, 142 h, 144 h (6 days), etc.

[0086] As an optional implementation, during the revival and activation of the *Schizophyllum commune*, the inoculation amount of the activated mycelium during the first shaking culture is 1-2 loops.

[0087] As an optional implementation, in the process of reviving and activating *Schizophyllum commune*: the temperature of the first shaking culture is 26-30℃, for example, 26℃, 27℃, 28℃, 29℃, 30℃, etc.; the rotation speed of the first shaking culture is 160-180 r / min, for example, 160 r / min, 162 r / min, 164 r / min, 166 r / min, 168 r / min, 170 r / min, 172 r / min, 174 r / min, 176 r / min, 178 r / min, 180 r / min, etc.; the duration of the first shaking culture is 2-4 days, for example, 48 h (2 days), 50 h, 52 h, 54 h, 56 h, 58 h, 60 h, 62 h, 64 h, 66 h, 68 h, 70 h, 72 h (3 days), 74 h, 76 h, 78 h, 80 h, 82 h, 84 h. h, 86 h, 88 h, 90 h, 92h, 94 h, 96 h (4 d), etc.

[0088] As an optional implementation, during the resuscitation and activation of the *Schizophyllum commune*, the inoculum volume during the second shaking culture is 8-12 vol.

[0089] As an optional implementation, during the resuscitation and activation of the *Schizophyllum commune*: the temperature of the second oscillation is 26-30°C, for example, 26°C, 27°C, 28°C, 29°C, 30°C, etc.; the rotation speed of the first oscillation culture is 180-200 r / min, for example, 180 r / min, 182 r / min, 184 r / min, 186 r / min, 188 r / min, 190 r / min, 192 r / min, 194 r / min, 196 r / min, 198 r / min, 200 r / min, etc.; and the time of the first oscillation culture is 12-36 h, for example, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h, 26 h, 28 h, 30 h, 32 h, 34 h, 36 h, etc.

[0090] As an optional implementation, in the process of reviving and activating the Saccharomyces cerevisiae: the solid culture medium is YEPD agar medium; the liquid culture medium is YEPD liquid medium.

[0091] As an optional implementation, during the revival and activation process of the Saccharomyces cerevisiae: during the first activation process, the mass-to-volume ratio of the Saccharomyces cerevisiae dry powder to water is 1 g:(8~10) mL, for example, it can be 1 g:8.0 mL, 1 g:8.2 mL, 1 g:8.4 mL, 1 g:8.6 mL, 1 g:8.8 mL, 1 g:9.0 mL, 1 g:9.2 mL, 1 g:9.4 mL, 1 g:9.6 mL, 1 g:9.8 mL, 1 g:10.0 mL, etc.

[0092] As an optional implementation, during the revival and activation process of the brewing yeast: the temperature of the first activation is 36~38℃, for example, it can be 36℃, 36.2℃, 36.4℃, 36.6℃, 36.8℃, 37℃, 37.2℃, 37.4℃, 37.6℃, 37.8℃, 38℃, etc., and the time of the first activation is 15~25 min, for example, it can be 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, etc.

[0093] As an optional implementation, during the revival and activation process of the *Saccharomyces cerevisiae*: the temperature for the second activation is 28~32℃, for example, it can be 28.0℃, 28.2℃, 28.4℃, 28.6℃, 28.8℃, 29.0℃, 29.2℃, 29.4℃, 29.6℃, 29.8℃, 30.0℃, 30.2℃, 30.4℃, 30.6℃, 30.8℃, 31.0℃, 31.2℃, 31.4℃, 31.6℃, 31.8℃, 32.0℃, etc., and the time for the second activation is 12~20 h, for example, it can be 12.0 h, 12.5 h, 13.0 h, 13.5 h, 14.0 h, 14.5 h, 15.0 h, 15.5 h, 16.0 h, 16.5 h, 17.0 h, 17.5 h, 18.0 h, etc. h, 18.5 h, 19.0 h, 19.5 h, 20.0 h, etc.

[0094] As an optional implementation, the temperature for the shaking culture is 28~32℃, for example, it can be 28.0℃, 28.2℃, 28.4℃, 28.6℃, 28.8℃, 29.0℃, 29.2℃, 29.4℃, 29.6℃, 29.8℃, 30.0℃, 30.2℃, 30.4℃, 30.6℃, 30.8℃, 31.0℃, 31.2℃, 31.4℃, 31.6℃, 31.8℃, 32.0℃, etc., and the shaking speed is 180~200 r / min, for example, it can be 180 r / min, 182 r / min, 184 r / min, 186 r / min, 188 r / min, 190 r / min, 192 r / min, 194 r / min, 196 r / min, 198 r / min, 200 r / min, etc. The oscillation incubation time is 18~24 h, for example, it can be 18.0 h, 18.5 h, 19.0 h, 19.5 h, 20.0 h, 20.5 h, 21.0 h, 21.5 h, 22.0 h, 22.5 h, 23.0 h, 23.5 h, 24.0 h, etc.

[0095] As an optional implementation, during the mixed culture process: the centrifugation temperature is 3~5℃, for example, 3℃, 3.5℃, 4℃, 4.5℃, 5℃, etc.; the centrifugation speed is 7000~9000 r / min, for example, 7000 r / min, 7200 r / min, 7400 r / min, 7600 r / min, 7800 r / min, 8000 r / min, 8200 r / min, 8400 r / min, 8600 r / min, 8800 r / min, 9000 r / min, etc.; and the centrifugation time is 5~15 min, for example, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, etc.

[0096] As an optional implementation, during the mixing process, the volume ratio of the brewing yeast seed liquid to the Schizophyllum commune fermentation liquid is (0.8~1.2):(0.8~1.2), for example, it can be 0.8:0.8, 0.8:0.9, 0.8:1, 0.8:1.1, 0.8:1.2, 0.9:0.8, 0.9:0.9, 0.9:1, 0.9:1.1, 0.9:1.2, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1.1:0.8, 1.1:0.9, 1.1:1, 1.1:1.1, 1.1:1.2, 1.2:0.8, 1.2:0.9, 1.2:1, 1.2:1.1, 1.2:1.2, etc.

[0097] As an optional implementation, the method further includes the following post-processing steps: The fermentation broth containing peach gum polysaccharide was centrifuged to obtain the supernatant. The supernatant was subjected to protein removal treatment to obtain a deproteinized polysaccharide solution; The deproteinized polysaccharide solution was subjected to dialysis to obtain a dialyzed polysaccharide solution; The polysaccharide solution after dialysis was subjected to alcohol precipitation, the precipitate was collected and dried to obtain fermented peach gum crude polysaccharide.

[0098] As an optional implementation, in the post-processing, the centrifugation speed is 9000~10000 r / min, for example, it can be 9000 r / min, 9100 r / min, 9200 r / min, 9300 r / min, 9400 r / min, 9500 r / min, 9600 r / min, 9700 r / min, 9800 r / min, 9900 r / min, 10000 r / min, etc., and the centrifugation speed is 10~30 min, for example, it can be 10 min, 12 min, 14 min, 16 min, 18 min, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, etc.

[0099] As an optional implementation, in the post-processing, the protein removal process is performed using the Sevage method.

[0100] Furthermore, in the post-processing: the dialysis treatment uses a dialysis bag with a molecular weight cutoff of 3500 Da.

[0101] As an optional implementation, in the post-processing, the alcohol precipitation treatment uses an ethanol solution of 90~100 vol% (for example, 90 vol%, 91 vol%, 92 vol%, 93 vol%, 94 vol%, 95 vol%, 96 vol%, 97 vol%, 98 vol%, 99 vol%, 100 vol%, etc.).

[0102] As an optional implementation, the method further includes the following purification step: The fermented peach gum crude polysaccharide was subjected to ion exchange column chromatography to obtain refined peach gum polysaccharide.

[0103] As an optional implementation, the purification process specifically includes the following steps: The fermented peach gum crude polysaccharide was dissolved in water to obtain a fermented peach gum crude polysaccharide solution; The fermented peach gum crude polysaccharide solution was loaded onto a chromatography column containing DEAE packing material, and then eluted sequentially with pure water, 0.1 M NaCl solution, 0.2 M NaCl solution, and 0.3 M NaCl solution, and the eluent was collected. The collected eluent was concentrated, then dialyzed and dried to obtain refined peach gum polysaccharides under different elution processes.

[0104] It should be noted that the different elution procedures for refined peach gum polysaccharides here specifically refer to the following: polysaccharides were separated and purified using DEAE-52 column chromatography, with gradient elution sequentially using distilled water, 0.1 M NaCl solution, 0.2 M NaCl solution, and 0.3 M NaCl solution. During this process, the concentration of purified polysaccharides was detected using the phenol-sulfuric acid method, thus obtaining elution curves. Three types of refined peach gum polysaccharides were obtained, namely the elution fractions from distilled water, 0.1 M NaCl solution, and 0.2 M NaCl solution, which were named PGP1, PGP2, and PGP3.

[0105] As an optional implementation, the DEAE packing material is DEAE-52 packing material during the purification process.

[0106] As an optional implementation, during the purification process, the concentration of the fermented peach gum crude polysaccharide solution is 10~30 mg / mL, for example, it can be 10 mg / mL, 12 mg / mL, 14 mg / mL, 16 mg / mL, 18 mg / mL, 20 mg / mL, 22 mg / mL, 24 mg / mL, 26 mg / mL, 28 mg / mL, 30 mg / mL, etc.

[0107] As an optional implementation, during the purification process, the elution flow rate is 0.5~1.5 mL / min, for example, it can be 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1 mL / min, 1.1 mL / min, 1.2 mL / min, 1.3 mL / min, 1.4 mL / min, 1.5 mL / min, etc.

[0108] As an optional implementation, the refined peach gum polysaccharide PGP1 is a white powder.

[0109] As an optional implementation, the refined peach gum polysaccharides PGP2 and PGP3 are in the form of yellow lumps.

[0110] As an optional embodiment, the molecular weight range of the refined peach gum polysaccharide PGP1 is 1.36 × 10⁻⁶. 7 ~4.25×10 6 Da; and its weight-average molecular weight (Mw) is 2164.655 kDa, and its polydispersity index (Mw / Mn) is 1.584.

[0111] As an optional embodiment, the molecular weight range of the refined peach gum polysaccharide PGP2 is 3.55 × 10⁻⁶. 6 ~1.19×10 3 Da; and its weight-average molecular weight (Mw) is 359.903 kDa, and its polydispersity index (Mw / Mn) is 32.704.

