Preparation method and application of beta-glucan

Abstract

The invention discloses a preparation method and application of beta-glucan, and belongs to the technical field of bioengineering.The preparation method comprises the steps that saccharomyces cerevisiae genetically engineered bacteria producing beta-glucan are treated in the mode that autolysis and high-temperature extraction are combined, mannan in yeast cell walls is removed, then specific ultrasonic treatment and impurity removal are conducted, and the beta-glucan is obtained. And the beta-glucan is prepared at a high extraction rate. After beta-glucan is prepared, carboxymethyl glucan prepared through carboxymethylation modification can be used for repairing and protecting ultraviolet radiation skin light aging, and is used for skin oxidation resistance, inflammation inhibition and skin collagen stability maintenance. The method is used for efficiently preparing the beta-glucan.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically a method for preparing β-glucan and its application. Background Technology

[0002] Saccharide β-glucan, a biomolecule with important physiological activities, is widely used in food, medicine, and health products. Its efficient preparation technology has always been a key focus of industry research. The cell wall of Saccharide yeast has a typical three-layered complex structure: an outer layer of mannan, a middle layer of protein, and an innermost layer of glucan. This structural feature causes β-glucan to be tightly encapsulated within the cell wall, binding closely to proteins, lipids, and other impurities. Therefore, the core challenge in extracting β-glucan from yeast cell walls lies in effectively removing these impurities while avoiding damage to the molecular structure of β-glucan.

[0003] Currently, common methods for preparing β-glucan from Saccharomyces cerevisiae include acid treatment, alkali treatment, combined acid-alkali methods, ultrasound-assisted methods, and composite methods combining multiple technologies. Acid treatment typically uses strong acids such as concentrated hydrochloric acid and dilute sulfuric acid to hydrolyze and disrupt the cell wall structure, removing some impurities. Alkali treatment relies on strong alkaline solutions such as sodium hydroxide to dissolve intermediate components like proteins. While combined acid-alkali methods can improve impurity removal efficiency to some extent, they are still essentially based on highly polar chemical reagents. Although physical methods such as ultrasound-assisted methods can enhance the extraction process, they often require the use of acids, alkalis, or organic solvents to achieve effective β-glucan release.

[0004] However, existing preparation technologies have significant drawbacks, specifically: Firstly, the product yield and activity are low. Strongly polar reagents such as acids, alkalis, and organic solvents not only remove impurities during the reaction process but also easily damage the glycosidic bonds of β-glucan, leading to molecular chain breakage and significantly reducing the product yield. Simultaneously, they destroy its unique physiologically active structure, affecting the application value of the final product. Secondly, there are significant operational risks and environmental hazards: strong acids and alkalis are highly corrosive, easily causing personal injury to operators during storage, transportation, and handling, and requiring specialized corrosion-resistant equipment, increasing production costs. Furthermore, improper treatment of the acid and alkali wastewater generated after the reaction can pollute soil, water bodies, and other environmental environments, which is inconsistent with the industry's trend towards green production. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing β-glucan and its application. The method involves treating genetically engineered bacteria that produce high levels of β-glucan using a combination of autolysis and high-temperature extraction, followed by ultrasonic and protease hydrolysis to achieve efficient extraction of β-glucan for the preparation of carboxymethyl glucan. To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing β-glucan includes the following steps performed sequentially: S1. Enrichment of yeast cells: Fermentation broth of genetically engineered Saccharomyces cerevisiae that produces β-glucan is collected and enriched by centrifugation; S2. Yeast autolysis and high-temperature extraction: Wash and resuspend the cells, perform autolysis reaction to obtain yeast autolysate precipitate; resuspend the yeast autolysate precipitate and extract at a high temperature of 110~130℃ to obtain wet yeast glucan. S3. Ultrasonic extraction: Treat the wet yeast glucan suspension with ultrasound at 200~300W for 30~50 minutes to obtain ultrasonic extract; S4. Enzymatic hydrolysis: Centrifuge the ultrasonic extract, resuspend the precipitate, add alkaline protease, and hydrolyze at pH 9.5-12 for 1.75-3 hours to obtain deproteinized yeast glucan. S5. Lipid removal: Lipids in the deproteinized yeast glucan were removed by Soxhlet extraction to obtain β-glucan.

[0006] As a limitation of the present invention, in the genetically engineered Saccharomyces cerevisiae... PGM2 Genes and UGP1 The gene promoter is replaced with a strong promoter, and the gene is knocked out. ALG5 Gene.

[0007] The promoter replacement and knockout are achieved using CRISPR-Cas9 gene editing technology.

