Beta-glucan produced by rhizobium fredii cc604-1 and its production method and application

Abstract

The application belongs to the technical field of microorganisms, and specifically discloses a beta-glucan produced by Bradyrhizobium sp. CC604-1 and a production method and application thereof. The production method comprises the following steps: (1) seed liquid preparation: inoculating activated Bradyrhizobium sp. CC604-1 into a seed culture medium for culture to obtain a seed liquid; the preservation number of the Bradyrhizobium sp. CC604-1 is GDMCC No. 68720; (2) fermentation culture: inoculating the seed liquid into a fermentation culture medium for two-stage culture to obtain a fermentation liquid; (3) post-treatment: performing centrifugation, purification and concentration treatment on the fermentation liquid to obtain the beta-glucan. Through a specific strain and a specific fermentation process, the application can effectively improve the yield of the beta-glucan, and the prepared beta-glucan has good moisturizing, soothing and repairing effects.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a β-glucan produced using Rhizobium pusa CC604-1, its production method, and its application. Background Technology

[0002] β-glucan is a polysaccharide composed of glucose molecules linked by β-glycosidic bonds. It is widely found in oats, yeast, fungi, and some bacteria. It possesses excellent thickening, gelling, and water-retention properties, and exhibits significant physiological activities such as enhancing immunity, anti-tumor activity, and lowering blood lipids. It is widely used in food additives, biomedicine, cosmetics, and animal feed.

[0003] Currently, industrial production mainly utilizes microbial fermentation methods using *Agrobacterium* species (such as *Alcaligenes faecalis* or *Agrobacterium radiodurans*) to produce microbial-derived β-glucan (e.g., the lectin *Curdlan*). However, existing production strains generally suffer from technical bottlenecks such as long fermentation cycles, excessively high viscosity of the fermentation solution leading to oxygenation difficulties in the later stages of fermentation, and low unit yields, significantly increasing energy consumption and extraction costs in industrial production. Therefore, screening novel fermentation strains capable of efficiently and efficiently producing β-glucan in high yields, while simultaneously matching them with optimal fermentation processes, is of significant practical importance for reducing production costs and improving product quality. Summary of the Invention

[0004] In view of the above, the present invention utilizes specific strains obtained through screening, combined with a two-stage fermentation control strategy, and simultaneously feeds in a specific manner to effectively promote cell growth and direct more metabolic flow toward the synthesis of β-glucan, thereby effectively increasing the yield of β-glucan. Furthermore, the prepared β-glucan has excellent moisturizing, soothing, and repairing effects.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for producing β-glucan using Rhizobium pusense CC604-1, the method comprising the following steps:

[0007] (1) Seed culture preparation: The activated Rhizobium pusense CC604-1 was inoculated into the seed culture medium for culture to obtain the seed culture; the Rhizobium pusense CC604-1 is classified as Rhizobium pusense, which is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with a deposit date of July 27, 2026 and a deposit number of GDMCCNo. 68720;

[0008] (2) Fermentation culture: The seed culture is inoculated into the fermentation medium and cultured in two stages to obtain the fermentation broth; wherein:

[0009] The first stage of cultivation conditions were: temperature 29–30℃, pH 6.8–7.0, aeration rate 0.6–0.8 vvm, stirring speed 250–280 rpm, and cultivation time 22–24 h; during the 8th–20th h of cultivation, Astragalus fermentation broth was added at a flow rate of 10–15 mL / h.

[0010] The second stage of cultivation conditions were as follows: temperature 26–27℃, pH 5.5–6.0, aeration rate 1.2–1.5 vvm, stirring speed 320–350 rpm, and cultivation time 40–44 h. During the 5th–30th h of cultivation, Astragalus fermentation broth and alginate oligosaccharide solution were added, with the Astragalus fermentation broth flowing at a rate of 1–2 mL / h and the alginate oligosaccharide solution flowing at a rate of 2–5 mL / h.

[0011] (3) Post-processing: Centrifuge the fermentation broth to obtain supernatant; purify and concentrate the supernatant sequentially to obtain the β-glucan.