[0112] As an optional embodiment, the molecular weight range of the refined peach gum polysaccharide PGP3 is 4.26 × 10⁻⁶. 6 ~1.45×10 5 It has a weight-average molecular weight (Mw) of 672.148 kDa and a polydispersity index (Mw / Mn) of 1.917.

[0113] As an optional implementation, the refined peach gum polysaccharides PGP1 and PGP3 have highly similar compositions, mainly consisting of arabinose and galactose, and containing small amounts of xylose, glucuronic acid and mannose. The high total sugar content indicates that they are relatively pure acidic polysaccharide components.

[0114] As an optional implementation, although the refined peach gum polysaccharide PGP2 is still mainly composed of arabinose and galactose, the mannose content is increased, and a small amount of glucose and fucose are detected. The total sugar content is low, suggesting that it contains more non-sugar impurities.

[0115] In a second aspect, the present invention provides a peach gum polysaccharide, which is prepared by the method of mixed bacterial fermentation and synergistic extraction of peach gum polysaccharide as described in the first aspect; Furthermore, the peach gum polysaccharide includes fermented peach gum crude polysaccharide and / or refined peach gum polysaccharide.

[0116] Thirdly, the present invention provides a method for preparing peach gum polysaccharide by co-extraction of peach gum polysaccharide through mixed fermentation as described in the first aspect, or the application of peach gum polysaccharide as described in the second aspect in the preparation of anti-inflammatory products, antioxidant products, moisturizing products, wound healing products, or anti-photoaging products.

[0117] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0118] The sources of some raw materials for the following embodiments and test examples are shown below:

[0119] Note: The peach gum was purchased from Guangzhou Kangmei Pharmaceutical Co., Ltd., and was identified as PRUNI RESINA. The certificate specimen (No. 20241210) is stored in the Department of Traditional Chinese Medicine Preparations, Southern Medical University.

[0120] Example 1 This embodiment provides a method for preparing fermented peach gum crude polysaccharide, the preparation method specifically including the following steps: S1. Preparation of mixed bacterial seed culture: Resuscitation and activation of *Schizophyllum commune*: *Schizophyllum commune* strains were inoculated onto PDA agar plates and cultured at 28℃ for 5 days. After the mycelium grew well, the plates were stored at 4℃ for later use. For seed culture, 1-2 loops of activated mycelium were inoculated into 250 mL Erlenmeyer flasks containing 100 mL of liquid seed culture medium (*Schizophyllum commune* medium, JC1831) and cultured at 28℃ and 170 r / min for 3 days with shaking. After 3 days, the seed culture was dispersed with sterile glass beads and inoculated into 250 mL Erlenmeyer flasks containing 90 mL of secondary culture medium (*Schizophyllum commune* medium, JC1831) at a 10% (v / v) inoculation rate. The flasks were cultured at 28℃ and 190 r / min for 24 h with shaking to obtain *Schizophyllum commune* fermentation broth. Revival and activation of Saccharomyces cerevisiae: Weigh the dried Saccharomyces cerevisiae powder and add it to warm water at a ratio of 1:9 (g / mL). Activate the Saccharomyces cerevisiae in a water bath at 37℃ for 20 min. Inoculate the activated culture solution onto YEPD agar medium and incubate at 30℃ for 16 h for further activation. Prepare the liquid culture medium according to the YEPD instructions, heat to dissolve, cool, and dispense into 250 mL Erlenmeyer flasks (100 mL per flask). Autoclave at 121℃ for 30 min for later use. Pick a single activated colony from the solid culture medium and inoculate it into YEPD liquid culture medium. Incubate at 30℃ and 190 r / min with shaking for 20 h to obtain the Saccharomyces cerevisiae seed culture. Centrifuge the above-mentioned brewer's yeast seed liquid at 4℃ and 8000 r / min for 10 min, collect the cell precipitate, add the cell precipitate to the Schizophyllum commune fermentation broth, mix, and obtain the mixed seed liquid.

[0121] S2, Mixed-culture fermentation: Peach gum was dried at 80℃ for 20 minutes, pulverized, and passed through a 60-mesh sieve to obtain peach gum powder. The peach gum powder was dissolved in water at a ratio of 1:50 (g / mL), and sterilized under high temperature and high pressure to obtain a peach gum powder solution. The mixed seed culture obtained in S1 was inoculated into the peach gum powder solution in a fermentation shake flask. The fermentation conditions were as follows: inoculum amount of 10% (v / v), pH of 4.0 (adjusted with citric acid), fermentation temperature of 28℃, shaking speed of 190 r / min, and fermentation time of 48 h to obtain a fermentation broth containing peach gum polysaccharides.

[0122] S3. Preparation of fermented peach gum crude polysaccharide: Centrifuge the fermentation broth containing peach gum polysaccharide obtained in S2 at 9700 r / min for 20 min and collect the supernatant. Mix chloroform and n-butanol at a volume ratio of 5:1 and store in a brown volumetric flask for later use to obtain the Sevage reagent solution. Mix the fermentation supernatant with the Sevage reagent solution at a ratio of 9:1 and shake thoroughly in a separatory funnel. Let stand for 40 min and collect the aqueous layer. Repeat this process 2-3 times until no denatured proteins are produced at the solution interface. Take a dialysis bag of suitable length and boil it in boiling water for 10 minutes to activate it. After activation, add a deproteinized polysaccharide solution to the dialysis bag. Place the dialysis bag containing the polysaccharide solution in a beaker containing distilled water, ensuring that the 3500 D dialysis bag is completely submerged. Dialyze at room temperature, changing the dialysis solution at 2-4 h, 6-8 h, and 10-14 h (the next morning) after the start of dialysis. After dialysis, collect the polysaccharide solution in the dialysis bag. Add 95% ethanol to the polysaccharide solution while stirring. Let it stand overnight in a refrigerator at 4°C. The next day, take the precipitate and dry it in an oven to constant weight to obtain the fermented peach gum crude polysaccharide.

[0123] Example 2 This embodiment provides a method for preparing fermented peach gum crude polysaccharide. The only difference from Example 1 is that the inoculation amount of the mixed culture seed liquid is 5% (v / v) during the S2 mixed culture fermentation process. The other steps are the same as in Example 1.

[0124] Example 3 This embodiment provides a method for preparing fermented peach gum crude polysaccharide. The only difference from Example 1 is that the inoculation amount of the mixed culture seed liquid is 15% (v / v) during the S2 mixed culture fermentation process. The other steps are the same as in Example 1.

[0125] Example 4 This embodiment provides a method for preparing fermented peach gum crude polysaccharide. The only difference from Example 1 is that the inoculation amount of the mixed culture seed liquid is 20% (v / v) during the S2 mixed culture fermentation process. The other steps are the same as in Example 1.

[0126] Example 5 This embodiment provides a method for preparing fermented peach gum crude polysaccharide. The only difference from Example 1 is that the inoculation amount of the mixed culture seed liquid is 25% (v / v) during the S2 mixed culture fermentation process. The other steps are the same as in Example 1.

[0127] Example 6 This embodiment provides a method for preparing fermented peach gum crude polysaccharide. The only difference from Example 1 is that the pH is 5.0 during the S2 mixed culture fermentation process, while the other steps are the same as in Example 1.

[0128] Example 7 This embodiment provides a method for preparing fermented peach gum crude polysaccharide. The only difference from Example 1 is that the pH is 6.0 during the S2 mixed culture fermentation process, while the other steps are the same as in Example 1.

[0129] Example 8 This embodiment provides a method for preparing fermented peach gum crude polysaccharide. The only difference from Example 1 is that the pH is 7.0 during the S2 mixed culture fermentation process, while the other steps are the same as in Example 1.

[0130] Example 9 This embodiment provides a method for preparing fermented peach gum crude polysaccharide. The only difference from Example 1 is that the fermentation temperature is 33°C during the S2 mixed culture fermentation process, while the other steps are the same as in Example 1.

[0131] Example 10 This embodiment provides a method for preparing fermented peach gum crude polysaccharide. The only difference from Example 1 is that the fermentation temperature is 38°C during the S2 mixed culture fermentation process, while the other steps are the same as in Example 1.

[0132] Example 11 This embodiment provides a method for preparing fermented peach gum crude polysaccharide. The only difference from Example 1 is that the fermentation temperature is 43°C during the S2 mixed culture fermentation process, while the other steps are the same as in Example 1.

[0133] Example 12 This embodiment provides a method for preparing fermented peach gum crude polysaccharide. The only difference from Example 1 is that the shaking speed is 170 r / min during the S2 mixed culture fermentation process, while the other steps are the same as in Example 1.

[0134] Example 13 This embodiment provides a method for preparing fermented peach gum crude polysaccharide. The only difference from Example 1 is that the shaking speed is 210 r / min during the S2 mixed culture fermentation process, while the other steps are the same as in Example 1.

[0135] Example 14 This embodiment provides a method for preparing fermented peach gum crude polysaccharide. The only difference from Example 1 is that the shaking speed is 230 r / min during the S2 mixed culture fermentation process, and the other steps are the same as in Example 1.

[0136] Example 15 This embodiment provides a method for preparing fermented peach gum crude polysaccharide. The only difference from Embodiment 1 is that the fermentation time is 0 h during the S2 mixed culture fermentation process, while the other steps are the same as in Embodiment 1.

[0137] Example 16 This embodiment provides a method for preparing fermented peach gum crude polysaccharide. The only difference from Example 1 is that the fermentation time in the S2 mixed culture fermentation process is 24 hours, while the other steps are the same as in Example 1.

[0138] Example 17 This embodiment provides a method for preparing fermented peach gum crude polysaccharide. The only difference from Example 1 is that the fermentation time in the S2 mixed culture fermentation process is 72 h, while the other steps are the same as in Example 1.

[0139] Example 18 This embodiment provides a method for preparing fermented peach gum crude polysaccharide. The only difference from Example 1 is that the fermentation conditions used in the S2 mixed culture fermentation process are as follows: inoculum amount of 20% (v / v), pH of 4.0, fermentation temperature of 28℃, shaking speed of 190 r / min, and fermentation time of 24 h. The other steps are the same as in Example 1.

[0140] Test Example 1 Phenol-sulfuric acid method for determining polysaccharide content in samples Test sample: Fermented peach gum crude polysaccharide prepared in Examples 1-17.