[0008] During promoter replacement PGM2 The sgRNA primer for the gene is an nucleotide sequence as shown in SEQ ID NO.1, sg- PGM2 -F and sg- as shown in SEQ ID NO.2 PGM2 -R; UGP1 The sgRNA primer for the gene is an sg-nucleotide sequence as shown in SEQ ID NO.3. UGP1 -F and sg- as shown in SEQ ID NO.4 UGP1 -R.

[0009] PGM2 The sgRNA primer for the gene is an nucleotide sequence as shown in SEQ ID NO.1, sg- PGM2 -F and sg- as shown in SEQ ID NO.2 PGM2 -R; UGP1 The sgRNA primer for the gene is an sg-nucleotide sequence as shown in SEQ ID NO.3. UGP1-F and sg- as shown in SEQ ID NO.4 UGP1 -R.

[0010] Promoter replacement was achieved by preparing donor DNA, wherein the donor DNA was Donor DNA-OE- PGM2 Or Donor DNA-OE- UGP1 ; wherein, the Donor DNA-OE- PGM2 The primer nucleotide sequences are shown in SEQ ID NO. 5 and SEQ ID NO. 6; the Donor DNA-OE- UGP1 The primer nucleotide sequences are shown in SEQ ID NO.7 and SEQ ID NO.8.

[0011] This method involves designing a donor DNA template, DonorDNA-EO, containing an enhanced promoter. Using the homologous recombination repair mechanism, DonorDNA-EO and a reconstructed plasmid are introduced into cells with DNA double-strand breaks, promoting the cell's gene repair process and achieving overexpression of the target gene.

[0012] To construct a complete expression vector containing both the sgRNA expression structure and the Cas9 gene, the original plasmid was first subjected to double restriction enzyme digestion using Bcl-I and Swa-I to obtain the vector fragment. Bcl-I digestion produces sticky ends, cleaving the four bases of "GATC"; while Swa-I digestion produces blunt ends. Therefore, in primer design, the nucleotide hybridization sequence was arranged with four bases of "GATC" from 5' to 3', and a 20bp guide RNA was designed based on the target gene. scaffold The first half of the RNA (5'- scaffold RNA (this fragment contains an sgRNA structure). Finally, the vector fragment is ligated with the designed primer nucleotide hybridization sequence using T4 ligase to form a complete expression vector.

[0013] Knockout ALG5 When processing genes, the primer nucleotide sequences of Donor DNA-KO are shown in SEQ ID NO.9 and SEQ ID NO.10.

[0014] The strong promoter is Saccharomyces cerevisiae. TDH3 The promoter of the gene, the brewer's yeast is Saccharomy cescerevisiae BY4741.

[0015] As a limitation of the present invention, the resuspension is a bacterial suspension with a mass-to-volume ratio of 15% obtained by adding water.

[0016] As another limitation of the present invention, the reaction conditions for the autolysis reaction are: temperature of 55°C, pH of 5.0, and reaction time of 40 h.

[0017] As a further limitation of the present invention, the high-temperature extraction is carried out at 121°C for 6 hours.

[0018] As a further limitation of the present invention, the temperature of the ultrasound is 30°C.

[0019] The present invention also provides an application of the above-mentioned method for preparing β-glucan, for preparing carboxymethyl glucan.

[0020] As a limitation of the present invention, the carboxymethyl dextran is prepared by adding chloroacetic acid in portions to an alkalized β-glucan solution and stirring the mixture at 60°C for 5 hours.

[0021] As a further limitation of the present invention, the degree of substitution of the carboxymethyl dextran is 0.429.

[0022] As a further limitation of the present invention, the method for preparing β-glucan is used to prepare skin care products or pharmaceuticals that resist skin photoaging.

[0023] The principle of this invention is to improve the extraction efficiency of β-glucan by combining a high-yield genetically engineered strain with a specific extraction method. Specifically, this genetically engineered strain utilizes CRISPR-Cas9 technology to... PGM2 , UGP1 The gene promoter was replaced with the strong promoter pTDH3 to construct a double overexpression strain, and the gene in the double overexpression strain was knocked out. ALG5 The gene blocks UDP-glucan shunting, thereby obtaining a high-yielding β-glucan-producing strain BY4741-pTDH3- PGM2 - UGP1 - ΔALG5 .

[0024] The inventors discovered that single-expression PGM2 Gene or UGP1 In genetically engineered bacteria with β-glucan genes, β-glucan synthesis increased slightly but not significantly, while when PGM2 Genes and UGP1 When genes are overexpressed synchronously, there is a synergistic effect between the two. Compared with overexpression alone, gene expression is significantly promoted, which in turn enhances the synthesis process of cell wall β-glucan and promotes the operation of the β-glucan synthesis pathway in the cell wall synthesis pathway.