[0012] Furthermore, in step (2), the preparation method of the Astragalus fermentation broth includes the following steps:

[0013] a) Preparation of Astragalus substrate: Astragalus powder and water were extracted twice in a water bath at a ratio of 1:10-15 (w / v), and the extracts were combined; glucose and yeast powder were added to the extract and mixed evenly to obtain the Astragalus substrate; the amount of glucose added was 4-6 g / L, and the amount of yeast powder added was 1-2 g / L.

[0014] b) Preparation of Bacillus hainanensis seed culture: Activated Bacillus hainanensis was inoculated into LB liquid medium and cultured to obtain Bacillus hainanensis seed culture;

[0015] c) Fermentation: The Bacillus hainanensis seed liquid was inoculated into the Astragalus membranaceus substrate at an inoculation rate of 4% to 5% (v / v), and fermented at 36 to 38°C and 180 to 190 rpm for 48 to 60 hours to obtain the fermented product;

[0016] d) Post-processing: The fermented product is heated to inactivate and then centrifuged. The supernatant is collected and filtered to obtain the Astragalus fermentation broth.

[0017] Furthermore, the Bacillus haynesii is Bacillus haynesii DXGZ2-6, which is classified as Bacillus haynesii. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on March 3, 2025, with accession number CGMCC No. 33710.

[0018] Furthermore, in step (2), the fermentation medium comprises the following raw material components at the following concentrations: glucose 45-60 g / L, tryptone 8-15 g / L, ammonium sulfate 1.5-2.0 g / L, potassium dihydrogen phosphate 1-1.5 g / L, dipotassium hydrogen phosphate 1-1.5 g / L, magnesium sulfate 0.5-0.7 g / L, calcium carbonate 2-3 g / L, and trace element solution 1-1.5 mL / L.

[0019] Furthermore, the trace element solution comprises the following raw material components at the following concentrations: ferrous sulfate heptahydrate 8-10 mg / L, manganese chloride tetrahydrate 8-10 mg / L, zinc sulfate heptahydrate 8-10 mg / L, and cobalt chloride hexahydrate 0.5-10 mg / L.

[0020] Furthermore, in step (2), the mass concentration of the brown algae oligosaccharide solution is 15-40 g / L.

[0021] The present invention also provides a β-glucan produced by the above method.

[0022] The present invention also provides the application of the above-mentioned β-glucan in the preparation of cosmetics with moisturizing, soothing and repairing effects.

[0023] Furthermore, the content of the β-glucan in the cosmetic is 0.1% to 10%.

[0024] The present invention has the following beneficial effects:

[0025] First, this invention utilizes *Rhizobium pusa* CC604-1, obtained through self-screening, and combines it with a two-stage dynamic fermentation strategy to effectively increase β-glucan production. In the first stage, mild environmental conditions promote rapid and abundant proliferation of *Rhizobium pusa*, quickly accumulating sufficient biomass. The second stage employs cooling and pH-lowering environmental stresses, coupled with high aeration and high rotation speed, to overcome the oxygen transfer challenges in the high-viscosity system of the later fermentation stage, thus shifting the metabolic focus of the bacteria towards polysaccharide synthesis. This "growth first, gum production later" environmental matching maximizes the synthesis efficiency and final potency of β-glucan.

[0026] Secondly, this method specifically added Astragalus fermentation broth and fucoidan during the fermentation process. The Astragalus fermentation broth, pre-decomposed by a specific Bacillus thuringiensis, provided the rhizobia with easily absorbed small-molecule active nutrients and metabolic precursors. Simultaneously, the fucoidan introduced in the second stage, as a highly efficient exogenous biological inducing factor, precisely activated the expression of polysaccharide synthesis-related enzymes within the bacteria. The synergistic addition of both broke through the yield bottleneck of the strain's conventional metabolism, significantly stimulating and promoting the excessive secretion of β-glucan.