[0141] Test method: Preparation of standard: Weigh 10 mg of standard glucose, dissolve it completely in distilled water and pour it into a volumetric flask. Then add distilled water to the volumetric flask to make up to 100 mL. The glucose standard solution is ready for use.

[0142] Selection of wavelength for determination: Accurately measure 2 mL of glucose reference solution and 1 mL of test solution into separate 10 mL graduated test tubes. Add water to a final volume of 2 mL, then add 1 mL of 5% phenol and 5 mL of concentrated sulfuric acid to each tube. Shake well and place in a hot water bath for 20 min, followed by a cold water bath for 20 min. The resulting sample is then scanned using ultraviolet-visible spectrophotometry in the wavelength range of 200–800 nm. Select the wavelength of maximum absorption and peak shape as the detection wavelength.

[0143] Linearity investigation: Prepare seven clean 10 mL test tubes. Add 0, 0.2, 0.4, 0.6, 0.8, 1.0 mL, and 1.2 mL of standard solution to the tubes in sequence, then add distilled water to a final volume of 2 mL and mix well. Next, add 1 mL of 5% phenol and 5 mL of concentrated sulfuric acid sequentially. After adding these solutions, place the tubes in a hot water bath for 20 min, then in a cold water bath for 20 min. Measure the absorbance of the sample in each tube at a wavelength of 490 nm, record the data, and plot a standard curve with glucose concentration on the x-axis and absorbance on the y-axis. Obtain the regression equation.

[0144] Weigh 10 mg of fermented peach gum polysaccharide sample, make up to 100 mL, take 1 mL, add distilled water to 2 mL, and measure absorbance using the same steps. Record the data and calculate the extraction rate using the following formula to calculate the proportion of polysaccharide after fermentation and degradation: Polysaccharide yield (%) = Total sugar content × Crude polysaccharide yield.

[0145] The specific test results are as follows: Figure 1 As shown in the figure: like Figure 1As shown, the inoculum size directly affects the initial biomass and metabolic rate of the cells in the fermentation system, thereby affecting the substrate degradation efficiency and polysaccharide synthesis and accumulation. The above examples set up five inoculum size gradients: 5% (Example 2), 10% (Example 1), 15% (Example 3), 20% (Example 4), and 25% (Example 5), to investigate their effects on the polysaccharide content in the fermentation broth. The experimental results show that with increasing inoculum size, the polysaccharide content exhibits a trend of first increasing and then decreasing. When the inoculum size increases from 5% to 20%, the polysaccharide content generally shows an upward trend; when the inoculum size reaches 20%, the polysaccharide content is the highest value among all groups; further increasing the inoculum size to 25% results in a decrease in polysaccharide content. This trend indicates that an appropriate inoculum size is beneficial for the cells to quickly establish growth advantage in the fermentation system, fully secrete extracellular enzyme systems to degrade the gum substrate, thereby promoting the dissolution and accumulation of polysaccharides. When the inoculum size is too low, cell growth is slow, metabolic product accumulation is insufficient, and substrate degradation efficiency is limited. When the inoculum size is too high, excessive cell proliferation may lead to competitive consumption of nutrients and accumulation of metabolic byproducts, which in turn inhibits the secretion activity of extracellular enzymes. Therefore, 20% is the optimal inoculum size for the fermentation process described in this invention.

[0146] like Figure 2 As shown, the pH of the culture medium is one of the key factors affecting microbial growth, metabolism, and extracellular enzyme activity, thus influencing the degradation efficiency of gum arabic substrate and the degree of polysaccharide dissolution. This valve was designed with pH gradients of 4.0 (Example 1), 5.0 (Example 6), 6.0 (Example 7), and 7.0 (Example 8) to investigate their effect on the polysaccharide content in the fermentation broth. The experimental results showed that the polysaccharide content in the fermentation broth gradually decreased with increasing pH. The polysaccharide content reached its highest value at pH 4.0; it decreased slightly when the pH increased to 4.5; and continued to decrease with increasing pH to 5.0, 5.5, 6.0, 6.5, and 7.0, reaching its lowest point at pH 7.0. This trend indicates that the mixed-culture fermentation system is more favorable for the dissolution and accumulation of gum arabic polysaccharides under slightly acidic conditions. The highest polysaccharide content was observed at pH 4.0, likely because the acidic environment facilitates the hydrolysis and release of pectin-like substances in gum arabic, promoting polysaccharide dissolution. Furthermore, *Saccharomyces cerevisiae* and *Schizophyllum commune* exhibit higher metabolic activity and more vigorous secretion of extracellular enzymes under slightly acidic conditions, resulting in higher degradation efficiency of gum arabic substrates. As the pH gradually approaches neutral, cell growth and metabolism may be inhibited, or extracellular enzyme activity may decrease, leading to a reduction in polysaccharide yield. Therefore, pH 4.0 was selected as the optimal initial pH for the fermentation process described in this invention.

[0147] like Figure 3As shown, temperature is one of the key factors affecting microbial growth and metabolism, as well as the activity of extracellular enzymes, thus influencing the dissolution and conversion efficiency of gum arabic polysaccharides. This invention established four temperature gradients: 28℃ (Example 1), 33℃ (Example 9), 38℃ (Example 10), and 43℃ (Example 11), to investigate their effects on the polysaccharide content in the fermentation broth. The experimental results showed that the polysaccharide content gradually decreased with increasing fermentation temperature. The polysaccharide content was highest at 28℃; it began to decrease at 33℃, and further decreased at 38℃ and 43℃. This trend indicates that higher temperatures may inhibit the growth and metabolism of *Saccharomyces cerevisiae* and *Schizophyllum commune*, or lead to the inactivation of some extracellular enzymes, thereby affecting the dissolution and accumulation of gum arabic polysaccharides; while 28℃ is more suitable for the growth and metabolic activities of this mixed microbial system, which is beneficial for polysaccharide synthesis and release. Therefore, 28℃ was selected as the optimal temperature for subsequent fermentation processes.

[0148] like Figure 4 As shown, the shaking speed directly affects the dissolved oxygen level and mass transfer efficiency in the fermentation system, thereby affecting the growth, metabolism, and extracellular enzyme secretion activity of microorganisms. This invention established four shaking speed gradients: 170 r / min (Example 12), 190 r / min (Example 1), 210 r / min (Example 13), and 230 r / min (Example 14), to investigate their effects on the polysaccharide content in the fermentation broth. The experimental results showed that with increasing shaking speed, the polysaccharide content initially increased and then stabilized. When the shaking speed increased from 170 r / min to 190 r / min, the polysaccharide content increased significantly, with 190 r / min being the highest value among all groups. Further increasing the shaking speed to 210 r / min and 230 r / min showed no significant difference compared to 190 r / min, and the overall level remained stable. This trend indicates that a suitable shaking speed is beneficial for increasing the dissolved oxygen level in the fermentation system, promoting cell growth and metabolic activity, thereby enhancing the dissolution and accumulation of gum polysaccharides. When the rotation speed exceeds 190 r / min, the polysaccharide content remains stable, possibly for two reasons: First, dissolved oxygen has reached its saturation threshold; yeast and Schizophyllum commune have an upper limit to their oxygen requirements, and further increasing the rotation speed will not further promote polysaccharide synthesis. Second, the fluid shear force generated by excessively high rotation speed may damage the cell structure, especially filamentous fungi like Schizophyllum commune, leading to growth restriction. Therefore, considering both fermentation efficiency and energy consumption, 190 r / min was selected as the optimal rotation speed for subsequent fermentation processes.

[0149] like Figure 5As shown, fermentation time directly affects the degree of degradation of the gum substrate by microorganisms and the efficiency of polysaccharide accumulation. This valve was set with four time gradients: 0 h (Example 15), 24 h (Example 16), 48 h (Example 1), and 72 h (Example 16) to investigate its effect on the polysaccharide content in the fermentation broth. The experimental results show that the polysaccharide content first increases and then decreases with the extension of fermentation time. At the initial stage of fermentation (0 h), the polysaccharide content was 29.0%, which may be due to the soluble polysaccharide components naturally present in the gum powder. At 24 h, the polysaccharide content increased, reaching its peak for the entire fermentation cycle. Further extending the fermentation time to 48 h and 72 h resulted in a decrease in polysaccharide content. This trend indicates that in the early stages of fermentation (0–24 h), *Saccharomyces cerevisiae* and *Schizophyllum commune* gradually adapt to the fermentation environment and secrete extracellular enzyme systems, effectively degrading the gum substrate and promoting the dissolution and accumulation of polysaccharides, reaching maximum yield at 24 h. However, when the fermentation time exceeded 24 hours, the polysaccharide content decreased significantly. This may be because the nutrients in the culture medium were gradually consumed in the later stages of fermentation, and the cells may have switched to using the dissolved polysaccharides as a carbon source for metabolism. Furthermore, some polysaccharides may have undergone enzymatic or chemical degradation in the continuous fermentation environment, leading to structural damage and reduced content. Therefore, 24 hours was selected as the optimal fermentation time for subsequent fermentation processes.

[0150] Test Example 2 Test on the effect of fermented peach gum crude polysaccharide on UVB-induced photoaging of mouse skin Test sample: Fermented peach gum crude polysaccharide prepared in Example 18 (fermented peach gum crude polysaccharide prepared in step S3).

[0151] (I) Animal grouping and construction of skin photoaging model Fifty-six 8-week-old female BALB / c mice were purchased from the Laboratory Animal Center of Southern Medical University. All mice were kept under conditions of 40–65% humidity, room temperature (24±1℃), and 12-hour light / dark cycles, and were provided with water and standard feed. The entire animal experiment was approved by the Animal Ethics Committee of Southern Medical University (Approval No.: SMUL20251021).

[0152] Before the experiment, mice were acclimatized for three days. Then, based on different treatment conditions, the mice were randomly divided into 6 groups of 8 mice each, including: (1) a blank control group that did not receive any drug treatment; (2) a model group treated only with UVB; (3) a positive control group treated with UVB and topical tretinoin cream (TC); (4) a low-dose group treated with UVB and topical fermented peach gum polysaccharide (30 mg / mL) (PGP 30 mg / mL); (5) a medium-dose group treated with UVB and topical fermented peach gum polysaccharide (60 mg / mL) (PGP 60 mg / mL); (6) a high-dose group treated with UVB and topical fermented peach gum polysaccharide (90 mg / mL) (PGP 90 mg / mL); and (7) a high-dose group treated with UVB and topical unfermented peach gum polysaccharide (90 mg / mL) (UP 90 mg / mL).