[0025] By adopting the above technical solution, the technical progress achieved by this invention compared with the prior art is as follows: This invention further optimizes the ultrasonic extraction parameters, enabling a β-glucan extraction rate of up to 5.79%. It utilizes the ultrasonic cavitation effect to efficiently disrupt the complex three-layer structure of the yeast cell wall (outer layer mannan, middle layer protein, and inner layer glucan), promoting the full release of β-glucan from the cell wall. This invention optimizes alkaline protease hydrolysis conditions and combines them with carboxymethylation modification to simultaneously improve the purity, enhance water solubility, and optimize functional activity of β-glucan. Under specific alkaline protease hydrolysis conditions, not only is the protein residue reduced to a minimum of 20.59%, but the β-glucan content also reaches a maximum of 51.63%. By precisely controlling the hydrolysis time and pH, the dynamic balance of the hydrolysis process is maintained, avoiding the reduction in extraction rate caused by enzyme inactivation or excessive substrate hydrolysis. This solves the contradiction between purity improvement and product loss in existing chemical purification methods. Furthermore, carboxymethyl glucan (CMG) is prepared through a chloroacetic acid substitution reaction, which significantly enhances molecular hydrophilicity and improves water solubility. This invention was verified through in vitro antioxidant experiments. The DPPH free radical scavenging rate of CMG reached 35.13%, and the ABTS⁺ scavenging rate reached 61.23%, which are 3.15 times and 2.62 times that of unmodified β-glucan, respectively. This significantly improved the antioxidant activity, making it more suitable for fields such as skin care products and pharmaceuticals that have high requirements for water solubility and bioactivity. Further mouse experiments showed that the carboxymethyl glucan of this invention can be used for the repair and protection of skin photoaging caused by ultraviolet radiation, and can be used for skin anti-oxidation, inhibition of inflammation and maintenance of skin collagen stability.

[0026] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings. Attached Figure Description

[0027] Figure 1 The graph shows the β-glucan extraction rate at a specific ultrasonic power in Example 1 of the efficacy verification of this invention. Figure 2 The graph shows the β-glucan extraction rate at a specific ultrasound time in Example 1 of the efficacy verification of this invention. Figure 3 The graph shows the β-glucan extraction rate at a specific enzymatic hydrolysis time in Example 2 of the efficacy verification of the present invention. Figure 4 The graph shows the β-glucan extraction rate at a specific enzymatic hydrolysis pH in Example 2 of the efficacy verification of this invention. Figure 5 The image shows the results of photographs taken on the back of mice in Example 3 of the efficacy verification of the present invention. In the image, (a) is the result of the blank group, (b) is the result of the model group, (c) is the result of the VE group, and (d) is the result of the CMG group. Figure 6 This is a diagram showing the skin characterization score results in Example 3 of the efficacy verification of the present invention; Figure 7 The image shows the results of the water content of the mouse back skin in Example 3, which is a verification example of the effectiveness of this invention. Figure 8 The following are the results of stained tissue sections in Example 3 of the efficacy verification of the present invention. In the figure, (a) is the result of the blank group, (b) is the result of the model group, (c) is the result of the VE group, and (d) is the result of the CMG group. Figure 9 The figure shows the results of the antioxidant index of skin tissue in Example 3 of the efficacy verification of the present invention. In the figure, (a) is the MDA content of mouse skin tissue, (b) is the result of the SOD enzyme activity of mouse skin tissue, (c) is the result of the CAT enzyme activity of mouse skin tissue, and (d) is the result of the GSH-Px enzyme activity of mouse skin tissue. Figure 10 The figure shows the detection results of inflammatory factors in skin tissue in Example 3 of the efficacy verification of the present invention. In the figure, (a) is the detection result of IL-6 concentration, (b) is the detection result of IL-1β concentration, and (c) is the detection result of TNF-α concentration. Figure 11 Real-time quantitative PCR in Example 3, which is a verification example of the effectiveness of this invention IL-6 , IL-1β and TNF-α The results of gene expression are shown in the figure. Figure (a) shows the expression of genes. IL-6 The gene expression results, (b) are as follows: IL-1β The gene expression results, (c) is TNF-α Results of gene expression; Figure 12 Real-time quantitative PCR in Example 3, which is a verification example of the effectiveness of this invention MMP1 and MMP9 The results of gene expression are shown in the figure. Figure (a) shows the expression of genes. MMP1 The gene expression results, (b) are as follows: MMP9 Results of gene expression. Detailed Implementation

[0028] The present invention will be further described in detail below through specific embodiments. It should be understood that the described embodiments are only for explaining the present invention and do not limit the present invention.