[0027] In summary, this invention deeply integrates specific strains, precise two-stage process control, and a specific feed-in strategy, which not only achieves high efficiency and high yield of β-glucan, but also optimizes the molecular structure of the product to a certain extent. This results in β-glucan with excellent film-forming, water-retaining, anti-inflammatory, and repairing capabilities, giving it extremely high application value in cosmetics with moisturizing, soothing, and repairing effects. Attached Figure Description

[0028] Figure 1 This is a colony diagram of the Rhizobium pusense CC604-1 strain of the present invention.

[0029] Figure 2 This is a microscopic image of the Rhizobium pusense CC604-1 strain of the present invention.

[0030] Figure 3 The figures show representative fluorescence staining areas of zebrafish embryos in each group according to the present invention. In the figures, (a) is a representative fluorescence staining area of ​​zebrafish embryos in the blank control group, (b) is a representative fluorescence staining area of ​​zebrafish embryos in the model control group, (c) is a representative fluorescence staining area of ​​zebrafish embryos in the positive control group, (d) is a representative fluorescence staining area of ​​zebrafish embryos in test group 1, and (e) is a representative fluorescence staining area of ​​zebrafish embryos in test group 2.

[0031] Figure 4 A comparison chart showing the skin stratum corneum moisture content of the subjects before and after using the β-glucan essence.

[0032] Figure 5 The skin redness index of subjects before and after using β-glucan serum. Value comparison chart.

[0033] Figure 6 The image shows the changes in facial redness before and after using the β-glucan serum on subject 2.

[0034] Figure 7 The image shows the changes in facial redness before and after using the β-glucan serum on subject number 8.

[0035] Information on the preservation of biological materials

[0036] The strain information deposited in this application is as follows:

[0037] The Rhizobium pusense is Rhizobium pusense CC604-1, which is classified as Rhizobium pusense. It is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on July 27, 2026, with accession number GDMCC No: 68720.

[0038] Bacillus haynesii DXGZ2-6, classified as Bacillus haynesii, is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on March 3, 2025, with accession number CGMCC No. 33710. Detailed Implementation

[0039] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments.

[0040] In this invention, unless otherwise specified, all reagents and consumables used are purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and techniques used are conventional methods and techniques in the field.

[0041] First, this invention provides a strain of Rhizobium pusense CC604-1. This Rhizobium pusense was isolated and screened from soybean rhizosphere soil samples from Wuming, Guangxi, and underwent biochemical and molecular biological identification, the specific process of which is shown below.

[0042] 1. Isolation and screening of strains

[0043] The *Pusa rhizobium* was isolated and screened from soybean rhizosphere soil samples from Wuming, Guangxi. The specific isolation and screening methods are as follows:

[0044] Weigh 5g of soil sample and add it to 45mL of sterile water to prepare a solution with a concentration of 10. -1 The soil sample bacterial suspension was diluted in a 10-fold gradient until the bacterial suspension concentration was 10.-1 ~10 -7 After vortexing to mix thoroughly, take 100 μL of the bacterial suspension from an appropriate dilution gradient and spread it onto selection agar plates. Perform two replicates for each dilution gradient and incubate at 35°C. Select representative strains based on colony color, elevation, edge shape, and size, and streak them onto selection agar plates, incubating at 35°C. After two subcultures, Gram staining and microscopic examination are performed on strains with uniform morphology observed visually. Strains with uniform microscopic examination are considered purified and designated as CC604-1, then stored at -40°C (final concentration 25% glycerol) for later use.

[0045] 2. Biochemical identification of the strain

[0046] Observe the morphology, color, etc. of the colonies of the strain on the surface of MRS medium according to Bergey's Manual of Bacteriological Identification (9th Edition). The colony morphology diagram is shown in the figure below. Figure 1 As shown; young cultures were selected, smeared, Gram-stained, and observed under a microscope for bacterial morphology, size, Gram staining reaction, and the presence, morphology, and attachment position of spores, etc. The microscopic images are shown below. Figure 2 As shown.

[0047] 3. Molecular biological identification of the strain

[0048] Taxonomic identification of the target strain CC604-1 was performed using 16S rDNA sequence analysis.