[0153] All mice had their back hair shaved off with a razor, creating an area of ​​2 × 2 cm². 2 The exposed areas were used. A UV irradiation chamber was constructed, and mice were irradiated three times a week with UVB lamps for six weeks. The distance between the animal's back and the lamp was 24 cm. Irradiation time was calculated using UV irradiance intensity and illumination distance. During the first week, the radiation dose administered was equivalent to a single minimum erythema dose (1MED), i.e., 160 mJ / cm². 2 The radiation dose was increased to 210, 280, and 370 mJ / cm² in weeks 2, 4, and 5, respectively. 2 The concentration remained at 370 mJ / cm² during weeks 5 and 6. 2 Drug administration was initiated after successful model establishment in the fourth week. The drug was applied to the mouse skin one hour after each irradiation. After model establishment, blood was collected from the ocular veins of mice, and the supernatant was collected by centrifugation (3000 r / min, 15 min). Mice were euthanized by cervical dislocation, and the exposed skin from the back was removed. A portion was fixed in 4% paraformaldehyde, and the remaining skin tissue was stored in clean, dry EP tubes at -80°C.

[0154] (II) Preparation of paraffin sections of mouse skin: Skin tissue fixed for 24 hours was removed, rinsed with running water, trimmed into a suitable shape, and placed in an embedding cassette. Prepare 80%, 90%, and 95% ethanol, anhydrous ethanol, xylene, and paraffin. Perform dehydration and paraffin-impregnation procedures on the tissue according to Table 1.

[0155] Table 1

[0156] (III) HE staining Perform the following procedures as per Table 2: dewaxing and rehydration, HE staining, and mounting.

[0157] Table 2

[0158] (IV) Masson staining Paraffin sections were sequentially immersed in xylene I, xylene II, xylene III, anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 90% ethanol, and 80% ethanol. The sections were then soaked in Masson A solution overnight and rinsed with running water. Next, the sections were immersed in a mixture of equal parts Masson B and Masson C solutions for 1 min, rinsed with running water, differentiated in differentiation solution for a few seconds, rinsed with running water, and then immersed in Masson D solution for 6 min. After rinsing with running water, the sections were immersed in Masson E solution for 1 min, without rinsing, slightly drained, and then directly immersed in Masson F solution for 2–30 s. After differentiation, the sections were rinsed with 1% acetic acid, dehydrated and cleared with anhydrous ethanol III, IV, and V, and xylene IV, V, and VI, mounted with neutral resin, and air-dried in a fume hood. The sections were then scanned and photographed using a scanning microtome to obtain Masson staining images of the skin.

[0159] (V) Immunofluorescence staining The slides were placed in the retrieval solution for antigen retrieval, taking care to prevent excessive evaporation of the buffer solution and drying of the slides. After natural cooling, the slides were washed three times with PBS (pH 7.4) on a decolorizing shaker for 5 min each time. Then, endogenous peroxidase was blocked by immersing the slides in 3% methanol-hydrogen peroxide solution and incubating at room temperature in the dark for 25 min. The slides were then washed three times with PBS for 5 min each time. For the serum blocking step, 3% BSA was evenly distributed on the tissue slides and blocked at room temperature for 30 min. After blocking, the blocking solution was gently shaken off, and the prepared primary antibody working solution was added. The slides were then placed flat in a humidified chamber and incubated overnight at 4°C. The next day, the slides were washed three times with PBS, and then HRP-labeled secondary antibody corresponding to the species of the primary antibody was added and incubated at room temperature for 50 min. After the secondary antibody incubation, the slides were washed again with PBS, and then freshly prepared DAB chromogenic solution was added. The chromogenic time was controlled under a microscope. When the positive signal turned brownish-yellow, the slides were rinsed with tap water to stop the chromogenic process. After staining, the sections were counterstained with hematoxylin for about 3 minutes, rinsed with running water, and then subjected to hematoxylin differentiation solution for a few seconds followed by blueing treatment. Finally, dehydration and mounting were performed: for paraffin sections, they were successively immersed in 75% alcohol, 85% alcohol, anhydrous ethanol I, anhydrous ethanol II, n-butanol, and xylene for 5 minutes each for dehydration and clearing, air-dried, and then mounted with mounting adhesive. All samples were then examined under a microscope for result interpretation.

[0160] The specific test results are shown below: Mouse skin appearance as Figure 6 As shown, after 6 weeks of modeling and group treatment, the skin on the backs of mice was photographed and observed. Compared with the smooth and healthy skin of the control group, the skin of the model group mice became redder, looser, and showed obvious dryness, desquamation, roughness, and even wrinkles after UVB irradiation. The skin condition of the positive control group improved, with reduced erythema and desquamation, and improved roughness and wrinkles compared to the model group. All dose groups of fermented peach gum polysaccharide showed different degrees of protective effect in a dose-dependent manner; the high-dose group showed the best effect, with skin roughness and color restored, dryness and desquamation alleviated, and wrinkles shallowed; the medium and low-dose groups showed similar improvement trends but were weaker. It is worth noting that although the unfermented high-dose group showed some improvement, its effect in alleviating skin roughness and deep wrinkles was weaker than that of the fermented group at the same dose, indicating that the fermentation process may enhance the anti-photoaging effect of peach gum polysaccharide.

[0161] Results of mouse skin elasticity Figure 7 As shown, the skin elasticity of the model group was generally lower than that of the blank group, indicating that successful modeling led to a decrease in skin elasticity. The elasticity value of the positive control group was between that of the model and the blank group, indicating that it had a certain improving effect. Among the high, medium and low dose groups of fermented peach gum, the elasticity value of the high dose group was relatively high, especially in the later stage, which was close to or slightly higher than that of the positive control group, showing a certain dose-dependent improvement trend. The elasticity value of the unfermented high dose group was generally lower than that of the fermented high dose group at the same time, suggesting that the fermentation process may have an enhancing effect on the skin elasticity improvement effect of peach gum.

[0162] Mouse skin moisture index results as follows Figure 8 As shown, the skin moisture level in the white group remained at a relatively high level throughout the experiment, indicating that its skin barrier function was not impaired. The skin moisture level in the model group was significantly lower than that in the control group at all time points, confirming the successful induction of dry skin. The moisture levels in the positive control group were generally higher than those in the model group, indicating that it had an effect on improving dry skin. Among the low, medium, and high dose groups of fermented peach gum, the high dose group showed more significant moisture recovery in the later stages of the experiment, approaching the level of the positive control group, and exhibited a certain dose-dependent improvement trend. Notably, the skin moisture levels in the unfermented high-dose group were lower than those in the fermented high-dose group at the same time point, with the difference becoming more pronounced in the later stages, suggesting that fermented peach gum may be more effective in increasing skin moisture content, and its effect on improving skin hydration is superior to that of unfermented peach gum.

[0163] HE staining results are as follows Figure 9 and Figure 10As shown, the epidermal thickness measurement results indicated that the skin tissue structure of the mice in the blank group was intact, and the epidermal layer was thin and uniform. Compared with the blank control group, the epidermal thickness of the model group was significantly increased, indicating that UVB induced significant abnormal epidermal proliferation, and the photoaging model was successfully established. Compared with the model group, the epidermal thickness of each dose group of fermented peach gum crude polysaccharide showed a decreasing trend, with the epidermal thickness of the high-dose group recovering to a level close to that of the blank control group. Although the epidermal thickness of the unfermented peach gum crude polysaccharide group decreased, the improvement effect was weaker than that of the fermentation group at the same high dose. The dermal thickness measurement results showed that the dermal collagen fibers of the blank group were tightly arranged. Compared with the blank control group, the dermal thickness of the model group was significantly increased, indicating that UVB irradiation caused abnormal thickening of the dermal layer, and the photoaging model was successfully established. Compared with the model group, the dermal thickness of each dose group of fermented peach gum crude polysaccharide was significantly decreased, showing a dose-dependent improvement trend, with the dermal thickness of the high-dose group approaching that of the positive drug retinoic acid group. The improvement effect of the dermal thickness of the unfermented peach gum crude polysaccharide group was weaker than that of the fermentation group at the same high dose. These results indicate that fermented peach gum crude polysaccharide can dose-dependently improve UVB-induced abnormal thickening of the epidermis and dermis in mice, restore skin tissue structure, and the fermentation process can enhance the improving effect of peach gum polysaccharide.

[0164] Masson staining results are as follows: Figure 11 As shown, in the control group, the dermal collagen fibers of mice were dark blue, with thick, tightly packed, and regularly arranged fiber bundles, exhibiting a typical woven structure, indicating dense collagen deposition. In the model group, after UVB irradiation, the dermal collagen fibers showed significantly lighter staining, with sparse, loosely arranged fiber bundles and some breakage, indicating that UVB induced collagen degradation and structural damage in photoaging. The positive control group (retinoic acid) showed good improvement, with collagen fiber staining depth and density superior to the model group, approaching the level of the control group. Compared to the model group, the dermal collagen fibers in each dose group of fermented peach gum crude polysaccharide showed increased staining depth, gradually thickening of fiber bundles, and a more compact arrangement. Among these, the medium and high dose groups showed the most significant improvement in collagen fiber morphology in terms of staining depth and fiber density. Although the high-dose group of unfermented peach gum crude polysaccharide showed some improvement, with collagen fiber staining being darker than the model group and fiber arrangement partially restored, its staining depth was shallower than that of the fermented group at the same dose, and the fiber arrangement was looser than that of the fermented group, indicating that the fermentation process can enhance the protective and repairing effect of peach gum polysaccharide on collagen fibers.

[0165] Immunofluorescence staining results as follows Figure 12As shown, p21 positive signals are mainly located in the cell nucleus, appearing as brownish-yellow to brownish-red granular distributions. In the control group, the number of p21 positive cells in the mouse skin tissue was extremely low, with light staining and only scattered weakly positive cells. After UVB irradiation, the model group showed a large number of p21 positive cells in the epidermis and superficial dermis, staining a deep brown color. These positive cells were densely distributed in patches, with a significantly expanded distribution area, indicating that UVB successfully induced the aging process of skin cells. The number of p21 positive cells in the retinoic acid positive control group was significantly reduced compared to the model group, with lighter staining and a more scattered distribution. Compared to the model group, the number of p21 positive cells in all dose groups of fermented peach gum crude polysaccharide decreased to varying degrees, with lighter staining. The high-dose fermented peach gum crude polysaccharide group showed extremely low numbers of p21 positive cells, with light staining and scattered distribution, approaching the level of the control group. Although the unfermented peach gum crude polysaccharide group showed some improvement, with a decrease in the number of p21 positive cells compared to the model group, its improvement effect was weaker than that of the fermented high-dose group at the same dose, suggesting that the fermentation process can enhance the inhibitory effect of peach gum polysaccharide on p21 expression.