[0029] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available. Experimental methods not specifically described in the embodiments are generally performed under standard conditions or as recommended by the manufacturer.

[0030] Example 1: A method for preparing β-glucan This embodiment describes a method for preparing β-glucan, comprising the following steps performed sequentially: S1. Enrichment of yeast cells: Using CRISPR-Cas9 gene editing technology, the Saccharomyces cerevisiae BY4741 ( Saccharomy cescerevisiae )middle PGM2 Genes and UGP1 The original promoter of the gene was replaced with Saccharomyces cerevisiae. TDH3 Gene promoter, knockout ALG5 Genes were used to produce the β-glucan-producing genetically engineered Saccharomyces cerevisiae strain BY4741- pPGM2 - pUGP1 - ΔALG5 .

[0031] Fermentation broth obtained by culturing β-glucan-producing Saccharomyces cerevisiae genetically engineered strains in YPD medium was centrifuged at 6000 rpm for 15 min, the supernatant was removed, and the precipitate was retained to enrich the bacterial cells. S2. Yeast autolysis and high-temperature extraction: The precipitate (yeast cells) was washed multiple times with deionized water and collected. 15g of cells were weighed and added to 100mL of deionized water to prepare a 15% (w / v) bacterial suspension. The pH of the suspension was adjusted to 5.0. The suspension was placed in a 55℃ water bath for 40 hours for autolysis. After autolysis, the suspension was centrifuged at 8000rpm for 10 minutes, and the precipitate was washed again to obtain yeast autolysate precipitate. Deionized water was added back to the yeast autolysate precipitate to prepare a 15% suspension again. The suspension was placed in an autoclave and extracted at 121℃ for 6 hours. After extraction, the precipitate was washed multiple times by centrifugation to obtain wet yeast glucan, which was stored at 4℃. S3. Ultrasonic extraction: Weigh 3g of wet yeast glucan into an Erlenmeyer flask, add 20mL of deionized water and mix thoroughly to prepare a bacterial suspension with a mass-volume ratio of 15%. Under 30℃, sonicate the wet yeast glucan suspension with 200W for 40min to obtain the ultrasonic extract. S4. Enzymatic hydrolysis with proteases: Centrifuge the ultrasonic extract, discard the supernatant, wash the precipitate several times with deionized water, resuspend it with deionized water, prepare a bacterial suspension with a mass-volume ratio of 15%, add alkaline protease, fix the enzyme activity to 800 U / ml, enzymatic hydrolysis temperature is 55℃, pH is 11, and the enzymatic hydrolysis reaction is carried out for 2 hours to obtain deproteinized yeast glucan. S5. Lipid removal: Freeze-dried deproteinized yeast glucan was ground into powder, wrapped in a paper package made of folded round filter paper, and placed in a Soxhlet extraction flask. Petroleum ether was slowly added from the top of the Soxhlet extractor until it was completely siphoned into the round-bottom flask. The sample was pre-soaked in petroleum ether for 20 minutes to initially dissolve the lipids. Then, about two-thirds of the extraction flask volume of petroleum ether was added to ensure sufficient solvent for reflux extraction. The assembled Soxhlet extractor was placed in a 70°C constant temperature water bath for extraction. When the solvent boiled, the rising vapor was condensed and dripped into the extraction tube to extract the sample. When the solvent level in the extraction tube reached the top of the siphon tube, the solvent containing lipids was siphoned back into the round-bottom flask. The frequency of condensation and reflux was controlled to remove the lipids from the deproteinized yeast glucan, yielding β-glucan.

[0032] Example 2: A method for preparing β-glucan This embodiment is basically the same as Embodiment 1, except that in step S3, the wet yeast glucan suspension is ultrasonically treated with 250W for 30 minutes to obtain ultrasonic extract.

[0033] Example 3: A method for preparing β-glucan This embodiment is basically the same as Embodiment 1, except that in step S3, the wet yeast glucan suspension is ultrasonically treated with 300W for 30 minutes to obtain ultrasonic extract.

[0034] Example 4: A method for preparing β-glucan This embodiment is basically the same as Embodiment 1, except that in step S3, the wet yeast glucan suspension is ultrasonically treated with 200W for 30 minutes to obtain ultrasonic extract.

[0035] Comparative Example 1: A method for preparing β-glucan This comparative example is basically the same as Example 1, except that in step S3, the wet yeast glucan suspension is ultrasonically treated with 100W for 30 minutes to obtain an ultrasonic extract.

[0036] Comparative Example 2: A method for preparing β-glucan This comparative example is basically the same as Example 1, except that in step S3, the wet yeast glucan suspension is ultrasonically treated at 150W for 30 minutes to obtain an ultrasonic extract.

[0037] Example 1 of effect verification (1) Verification of the effect of specific ultrasonic power on improving the extraction rate of β-glucan from Saccharomyces cerevisiae: Using a glucose kit and the GOPOD double enzyme method, the absorbance was measured on a UV spectrophotometer, and the glucan content in the sample was calculated to calculate the extraction rate of β-glucan from Saccharomyces cerevisiae in the preparation methods of Examples 2-4 and Comparative Examples 1-2. The calculation formula is as follows: The calculation results of β-glucan extraction rate are as follows: Figure 1 As the ultrasonic power increased from 100W to 250W, the extraction rate of yeast glucan slowly increased, reaching a maximum of 5.79% at 250W. Subsequently, the extraction rate decreased from 250W to 300W. There was a significant difference between 200W and other powers (different letters indicate p < 0.05), indicating that the extraction rate of yeast glucan was different under different ultrasonic powers, and the degree of cavitation effect was different.

[0038] (2) Verification of the effect of specific ultrasonic time on improving the extraction rate of β-glucan from Saccharomyces cerevisiae: The ultrasonic power was fixed at 200W, the ultrasonic temperature at 30℃, and the bacterial suspension was 15%. The ultrasonic time was set at 10min, 20min, 30min, 40min, and 50min respectively. The extraction rate of β-glucan from Saccharomyces cerevisiae prepared by the corresponding β-glucan preparation method was detected. The results are as follows: Figure 2 . Figure 2 The results showed that the dextran extraction rate increased significantly from 30 min to 40 min, and reached a maximum of 5.34% at 40 min.

[0039] The above results indicate that the extraction rate of yeast glucan prepared under ultrasonic conditions according to the present invention is higher.

[0040] Example 5: A method for preparing β-glucan This embodiment is basically the same as that of Embodiment 1, except that in step S4, the enzymatic hydrolysis reaction takes 2.5 hours.

[0041] Example 6: A method for preparing β-glucan This embodiment is basically the same as that of Embodiment 1, except that in step S4, the enzymatic hydrolysis reaction takes 3 hours.

[0042] Comparative Example 3: A method for preparing β-glucan This embodiment is basically the same as that of Embodiment 1, except that in step S4, the enzymatic hydrolysis reaction takes 1 hour.

[0043] Comparative Example 4: A method for preparing β-glucan This embodiment is basically the same as that of Embodiment 1, except that in step S4, the enzymatic hydrolysis reaction is carried out for 1.5 hours.

[0044] Example 2 of effect verification (1) Verification of the effect of specific enzymatic hydrolysis time on improving the extraction rate of Saccharin from Saccharomyces cerevisiae: Using the BCA protein content assay, the absorbance of the BCA-detected samples of Examples 1, 5, 6, Comparative Example 3, and Comparative Example 4 at 562 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The protein concentration was calculated based on the standard curve. The extraction rate of Saccharin from Saccharomyces cerevisiae was then detected using the above method. The results are as follows: Figure 3 As the enzymatic hydrolysis time of Saccharomyces cerevisiae increases, the protein content gradually decreases, while the β-glucan content gradually increases. When the hydrolysis time increases from 1 hour to 2 hours, the protein content decreases significantly, reaching a minimum of 20.59% in the second hour, while the β-glucan content reaches a maximum of 48.76%.

[0045] (2) Verification of the effect of specific enzymatic hydrolysis pH on improving the extraction rate of dextran from Saccharomyces cerevisiae: The enzyme activity was fixed at 800 U / ml, the hydrolysis temperature was 55℃, and the hydrolysis time was 3h. The pH values ​​were set at 8, 9, 10, 11, and 12, and the extraction rate of dextran was detected at different pH values. The results are as follows: Figure 4 . Figure 4 The results showed that as pH increased, especially from pH 8 to 10, protein content decreased significantly while dextran content increased significantly. At pH 10, protein content reached its lowest point at 20.81%, while dextran content reached its highest point at 51.63%. Further increasing pH, with the increase in alkalinity, suppressed the trends in protein and sugar content.

[0046] The above results indicate that the enzymatic hydrolysis conditions of the present invention result in a higher extraction rate of yeast glucan.