[0166] Example 19 This embodiment provides a method for preparing refined polysaccharide from fermented peach gum, the preparation method specifically including the following steps: S1. Preparation of mixed bacterial seed culture: Resuscitation and activation of *Schizophyllum commune*: *Schizophyllum commune* strains were inoculated onto PDA agar plates and cultured at 28℃ for 5 days. After the mycelium grew well, the plates were stored at 4℃ for later use. For seed culture, 1-2 loops of activated mycelium were inoculated into 250 mL Erlenmeyer flasks containing 100 mL of liquid seed culture medium (*Schizophyllum commune* medium, JC1831) and cultured at 28℃ and 170 r / min for 3 days with shaking. After 3 days, the seed culture was dispersed with sterile glass beads and inoculated into 250 mL Erlenmeyer flasks containing 90 mL of secondary culture medium (*Schizophyllum commune* medium, JC1831) at a 10% (v / v) inoculation rate. The flasks were cultured at 28℃ and 190 r / min for 24 h with shaking to obtain *Schizophyllum commune* fermentation broth. Revival and activation of Saccharomyces cerevisiae: Weigh the dried Saccharomyces cerevisiae powder and add it to warm water at a ratio of 1:9 (g / mL). Activate the Saccharomyces cerevisiae in a water bath at 37℃ for 20 min. Inoculate the activated culture solution onto YEPD agar medium and incubate at 30℃ for 16 h for further activation. Prepare the liquid culture medium according to the YEPD instructions, heat to dissolve, cool, and dispense into 250 mL Erlenmeyer flasks (100 mL per flask). Autoclave at 121℃ for 30 min for later use. Pick a single activated colony from the solid culture medium and inoculate it into YEPD liquid culture medium. Incubate at 30℃ and 190 r / min with shaking for 20 h to obtain the Saccharomyces cerevisiae seed culture. Centrifuge the above-mentioned brewer's yeast seed liquid at 4℃ and 8000 r / min for 10 min, collect the cell precipitate, add the cell precipitate to the Schizophyllum commune fermentation broth, mix, and obtain the mixed seed liquid.

[0167] S2, Mixed-culture fermentation: Peach gum was dried at 80℃ for 20 minutes, pulverized, and passed through a 60-mesh sieve to obtain peach gum powder. The peach gum powder was dissolved in water at a ratio of 1:50 (g / mL), and sterilized under high temperature and high pressure to obtain a peach gum powder solution. The mixed seed culture obtained in S1 was inoculated into the peach gum powder solution in a fermentation shake flask. The fermentation conditions were as follows: inoculum amount of 10% (v / v), pH of 4.0 (adjusted with citric acid), fermentation temperature of 28℃, shaking speed of 190 r / min, and fermentation time of 48 h to obtain a fermentation broth containing peach gum polysaccharides.

[0168] S3. Preparation of fermented peach gum crude polysaccharide: The fermentation broth containing peach gum polysaccharide obtained in S2 was centrifuged at 9700 r / min for 20 min, and the supernatant was collected. Protein was removed by repeated extraction using the Sevage method (chloroform:n-butanol = 5:1, v / v), and the aqueous phase was collected. The deproteinized polysaccharide solution was placed in a pre-treated dialysis bag (molecular weight cutoff 3500 Da) and dialyzed in distilled water at 4℃ for 48 h, changing the dialysis solution every 6-8 h to remove small molecule impurities. After dialysis, the solution in the bag was collected, concentrated under reduced pressure to an appropriate volume, ethanol was added, and the mixture was allowed to stand overnight at 4℃ to precipitate the polysaccharide. The precipitate was collected by centrifugation, freeze-dried, and the fermented peach gum crude polysaccharide was obtained and stored in a dry place for later use.

[0169] S4. Ion exchange column chromatography purification: DEAE packing material pretreatment: Weigh an appropriate amount of DEAE-52 packing material, soak it in distilled water, and let it stand in a refrigerator at 4℃ for 24 hours. Pour off the supernatant, remove impurities, and soak in 0.5 M hydrochloric acid solution for 30 minutes. Pour off the supernatant acid solution and filter until dry. Wash repeatedly with deionized water until the filtrate is neutral. Then soak in 0.5 M NaOH solution for 30 minutes, pour off the supernatant alkaline solution, filter until dry, and wash repeatedly with deionized water until the filtrate is neutral. Place the cleaned packing material in a beaker and place it in a refrigerator for at least 2-3 hours to avoid the generation of bubbles due to temperature changes during column packing.

[0170] Column packing: Rinse the chromatography column repeatedly with distilled water until clean, fix it to the iron stand, add distilled water, open the outlet, maintain a smooth water flow, ensure the stability of the flow system, and remove air bubbles. Stir the DEAE-52 packing material thoroughly, and use a glass rod to guide it evenly into the chromatography column, allowing it to settle. Use a constant flow pump to deliver distilled water to equilibrate the packing material after loading. Once the liquid surface is balanced, adjust the flow rate to 1 mL / min.

[0171] Preparation of polysaccharide solution: Weigh 500 mg of peach gum polysaccharide sample, add 25 mL of distilled water, and stir magnetically at room temperature until fully dissolved to obtain a 20 mg / mL peach gum polysaccharide solution.

[0172] Sample loading and elution program initiation: After adjusting the flow rate, begin sample loading. Turn on the constant flow pump. When the distilled water level above the packing material is approximately 1 cm, slowly add the polysaccharide solution along the column wall using a dropper. Then elute sequentially with distilled water and 0.1 M, 0.2 M, and 0.3 M NaCl solutions. The flow rate is 1 mL / min. Collect the eluent using an automated separator and collector, collecting 5 mL from each of 100 tubes.

[0173] Polysaccharide content detection: Select 1, 5, 10, 15, 20, ... 100 test tubes from the fully automated collector, and use the phenol-concentrated sulfuric acid method to detect the polysaccharide content, and plot the elution curve based on the absorbance values.

[0174] Sample processing: The collected eluents were concentrated to a suitable volume using a rotary evaporator, then placed in dialysis bags for dialysis, and freeze-dried in a vacuum freeze dryer for 48 hours to obtain the freeze-dried components of peach gum polysaccharide under different elution programs.

[0175] Test Example 3 Physicochemical properties and structural characterization of purified peach gum polysaccharides Test sample: The freeze-dried component of peach gum polysaccharide prepared under the same elution procedure as in Example 19.

[0176] Test method: (I) Determination of molecular weight and purity: The specific chromatographic column and elution conditions were as follows: Ohpak SB-805 HQ (300×8mm) and Ohpak SB-803 HQ (300×8mm) gel size exclusion columns were used in series. The differential detector was Optilab T-rEX, the laser light scattering detector was DAWN HELEOS Ⅱ, the column temperature was 45℃, the injection volume was 100 μL, the mobile phase was A (0.02% NaN3, 0.1M NaNO3), the flow rate was 0.6 mL / min, and the elution gradient was isocratic for 75 min.

[0177] Sample pretreatment: Dissolve the sample in 0.1M NaNO3 aqueous solution (containing 0.02% NaN3, w / w) to a final concentration of 1 mg / mL, and filter through a 0.45 μm filter before analysis. Record each chromatogram. Calculate the relative molecular mass of each purified polysaccharide of peach gum based on the standard curve.

[0178] (II) Monosaccharide composition analysis Sample pretreatment: Take a clean chromatographic vial, weigh an appropriate amount of purified peach gum polysaccharide sample, add 1 mL of 2M TFA acid solution, and heat at 121℃ for 2 hours. Purge with nitrogen and dry. Wash with 99.99% methanol, then dry again, repeating the methanol washing 2-3 times. Dissolve in sterile water and transfer to a chromatographic vial for analysis.

[0179] Preparation of monosaccharide standards: After accurately weighing the required standards, add water to prepare a 10 mg / mL standard solution stock solution. Then, take an appropriate amount of the stock solution and mix them to prepare a mixed standard with a maximum index concentration of 60 μg / mL, 50 μg / mL or 40 μg / mL. Prepare a series of standards for use according to the following concentration gradient.

[0180] Chromatographic conditions: A Thermo ICS 5000+ ion chromatography system was used, and an electrochemical detector was employed for the analysis and detection of monosaccharide components. A Dionex analyzer was used. TM CarboPac TM PA20 (150×3.0 mm, 10 μm) liquid chromatography column; injection volume: 5 μL. Mobile phase A (H2O), mobile phase B (0.1M NaOH), mobile phase C (0.1M NaOH, 0.2M NaAc), flow rate 0.5 ml / min; column temperature 30℃; elution gradient: 0 min A / B / C (95:5:0, V / V), 26 min A / B / C (85:5:10, V / V), 42 min A / B / C (85:5:10, V / V), 42.1 min A / B / C (60:0:40, V / V), 52 min A / B / C (60:40:0, V / V), 52.1 min A / B / C (95:5:0, V / V), 60 min A / B / C (95:5:0, V / V).

[0181] The monosaccharide mixed standard gradient concentration information is shown in Table 3 below: Table 3

[0182] (III) Infrared spectral scanning A small amount of purified peach gum polysaccharide sample was weighed and mixed with 200 mg of potassium bromide, then pressed into 1 mm thick sheets for analysis. The analysis was performed using a Nicolet iS 5 Fourier transform infrared spectrometer with a resolution of 4.00 cm⁻¹. -1 The scanning range is 4000-400cm. -1 Scan count: 32. Sampling gain: 8.0; Moving mirror speed: 0.4747; Aperture: 80.00; DTGSKBr detector; KBr beam splitter; Infrared light source.

[0183] (IV) Scanning electron microscopy analysis Take an appropriate amount of dried purified peach gum polysaccharide sample, pass it through a 100-mesh sieve, adhere it to a sample stage with conductive adhesive, cover it with a layer of conductive gold film in an ion sputtering instrument, observe it under an electron microscope, select an appropriate magnification, and take pictures of representative fields of view.