[0047] Example 7: Application of a method for preparing β-glucan in the preparation of carboxymethyl glucan. After preparing β-glucan according to the method of Example 1, the β-glucan was dissolved in sodium hydroxide solution to dissociate the hydroxyl groups in the dextran molecules into sodium alkoxide, thereby increasing its nucleophilicity and completing the alkalization. Chloroacetic acid was added to the alkalized dextran solution (slowly added in multiple portions), and the reaction was stirred at a constant temperature of 60°C for 5 hours. During the reaction, the carboxyl methyl group of chloroacetic acid replaced the hydrogen atom on the hydroxyl group of the dextran to form carboxymethyl dextran (CMG).

[0048] Calculation of the degree of substitution of carboxymethyl glucan: Weigh 0.1 g of CMG into a 100 mL beaker. After adding 1 mL of ethanol to moisten the sample, successively add 20 mL of water and 20 mL of ammonium chloride buffer solution, and adjust the pH to 8.0 with 0.1 mol / L hydrochloric acid or sodium hydroxide solution. Then transfer the solution to a 250 mL volumetric flask, add 20 mL of copper sulfate solution, shake well, let it stand for 20 min, then dilute to the mark and shake well and filter. Take 20 mL of the filtrate, use ammonium purpurate as an indicator, and titrate with the EDTA standard solution to the end point. At the same time, conduct a blank control experiment with copper sulfate under the same conditions, and calculate the degree of substitution (DS) according to the following formula.

[0049] In the formula: C is the concentration of the EDTA standard solution; V0 is the volume of EDTA used in the copper sulfate blank; V is the volume of EDTA used for the sample; m is the mass of the sample.

[0050] After calculation, the degree of substitution of the obtained carboxymethyl glucan is 0.429.

[0051] Effect verification example 3 Establish a skin damage model by irradiating the dorsal skin of mice with ultraviolet light. Apply the extracted gene-edited and carboxymethyl-modified β-glucan, that is, CMG, to the damaged area, observe the repair situation of the mouse skin, and verify the biological activity of CMG through methods such as skin state analysis, histological analysis, antioxidant indexes of skin tissue, detection of skin tissue inflammatory factors, and relative expression levels of key genes, so as to verify that the preparation method of the present invention can be used to prepare skin care products or drugs for anti-skin photoaging.

[0052] Experimental animals: 36 healthy SPF-grade male Balb / c mice, 6 - 8 weeks old, with a body weight of about 20 g (provided by the Experimental Animal Center of Hebei Medical University, and has passed the review of the Experimental Animal Ethics Committee of the Experimental Animal Center of Hebei Medical University, license number: SCXK(Hebei)2022 - 001).

[0053] Experimental grouping: After placing 36 mice in an environment of 24°C for one week of adaptive feeding, randomly divide them into 4 groups, with 9 mice in each group, namely the blank group, the model group, the vitamin E (VE) group, and the CMG group. The mice in the blank group neither receive ultraviolet irradiation nor apply any drugs; the mice in the model group only receive ultraviolet irradiation and do not apply drugs; the mice in the VE group are smeared with VE (vitamin E) at a dose of 200 μl / day 1 h before receiving ultraviolet irradiation; the mice in the CMG group are smeared with CMG at a concentration of 10 mg / ml at a dose of 200 μl / day 1 h before ultraviolet irradiation.

[0054] The experimental results are as follows: (1) Skin condition analysis: The skin condition of mice was analyzed by observing the appearance of the skin, scoring the skin characterization, and analyzing the skin moisture content.

[0055] ① Skin Appearance Morphology Observation and Characterization Scoring: After three weekly UV irradiations, photographs were taken of the skin on the backs of mice to observe its appearance morphology. Typical results for each group are shown below. Figure 5 .

[0056] Skin appearance score: 0 points for skin that is pinkish-red, delicate, smooth, and shiny; 1 point for skin with a few red spots, wrinkles, and roughness; 2 points for skin with a lot of red spots, wrinkles, and roughness; 3 points for skin that is dry, peeling, and crusted with mild skin damage; and 4 points for skin with a lot of deep wrinkles, dryness, peeling, and crusting with severe skin damage.

[0057] Skin characterization score results as follows Figure 6 Combining observation records and Figures 5-6 The results showed that UV irradiation accelerated oxidation of mouse skin, leading to aging. Treatment with carboxymethyl dextran demonstrated the ability to inhibit wrinkles and reduce erythema. The control group mice had healthy red skin on their backs, with a rosy and glossy appearance. Fine wrinkles were present on the skin surface, but these disappeared during movement. The skin exhibited good elasticity and no erythema was observed. The model group mice, exposed only to UV irradiation without any medication, showed significant skin damage, with numerous obvious erythema, rough skin with white scales, deeper wrinkles, and significantly increased skin thickness. Their scores were much higher than the control group, demonstrating successful simulation of skin damage. Compared to the model group, the VE and CMG treatment groups had much lower scores, indicating significant improvement in skin condition. Wrinkles became shallower, only a few small erythema appeared, skin dryness was reduced, and white scales were significantly decreased, suggesting that both the VE and CMG groups had skin repair effects. Different letters above the bars in the figure indicate significant differences (p<0.05).