[0184] (V) Congo Red Experiment A small amount of polysaccharide sample was weighed and dissolved in pure water to prepare a 5 mg / mL polysaccharide solution. The polysaccharide solution was mixed with an equal volume of 100 μmol / L Congo red solution, and sodium hydroxide solution of different concentrations was gradually added to achieve final sodium hydroxide concentrations of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L in the mixed solution. After mixing, the solution was incubated at room temperature for 10 min, and the maximum absorption wavelength was scanned and recorded in the wavelength range of 400–600 nm. A curve was plotted with NaOH concentration on the x-axis and the maximum absorption wavelength on the y-axis.

[0185] (VI) Protein content determination Protein Standard Curve: Following the instructions of the BCA protein assay kit, BCA reagent A and reagent B were thoroughly mixed at a ratio of 50:1 to prepare an appropriate amount of BCA working solution. Then, different volumes of bovine serum albumin standard and the sample to be tested were added to each well of a 96-well plate, and the volume was brought up to 20 μL with PBS. 200 μL of freshly prepared BCA working solution was added to each well, gently shaken to mix, and then incubated at 37°C for 30 min to allow the protein to convert Cu to Cu. 2+ Reduced to Cu + It then undergoes a chelation reaction with BCA reagent to form a stable blue-purple complex. After incubation, the sample is cooled to room temperature, and the absorbance of each well is measured at 562 nm using a microplate reader. A standard curve is plotted with the standard protein concentration on the x-axis and the corresponding absorbance value on the y-axis.

[0186] Detection of protein content in purified polysaccharides: Purified peach gum polysaccharide was prepared into a solution of a specific concentration. 20 μL was added to a 96-well plate, followed by the rapid addition of 200 μL of BCA working reagent mixture. The plate was incubated at 37°C in the dark for 30 min, and the absorbance of the polysaccharide sample was measured at 562 nm. The protein content in the purified polysaccharide was calculated based on the polysaccharide solution concentration and the protein standard curve.

[0187] The specific test results are shown below: like Figure 13 As shown, polysaccharides were separated and purified using DEAE-52 column chromatography, with gradient elution using distilled water, 0.1 M, 0.2 M, and 0.3 M NaCl solutions. During this process, the concentration of purified polysaccharides was detected using the phenol-sulfuric acid method, resulting in elution curves for the DEAE-52 column. The results showed that three purified peach gum polysaccharide fractions were obtained, eluted with distilled water, 0.1 M, and 0.2 M NaCl solutions, respectively, and named PGP1, PGP2, and PGP3.

[0188] like Figure 14 As shown, after freeze-drying, PGP1 is a white powder, while PGP2 and PGP3 appear as yellow lumps.

[0189] like Figures 15A-15C , Figures 16A-16C As shown in Table 4, the elution peak shapes and elution times of the three components differ significantly. In the figure, the black line represents the multi-angle laser light scattering (LS) signal, and the red line represents the differential refractive index (dRI) signal. The consistency of the peak shapes of the two signals reflects the uniformity of the sample's molecular weight distribution. The differential refractive index (RI) detector signal is used to reflect the polysaccharide concentration distribution, and the laser light scattering (LS) detector signal is used to reflect the molecular weight. The absolute molecular weight (Molar Mass) at each time point is calculated by combining the two. Analysis is performed in conjunction with the molecular weight parameters in Table 4. The three samples show certain differences in molecular weight distribution. The PGP1 component has the earliest elution time (17.70–24.00 min), and the LS and dRI signal peaks are symmetrical and overlap well. From the absolute molecular weight distribution, the molecular weight range of PGP1 is approximately 1.36 × 10⁻⁶. 7 Up to 4.25×10 6The molecular weight of PGP3 gradually decreased with increasing elution time, exhibiting a narrow overall distribution dominated by high molecular weight components and no obvious low molecular weight tails. Its weight-average molecular weight (Mw) was the highest, reaching 2164.655 kDa, and its polydispersity index (Mw / Mn) was 1.584, close to 1, indicating that this component is a high molecular weight polysaccharide with a relatively uniform molecular weight distribution. The elution time of the PGP3 component ranged from 19.50 to 33.70 min, with a relatively concentrated peak shape. Absolute molecular weight analysis showed that the molecular weight range of PGP3 was approximately 4.26 × 10⁻⁶. 6 Up to 1.45×10 5 Da showed a significant decrease in molecular weight with prolonged elution time, exhibiting a broad molecular weight distribution, indicating the presence of a certain proportion of low to medium molecular weight components in the sample. Mw was 672.148 kDa, and the Mw / Mn ratio was 1.917, falling within the medium molecular weight range, with a relatively uniform distribution. However, the PGP2 component had the longest elution time span (19.50–34.00 min), a broad peak shape, and asymmetrical peak shapes in both LS and dRI signals. Absolute molecular weight analysis showed that the molecular weight range of PGP2 was approximately 3.55 × 10⁻⁶ kDa. 6 Up to 1.19×10 3 Da exhibits the most significant downward trend, with a very wide distribution, and a large number of low molecular weight components (below 2×10⁻⁶) appear at the tail end. 3 The molecular weight (Mw) of PGP2 is 359.903 kDa, but the molecular weight (Mn) is only 11.005 kDa, resulting in a polydispersity index as high as 32.704, which is much greater than 2. This result indicates that PGP2 has an extremely broad molecular weight distribution and poor homogeneity, suggesting that PGP2 may be a complex mixture system. This result is also consistent with the broad peak shape of PGP2 in the DEAE-52 elution curve.

[0190] Table 4

[0191] like Figures 17A-17D As shown in Table 5, the main monosaccharides of the three fermented peach gum polysaccharide components are arabinose and galactose, but the proportions of each component differ significantly. PGP1 and PGP3 have highly similar compositions, both mainly composed of arabinose and galactose, with small amounts of xylose, glucuronic acid, and mannose, resulting in high total sugar content, indicating that they are relatively pure acidic polysaccharide components. Although PGP2 is still mainly composed of arabinose and galactose, the mannose content is increased, and small amounts of glucose and fucose are detected, resulting in a lower total sugar content, suggesting the presence of more non-sugar impurities. Molar ratio analysis further confirmed the above differences.

[0192] Table 5

[0193] like Figures 18A-18CAs shown, the infrared spectra of the three polysaccharide components have similar overall profiles, all exhibiting typical polysaccharide characteristic absorption peaks, indicating that their main structure is polysaccharide. The three samples showed similar absorption peaks at 3400 cm⁻¹. -1 Strong and broad absorption peaks appeared in the vicinity, attributed to the stretching vibration of OH, which is the result of the combined action of intermolecular and intramolecular hydrogen bonds in polysaccharides, indicating the presence of a large number of hydroxyl groups in the sample; at 2930 cm⁻¹ -1 The weak absorption peaks nearby are attributed to CH stretching vibrations, suggesting the presence of alkyl chains in the polysaccharide molecule; carboxylate ions (-COO) - Asymmetric and symmetric stretching vibrations typically occur at 1610–1550 cm⁻¹. -1 and 1420~1350 cm -1 The region is characteristic of the presence of uronic acid. It is located at 1612–1641 cm⁻¹. -1 and 1407~1419 cm -1 Absorption peaks appeared in the vicinity, which is consistent with the characteristic absorption of uronic acid, indicating that uronic acid is present in all three components. This result is corroborated by the detection of glucuronic acid (Glc-UA) in the monosaccharide composition analysis; 1040 cm⁻¹ -1 The strong absorption peaks nearby indicate the presence of stretching vibrations of COC and COH, which are characteristic absorptions of sugar ring ether bonds and also mark the fingerprint region of polysaccharides; the peaks at 895–898 cm⁻¹... -1 A distinct absorption peak appears near the point where the peak is located. This peak is attributed to the out-of-plane bending vibration of CH, which is closely related to the configuration of the glycosidic bond, suggesting that the glycosidic bond may be mainly in the β-configuration.

[0194] Although the overall spectra of the three samples were similar, significant differences existed in some regions: PGP1 showed a difference at 1239.98 cm⁻¹. -1 and 1369.30 cm -1 A characteristic absorption peak appeared at 1239.98 cm⁻¹, while it was not detected in PGP2 and PGP3. -1 This can be attributed to CO stretching vibrations, possibly related to a specific type of glycosidic bond or branched structure in PGP1. PGP2 is located at 1308.85 cm⁻¹. -1 A characteristic peak appears at 1641.40 cm⁻¹, which can be attributed to the in-plane bending vibration of OH or the stretching vibration of CN. Considering the complex monosaccharide composition and low total sugar content of PGP2, this peak may be related to the non-sugar components present in PGP2. PGP3 shows a peak at 1641.40 cm⁻¹. -1 The absorption peak at [location] is higher than that of PGP1 (1612.15 cm⁻¹). -1 ) and PGP2 (1613.52 cm) -1 The significant blue shift may be related to differences in uronic acid content or bound water.

[0195] like Figures 19A-19C As shown, at magnifications of 2.00 K, 5.00 K, and 10.00 K, the surface of polysaccharide sample PGP1 was observed to be relatively rough, exhibiting a directional ordered arrangement. The formation of this microstructure may be related to the aggregation behavior of its molecular chains. Some polysaccharides can form ordered microstructures through intermolecular interactions in the solid state, which is generally beneficial for improving the mechanical properties of materials. PGP2 and PGP3 polysaccharide samples exhibit numerous interconnected pores, presenting a loose, porous network structure similar to a sponge. The formation of this porous structure is usually related to the sublimation of ice crystals during freeze-drying. Polysaccharide molecules extend in a chain-like manner in water, resulting in a high concentration of polysaccharides densely distributed in the solution. The main chain and branches of the polysaccharide connect to form a network, and water sublimation causes the polysaccharide to form a foam-like porous structure.

[0196] like Figure 20 As shown, the λmax of the purified peach gum polysaccharide-Congo red complex showed a trend consistent with the blank control when the NaOH concentration increased. No characteristic increase in λmax was observed in typical triple-helix polysaccharides under low NaOH concentrations. Therefore, the purified peach gum polysaccharide may not possess a triple-helix structure.

[0197] like Figure 21 As shown, a standard curve was plotted with the concentration of the protein standard solution on the x-axis and the absorbance at 562 nm on the y-axis, yielding the equation: y = 0.0006x + 0.0085, R0 2 =0.9991. Based on this equation, the protein content of PGP1, PGP2, and PGP3 was calculated. The results showed that the protein content of PGP1 was 8.975 mg / g, the protein content of PGP2 was 55.132 mg, and the protein content of PGP3 was 17.884 mg.