[0058] ② Skin moisture content analysis: The skin moisture content on the back of mice was measured using the RealBubee skin moisture meter. The results are as follows: Figure 7 The results showed that the skin water content of the normal control group mice remained at around 30%. Compared with the control group, the skin water content of the model group mice decreased significantly to 17.9%. Compared with the model group, the positive control group (VE group) and the experimental group (CMG group) increased the skin water content of mice by 26.5% and 23.3%, respectively, demonstrating that both have hydrating functions. Moreover, the improvement effect of the CMG group in increasing skin water content was close to that of the positive control group (VE group). Different letters above the bars in the figure indicate significant differences (p<0.05).

[0059] (2) Histological analysis: Skin samples were collected from each group after cervical dislocation of mice, and HE staining was performed to prepare stained tissue sections. The results are as follows: Figure 8 The VE and CMG groups demonstrated the ability to repair skin damage and restore the skin structure to its original state. In the control group, the skin cells in each layer were neatly arranged, clearly layered, with regular cell morphology, uniform nucleus size, and a normal nucleoplasm ratio. Collagen fiber bundles were uniform in thickness, tightly packed, and parallel to each other, exhibiting a wavy pattern. Skin appendages such as hair follicles, sebaceous glands, and sweat glands remained intact. In contrast, the epidermis in the model group suffered significant damage, with uneven epidermal thickness, disordered cell arrangement, abnormal cell morphology in each layer, excessive keratinization in the granular layer and stratum corneum, and extensive inflammatory cell infiltration. The connection between the dermis and epidermis was relatively straight, losing its original wavy pattern. Compared to the model group, the VE and CMG groups had slightly thinner epidermis, slightly better structural condition, reduced irregularity in cell morphology, and looser interstitial spaces, but still contained a small number of inflammatory cells, effectively reducing skin hyperplasia.

[0060] (3) Antioxidant indicators of skin tissue: Skin samples from each group were taken, added to physiological saline or PBS, and ground using a high-speed tissue homogenizer to prepare a 10% skin tissue homogenate. The malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and trace amounts of reduced glutathione (GSH-Px) in the skin tissue were measured according to the test kit instructions. MDA is a peroxidation product after oxidative damage to cells. After exposure to ultraviolet radiation, oxidative stress is enhanced, and the more severe the intracellular oxidative damage, the more MDA is generated. SOD, CAT, and GSH-Px are key antioxidant enzymes in cells to resist ROS. When the amount of ROS generated far exceeds their scavenging capacity, they will not only be continuously consumed, but also have their protein structure altered and active sites damaged in an oxidative stress environment, resulting in reduced enzyme activity.

[0061] The results are as follows Figure 9 Compared to the other three groups, the MDA content in the model group was significantly increased to 2.06 times that of the control group, while the MDA content in the VE and CMG groups was significantly decreased and close to that of the control group. Compared to the model group, the enzyme activities of SOD, CAT, and GSH-Px in the CMG group increased by 43.38%, 25.69%, and 12.99%, respectively, while the MDA content decreased by 36.9%. The activities of these three enzymes in the model group were significantly lower than those in the other three groups, and all differences were statistically significant (p < 0.05).

[0062] Under oxidative stress, both the VE and CMG groups significantly reduced the production of the peroxidation product MDA, effectively inhibiting its formation. Although the enzyme activities of the VE and CMG groups did not reach the levels of the unexposed UV control group, they both significantly improved the enzyme activity decline caused by oxidative stress in the model group, increasing enzyme activity. This indicates that the CMG group upregulated the activities of SOD, CAT, and GSH-Px enzymes, reduced MDA levels, and exhibited strong antioxidant capacity in response to oxidative stress.

[0063] (4) Detection of inflammatory factors in skin tissue: Take 10% skin tissue homogenate and use ELISA kit to determine the content of IL-6, IL-1β and TNF-α in the skin sample.

[0064] The results are as follows Figure 10 The levels of cytokines such as IL-6, IL-1β, and TNF-α in the skin tissue of mice in the model group were significantly higher than those in the control group, indicating that the stimulation was more severe and inflammatory signaling pathways had been activated, leading to the release of inflammatory factors. Compared with the model group, the levels of IL-6, IL-1β, and TNF-α in the skin tissue of mice in the CMG group were reduced by 10.68%, 15.95%, and 23.99%, respectively. The levels of anti-inflammatory factors in the VE group were also significantly reduced, approaching the levels of the control group. This indicates that the VE group and the CMG group can effectively inhibit the expression of inflammatory factors IL-6I, IL-1β, and TNF-α, and have a good effect on improving the inflammatory state, demonstrating significant anti-inflammatory effects.

[0065] (5) Relative expression levels of key genes: RNA was extracted from mouse skin tissue and subjected to real-time quantitative PCR to determine the relative expression levels of key genes. GAPDH Genes used as internal reference genes to detect matrix metalloproteinases MMP1 , MMP9 , IL-6 , IL-1β and TNF-α Gene expression status.

[0066] The results are as follows Figures 11-12Compared with the blank control group, the relative expression levels of inflammatory factors IL-6, IL-1β, and TNF-α in the skin tissue of mice in the model group showed a highly significant increase, proving that the skin injury simulation was successful. In particular, the mice in the model group suffered severe injury, and the expression levels of IL-6, IL-1β, and TNF-α in their bodies increased by 4.31 times, 2.47 times, and 2.85 times, respectively, compared with the blank group. Compared with the model group, the expression levels of these inflammatory factors in mice in the VE group and CMG group were significantly reduced, with the CMG group showing a reduction of 37.32%, 27.1%, and 33.89%, respectively. This result is highly consistent with the experimental data of ELISA measurement of inflammatory factors in mouse skin tissue, further confirming that the CMG prepared in this invention effectively inhibits the expression of inflammatory factors. All differences were statistically significant (p < 0.05).

[0067] The above results indicate that the CMG prepared by the method of the present invention has a repair and protective effect on photoaging of mouse skin under ultraviolet radiation, and can be used to prepare skin care products or pharmaceuticals that resist photoaging of the skin.

[0068] In other embodiments, step S4, where the enzymatic hydrolysis reaction is carried out for 1.75 hours or at pH 9.5, yields β-glucan, and both methods successfully produce β-glucan.

[0069] It should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing β-glucan, characterized in that, This includes the following steps performed sequentially: S1. Enrichment of yeast cells: Fermentation broth of genetically engineered Saccharomyces cerevisiae that produces β-glucan is collected and enriched by centrifugation; S2. Yeast autolysis and high-temperature extraction: Wash and resuspend the cells, perform autolysis reaction to obtain yeast autolysate precipitate; resuspend the yeast autolysate precipitate and extract at a high temperature of 110~130℃ to obtain wet yeast glucan. S3. Ultrasonic extraction: Treat the wet yeast glucan suspension with ultrasound at 200~300W for 30~50 minutes to obtain ultrasonic extract; S4. Enzymatic hydrolysis: Centrifuge the ultrasonic extract, resuspend the precipitate, add alkaline protease, and hydrolyze at pH 9.5-12 for 1.75-3 hours to obtain deproteinized yeast glucan. S5. Lipid removal: Lipids in the deproteinized yeast glucan were removed by Soxhlet extraction to obtain β-glucan.

2. The method for preparing β-glucan according to claim 1, characterized in that, In the genetically engineered Saccharomyces cerevisiae strain PGM2 Genes and UGP1 The gene promoter is replaced with a strong promoter, and the gene is knocked out. ALG5 Gene.

3. The method for preparing β-glucan according to claim 2, characterized in that, The resuspension is a bacterial suspension with a mass-to-volume ratio of 15% obtained by adding water.

4. The method for preparing β-glucan according to claim 3, characterized in that, The autolysis reaction conditions are: temperature 55℃, pH 5.0, and reaction time 40h.

5. The method for preparing β-glucan according to claim 4, characterized in that, The high-temperature extraction was carried out at 121°C for 6 hours.

6. The method for preparing β-glucan according to claim 5, characterized in that, The temperature of the ultrasound is 30°C.

7. The application of a method for preparing β-glucan according to any one of claims 2 to 6, characterized in that, Used to prepare carboxymethyl dextran.

8. The application of the method for preparing β-glucan according to claim 7, characterized in that, The carboxymethyl dextran was prepared by adding chloroacetic acid in portions to an alkalized β-glucan solution and stirring the mixture at 60°C for 5 hours.

9. The application of the method for preparing β-glucan according to claim 8, characterized in that, The degree of substitution of the carboxymethyl dextran is 0.

429.

10. The application of the method for preparing β-glucan according to claim 9, characterized in that, Used to prepare skin care products or pharmaceuticals that combat photoaging of the skin.

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