[0198] In summary, compared with the conventional water extraction, enzymatic hydrolysis, and ultrasound-assisted extraction methods reported in the literature, the mixed fermentation method of *Schizophyllum commune* and *Saccharomyces cerevisiae* used in this valve altered the molecular weight distribution characteristics of peach gum polysaccharides. This valve found that fermentation treatment actually yielded peach gum polysaccharide components with higher molecular weight (2164.655 kDa) and more uniform distribution (Mw / Mn = 1.584). This difference may stem from the preferential utilization of low molecular weight components and selective retention of high molecular weight components by microorganisms during fermentation. Additionally, extracellular polysaccharides synthesized by microorganisms in the later stages of fermentation may also have contributed to the high molecular weight components. Furthermore, scanning electron microscopy revealed that the fermented peach gum polysaccharides exhibited a loose and porous microstructure, differing from the plate-like or fibrous structure of conventionally extracted polysaccharides. This suggests that the fermentation process can effectively alter the spatial conformation of polysaccharides, enhancing their specific surface area and water absorption and swelling properties.

[0199] Test Example 4 Cellular test of fermented peach gum polysaccharide against skin photoaging Test sample: The freeze-dried component of peach gum polysaccharide prepared under the same elution procedure as in Example 19.

[0200] Test method: (I) Cell resuscitation and culture Remove the frozen cells from the liquid nitrogen tank and thaw them rapidly in a 37°C water bath, ensuring the cell cryopreservation solution is completely thawed within 1 minute. Immediately transfer the cell culture to a centrifuge tube containing 1% antibiotics and 10% FBS in DMEM complete medium and gently pipette to mix. Centrifuge at 800 rpm for 3 minutes. Discard the supernatant from the centrifuge tube, add DMEM complete medium, mix well, and then transfer the HaCaT cells to a cell culture flask. Place the flask in a cell culture incubator at 37°C and 5% CO2. Observe the cell condition and replace the complete medium with fresh medium as needed.

[0201] (II) Cell passage When the cells reach approximately 80-90% confluence with the bottom of the culture flask, discard the old culture medium. Wash twice with PBS buffer, then add 0.25% trypsin to digest the cells. Incubate in a cell culture incubator for 1-2 minutes. Observe the cell state under an inverted optical microscope. After the cells shrink and become rounded, tap the culture flask until the cells detach from the bottom and resemble quicksand. Quickly add DMEM complete culture medium to stop digestion. Transfer the cell culture to a centrifuge tube and centrifuge at 800 rpm for 3 minutes. Discard the supernatant in the centrifuge tube, add PBS, and mix the bottom precipitate by pipetting. Continue centrifuging at 800 rpm for 3 minutes. Then, discard all the supernatant in the centrifuge tube, add culture medium, mix the bottom precipitate by pipetting, and transfer to new cell culture flasks. Shake well to evenly disperse the cells in the culture flasks and incubate in a cell culture incubator.

[0202] (III) Cell cryopreservation HaCaT cells in the growth phase were removed, passaged, and then resuspended in DMEM medium for counting. 1 μL of cell suspension was placed on a cell counting plate with a coverslip, and the cell count was performed under an inverted optical microscope. The cell density was adjusted to 1 × 10⁻⁶ cells / mL. 6 ~10 7 / mL. Centrifuge at 800 r / min for 3 min, discard the supernatant, add cell cryopreservation solution, gently resuspend, transfer to sterile cryovials, and label with cell name and cryopreservation date. Seal the cryovials with sealing film, place in a programmed cooling box, and store at -80℃ for 24 h. After 24 h, transfer to liquid nitrogen.

[0203] (IV) Dosage screening of a UVB-induced HaCaT cell photoaging model HaCaT cells at 5×10 3 Cells were seeded per well in 96-well cells, with 6 replicates per group. Cells were cultured in DMEM complete medium until adherent. The old medium was discarded, and 1.0 mL of PBS was added to each well. The cells were then placed in a UV irradiation chamber for UV irradiation. UVB doses of 20, 30, 40, 50, 60, and 70 mJ / cm² were set. 2 And the unirradiated group. Immediately after UVB irradiation, the medium was replaced with DMEM complete medium and cultured for 24 h. After 24 h, CCK-8 working reagent was prepared by adding 10 μL CCK-8 to 100 μL of medium. After incubation for 40 min, the absorbance was measured at 450 nm, and cell viability was calculated using the following formula: Cell viability (%) = (A model -A k ) / (A0-A k ) × 100%; Among them, A mode A1 represents the absorbance value of cells after UVB treatment, A2 represents the absorbance value of cells in the control group, and A3 represents the absorbance value of cells in the control group. k This represents the absorbance value of the blank well.

[0204] (V) Screening of safe concentrations of purified peach gum polysaccharide for HaCaT cells HaCaT cells were processed at a rate of 5 × 10 3 The cells were seeded at a density of 6 wells into 96-well cell culture plates, with 6 replicates per group. After cell attachment, purified gum arabic (7.8125, 15.625, 31.25, 62.5, 125, 250, 500, 1000, and 2000 μg / mL) prepared in DMEM complete medium (filtered through a 0.22 μm filter) was added, and the cells were cultured in a cell incubator for 24 h. CCK-8 working reagent was prepared by adding 10 μL of CCK-8 to 100 μL of medium. After removing the old medium and washing with PBS, CCK-8 working reagent was added, and the cells were incubated for 40 min. The absorbance was measured at 450 nm, and cell viability was calculated using the following formula: Cell viability (%) = (A model -A k ) / (A0-A k ) × 100%; Among them, A model A1 represents the absorbance value of cells after UVB treatment, A2 represents the absorbance value of cells in the control group, and A3 represents the absorbance value of cells in the control group. k This represents the absorbance value of the blank well.

[0205] (VI) Protective effect of purified peach gum polysaccharide and positive control drug retinoic acid on UVB-induced cells HaCaT cells were processed at a rate of 5 × 10 3 The cells were seeded at a density of / wells into 96-well cell culture plates. After cell attachment, the cells were irradiated with an intensity of 60 mJ / cm². 2 The cells were cultured under UV light. After washing with PBS, each group was given 62.5, 125, 250, 500, and 1000 μg / mL of purified gum arabic polysaccharide prepared in DMEM complete medium, respectively. For the positive control group, 4 mg of retinoic acid was dissolved in 13.33 mL of DMSO to obtain a 1 mM stock solution, which was then diluted to 10 µM, 5 µM, 1 µM, 0.5 µM, 0.1 µM, and 0.01 µM. The cells were cultured in a cell culture incubator for 24 h. After 24 h, CCK-8 reagent was prepared by adding 10 μL of CCK-8 to 100 μL of medium. The old medium was removed, and the cells were washed with PBS. CCK-8 reagent was then added, and the cells were incubated for 40 min. The absorbance was measured at 450 nm, and the cell viability was calculated using the above formula.

[0206] The specific test results are shown below: like Figure 22 As shown, 60 mJ / cm was selected. 2 As a modeling dose for photoaging, this dose resulted in a cell survival rate of approximately 63.81%, indicating that the cells suffered significant photodamage but still retained sufficient activity to observe the protective effect of the drug. This dose falls between moderate and severe damage, which is beneficial for differentiating drug effects and avoiding unstable results due to excessively mild or severe damage.

[0207] like Figure 23 As shown, none of the three polysaccharide components exhibited significant cytotoxic effects on HaCaT cells within their respective concentration ranges. The cell survival rate of component PGP1 was above 87% at all concentrations, with the highest survival rate (137.05%) at 125 μg / mL, and it showed a trend of promoting cell proliferation at all concentrations within the range of 62.5–2000 μg / mL. The cell survival rate of component PGP2 was above 72% within the concentration range of 7.8–125–2000 μg / mL, and above 100% within the range of 125–1000 μg / mL. The cell survival rate of component PGP3 was above 89% within the concentration range of 7.8–125–2000 μg / mL, and exceeded 115% at 250–500 μg / mL. Therefore, this study selected a concentration range of 62.5–1000 μg / mL to further investigate the protective effect of the three fermented peach gum polysaccharide components against UVB-induced photoaging damage in HaCaT cells.

[0208] like Figure 24As shown, the cell survival rate in the model group decreased significantly after UVB irradiation, indicating successful modeling of photoaging damage. All three peach gum polysaccharide components exhibited certain protective effects at different concentrations. PGP1 showed a stable protective effect at all prepared concentrations. In contrast, PGP2 and PGP3 showed weaker protective effects. Therefore, PGP1 was selected as the subject of subsequent efficacy and mechanism studies. It exhibited strong protective effects at medium-to-high concentrations (250–500 µg / mL), with 250 μg / mL selected as the medium dose, 500 μg / mL as the high dose, and 125 μg / mL as the low dose. After UVB irradiation, the cell survival rate in the positive control group decreased, indicating successful modeling of photoaging damage. Retinoic acid showed significant protective effects at different concentrations. At a concentration of 1 µM, retinoic acid showed the best protective effect, at which concentration it could repair UVB-induced cell damage. While lower concentrations (0.01–0.5 µM) also showed protective effects, the efficacy was relatively weak; higher concentrations (5–10 µM) resulted in a decrease in protective efficacy, possibly related to the potential cytotoxicity of high concentrations. Therefore, 1 µM was chosen as the concentration for subsequent positive control drug administration.

[0209] Test Example 5 Test sample: The freeze-dried component of peach gum polysaccharide prepared under the same elution procedure as in Example 19.

[0210] Test method: (1) Glutathione peroxidase (GSH-Px) activity detection: HaCaT cells were processed at a rate of 4 × 10 5 Seeds were placed into 6-well cell culture plates at a density of / wells. Irradiation intensity was 60 mJ / cm². 2 After modeling with UV light, except for the blank control group and the model group, cells were cultured for 24 h with PGP1 (125, 250, 500 μg / mL) or retinoic acid (1 µM). After washing 1-2 times with PBS, cells were extracted and lysed according to the kit instructions: a small amount of 2% Triton X-100 lysis buffer was added, and cells were lysed on ice for 30-40 minutes, followed by sonication (20% power, 3 s sonication, 7 s interval, repeated 6 times). Cells were centrifuged at 4℃ for 10 min, and the supernatant was collected as the cell sample and placed on ice for analysis.

[0211] Prepare the reagent solutions according to the instructions for the glutathione peroxidase (GSH-Px) test kit, and perform the assay according to the instructions. Calculate the GSH-PX activity in cells using the following formula: GSH-PX activity (U / mL) = (A... 非酶管 -A 酶管 ) ÷ (A 标准管 -A 酶管 )×C标准 ×N÷T÷(V 样 ×Cpr); Among them, C 标准 N is the concentration of the GSH standard solution in the colorimetric reaction, N is the dilution factor of the enzyme reaction system, T is the enzyme reaction time, and V is the concentration of the standard solution in the colorimetric reaction system. 样 The sample volume for the enzyme-catalyzed reaction is Cpr, which represents the protein concentration in the homogenate (mg / mL).

[0212] (2) Detection of superoxide dismutase (SOD) activity: The experiment was conducted according to the instructions for preparing the reagent solutions and the measurement procedure of the superoxide dismutase (SOD) test kit. The SOD inhibition rate was calculated using the following formula: SOD inhibition rate (%) = 1 - (A 测定 -A 测定空白 )÷(A 对照 -A 对照空白 SOD activity (U / mg protein) = SOD inhibition rate (%) ÷ 50% × V 反总 ÷V_sample × N ÷ Cpr; Among them, V 反总 The total volume of the reaction system is given by N, the dilution factor of the sample before testing is given by V_sample, the amount of sample added in the operation table is given by Cpr, and the protein concentration of the sample homogenate is given by Cpr (mg / mL).

[0213] (3) Catalase (CAT) activity assay: The experiment was conducted according to the instructions for preparing the reagent solutions and the measurement procedure table of the catalase (CAT) test kit. The operation steps are shown in Table 6. Table 6

[0214] After mixing, transfer 200 µL of the reaction solution to each well of a 96-well plate. Preheat the microplate reader and obtain the absorbance (A) of each well at 405 nm. The calculation formula is as follows: CAT activity in cells (U / mgprot) = (A... 测定 -Α 对照 )×271÷V 样 ÷T÷Cpr; where, V 样 The sample size is T, the reaction time is Cpr, and the protein concentration of the homogenate is Cpr (mg / mL).

[0215] (4) Measurement of inflammatory factor content The levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in cell supernatant were detected using enzyme-linked immunosorbent assay (ELISA). The experimental procedures were strictly performed according to the respective kit instructions. The specific steps are as follows: Sample preparation: The cell supernatant from the culture plate was centrifuged at 1000×g for 20 minutes to remove impurities and cell debris. The supernatant was collected from the centrifuge tubes for analysis.

[0216] The specific test results are shown below: like Figure 25 As shown, compared with the blank control group, the activities of SOD, GSH-Px, and CAT in HaCaT cells of the model group were significantly decreased after UVB irradiation (p<0.001), indicating that UVB irradiation successfully induced oxidative stress in cells, leading to damage to the intracellular antioxidant defense system. Compared with the model group, the activities of intracellular antioxidant enzymes all showed varying degrees of recovery after pretreatment with different concentrations of PGP1. The effect of PGP1 on enhancing the activities of the three antioxidant enzymes showed a certain dose-dependent trend, with the 250 μg / mL medium-dose group showing the best improvement effect in all indicators, and some indicators approaching or reaching the level of the positive control drug ATRA. These results indicate that purified peach gum polysaccharide PGP1 can enhance the antioxidant defense capacity of HaCaT cells by increasing the activities of antioxidant enzymes such as SOD, GSH-Px, and CAT induced by UVB, thereby alleviating oxidative stress damage caused by UVB irradiation and exerting an anti-photoaging effect.

[0217] like Figure 26 As shown, compared with the blank control group, the levels of IL-1β, IL-6, and TNF-α in the supernatant of model group cells after UVB irradiation were significantly increased, indicating that UVB successfully induced cellular inflammatory response. After pretreatment with different concentrations of PGP1, the levels of the three inflammatory factors decreased to varying degrees in each dose group, showing a certain dose-dependent trend. The high-dose group showed the most significant decrease in inflammatory factor levels. The medium and low-dose groups also showed some anti-inflammatory effects, but the improvement was weaker than that of the high-dose group. This indicates that purified peach gum polysaccharide PGP1 can dose-dependently inhibit the overexpression of IL-1β, IL-6, and TNF-α in UVB-induced HaCaT cells, reduce cellular inflammatory response, and thus exert an anti-photoaging effect.

[0218] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for synergistic extraction of peach gum polysaccharide through mixed-culture fermentation, characterized in that, The method includes: Mixed bacterial seed liquid was inoculated into a peach gum powder solution and fermented in a shaker to obtain a fermentation broth containing peach gum polysaccharide; The mixed seed culture includes yeast and Schizophyllum commune, and the inoculation amount of the mixed seed culture is 5-25 vol%. The fermentation culture is conducted at a pH of 4-7, at a temperature of 28-43°C, on a shaking incubator at a speed of 170-230 r / min, and for a duration of 0-72 h.

2. The method for synergistic extraction of peach gum polysaccharide by mixed-culture fermentation according to claim 1, characterized in that, The inoculation volume of the mixed seed culture was 18-22 vol%. And / or, the pH of the fermentation culture is 3.5~4.5, the temperature of the fermentation culture is 26~30℃, the shaking speed of the fermentation culture is 180~200 r / min, and the fermentation culture time is 18~30 h.

3. The method for synergistic extraction of peach gum polysaccharide by mixed-culture fermentation according to claim 1, characterized in that, The method for preparing the mixed bacterial seed solution includes the following steps: Revival and activation of Schizophyllum commune: Schizophyllum commune strains were inoculated into solid culture medium for activation to obtain activated mycelia; the activated mycelia were inoculated into primary liquid culture medium for a first shaking culture, and after being dispersed, they were inoculated into secondary liquid culture medium for a second shaking culture to obtain Schizophyllum commune fermentation broth; Revival and activation of Saccharomyces cerevisiae: Saccharomyces cerevisiae dry powder and water were mixed for the first activation to obtain an activated bacterial solution; the activated bacterial solution was inoculated into a solid culture medium for the second activation to obtain activated single colonies; the activated single colonies were inoculated into a liquid culture medium and cultured with shaking to obtain Saccharomyces cerevisiae seed culture; Mixed culture: The brewer's yeast seed liquid is centrifuged and the brewer's yeast cell precipitate is collected; the brewer's yeast cell precipitate is mixed with the Schizophyllum commune fermentation broth to obtain the mixed culture seed liquid.

4. The method for synergistic extraction of peach gum polysaccharide by mixed microbial fermentation according to claim 3, characterized in that, During the resuscitation and activation process of the aforementioned Schizophyllum commune: The solid culture medium is a PDA plate culture medium; the primary liquid culture medium is Schizophyllum commune liquid culture medium; the secondary liquid culture medium is Schizophyllum commune liquid culture medium; And / or, the activation temperature is 26~30℃, and the activation time is 4~6 days; And / or, during the first shaking culture, the inoculation amount of the activated mycelium is 1-2 shovels; the temperature of the first shaking culture is 26-30℃; the rotation speed of the first shaking culture is 165-175 r / min; and the time of the first shaking culture is 3-4 days. And / or, during the second shaking culture, the inoculum volume is 9-11 vol%; the temperature of the second shaking is 26-30℃; the rotation speed of the first shaking culture is 185-195 r / min; and the time of the first shaking culture is 22-30 h.

5. The method for synergistic extraction of peach gum polysaccharide by mixed-culture fermentation according to claim 3, characterized in that, During the resuscitation and activation process of the aforementioned Saccharomyces cerevisiae: The solid culture medium is YEPD agar medium; the liquid culture medium is YEPD liquid medium; And / or, during the first activation process, the mass-to-volume ratio of the brewer's yeast dry powder to water is 1 g:(8~10) mL; the temperature of the first activation is 36~38℃; and the time of the first activation is 18~25 min. And / or, the temperature of the second activation is 28~32℃, and the time of the second activation is 15~20 h; And / or, the temperature of the shaking culture is 28~32℃, the shaking speed is 180~200 r / min, and the shaking culture time is 18~24 h.

6. The method for synergistic extraction of peach gum polysaccharide by mixed-culture fermentation according to claim 3, characterized in that, During the process of mixing bacteria: The centrifugation process is carried out at a temperature of 3-5°C, at a speed of 7000-10000 r / min, and for a time of 10-15 min. And / or, the volume ratio of the brewer's yeast seed liquid to the Schizophyllum commune fermentation broth is (0.9~1.1):(0.9~1.1).

7. The method for synergistic extraction of peach gum polysaccharide by mixed-culture fermentation according to claim 1, characterized in that, The method further includes the following post-processing steps: The fermentation broth containing peach gum polysaccharide was centrifuged to obtain the supernatant. The supernatant was subjected to protein removal treatment to obtain a deproteinized polysaccharide solution; The deproteinized polysaccharide solution was subjected to dialysis to obtain a dialyzed polysaccharide solution; The polysaccharide solution after dialysis was subjected to alcohol precipitation, the precipitate was collected and dried to obtain fermented peach gum crude polysaccharide.

8. The method for synergistic extraction of peach gum polysaccharide by mixed-culture fermentation according to claim 7, characterized in that, The centrifugation speed is 9000~10000 r / min, and the centrifugation speed is 10~30 min; And / or, the protein removal process is performed using the Sevage method; And / or, the molecular weight cutoff of the dialysis bag used in the dialysis treatment is 3500 Da; And / or, the alcohol precipitation treatment uses a 90-100 vol% ethanol solution; And / or, the method further includes the following purification step: subjecting the fermented peach gum crude polysaccharide to ion exchange column chromatography to obtain refined peach gum polysaccharide.

9. A peach gum polysaccharide, characterized in that, The peach gum polysaccharide was prepared by the method of mixed-culture fermentation and synergistic extraction of peach gum polysaccharide as described in any one of claims 1 to 8; Furthermore, the peach gum polysaccharide includes fermented peach gum crude polysaccharide and / or refined peach gum polysaccharide.

10. A method for preparing peach gum polysaccharide by mixed-culture fermentation and synergistic extraction according to any one of claims 1 to 8, or the application of the peach gum polysaccharide according to claim 9 in the preparation of anti-inflammatory products, antioxidant products, moisturizing products, wound-healing products, or anti-photoaging products.

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