[0049] Strain CC604-1 was sent to the Sequencing and Identification Department of Shanghai Sangon Biotech for sequencing identification. The sequencing results are as follows:

[0050]

[0051] The sequencing results were compared and analyzed in the NCBI ribosome database. 16S rDNA sequence analysis showed that the strain of this invention is *Rhizobium pusense*.

[0052] The strain was deposited, and the specific deposit information is as follows: the Rhizobium pusense is Rhizobiumpusense CC604-1, and the taxonomic name of Rhizobium pusense CC604-1 is Rhizobiumpusense. It is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on July 27, 2026, with the deposit number GDMCC No: 68720.

[0053] Secondly, this invention provides a *Bacillus haynesii* DXGZ2-6 strain, classified as *Bacillus haynesii*, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on March 3, 2025, with accession number CGMCC No. 33710. Detailed information about this strain is disclosed in Chinese Patent CN120966725B.

[0054] Furthermore, the present invention provides a β-glucan. The β-glucan is prepared using the aforementioned Rhizobiumpusense CC604-1, and the specific preparation method includes the following steps:

[0055] (1) Seed culture preparation: The activated Rhizobium pusense CC604-1 was inoculated into the seed culture medium and cultured to obtain the seed culture;

[0056] (2) Fermentation culture: The seed culture is inoculated into a fermentation medium and cultured in two stages to obtain a fermentation broth; wherein:

[0057] The first stage of cultivation conditions were: temperature 29–30℃, pH 6.8–7.0, aeration rate 0.6–0.8 vvm, stirring speed 250–280 rpm, and cultivation time 22–24 h; during the 8th–20th h of cultivation, Astragalus fermentation broth was added at a flow rate of 10–15 mL / h.

[0058] The second stage of cultivation conditions were as follows: temperature 26–27℃, pH 5.5–6.0, aeration rate 1.2–1.5 vvm, stirring speed 320–350 rpm, and cultivation time 40–44 h. During the 5th–30th h of cultivation, Astragalus fermentation broth and alginate oligosaccharide solution were added, with the Astragalus fermentation broth flowing at a rate of 1–2 mL / h and the alginate oligosaccharide solution flowing at a rate of 2–5 mL / h.

[0059] (3) Post-processing: Centrifuge the fermentation broth to obtain supernatant; purify and concentrate the supernatant sequentially to obtain the β-glucan.

[0060] Preferably, in step (1), the seed culture medium comprises the following raw material components at the following concentrations: glucose 8-12 g / L, tryptone 3-5 g / L, dipotassium hydrogen phosphate 0.5-1 g / L, magnesium sulfate heptahydrate 0.1-0.3 g / L; the culture conditions are: temperature 28-30℃, rotation speed 180-200 rpm, and culture time 24-48 h.

[0061] For example, in the seed culture medium, the concentration of glucose can be 8 g / L, 10 g / L, or 12 g / L, etc.; the concentration of tryptone can be 3 g / L, 4 g / L, or 5 g / L, etc.; the concentration of dipotassium hydrogen phosphate can be 0.5 g / L, 0.8 g / L, or 1.0 g / L, etc.; and the concentration of magnesium sulfate heptahydrate can be 0.1 g / L, 0.2 g / L, or 0.3 g / L, etc. The temperature can be 28℃, 29℃, or 30℃, etc.; the rotation speed can be 180 rpm, 190 rpm, or 200 rpm, etc.; and the culture time can be 24 h, 36 h, or 48 h, etc.

[0062] Preferably, in step (2), the fermentation medium comprises the following raw material components at the following concentrations: glucose 45-60 g / L, tryptone 8-15 g / L, ammonium sulfate 1.5-2.0 g / L, potassium dihydrogen phosphate 1-1.5 g / L, dipotassium hydrogen phosphate 1-1.5 g / L, magnesium sulfate 0.5-0.7 g / L, calcium carbonate 2-3 g / L, and trace element solution 1-1.5 mL / L.

[0063] For example, in the fermentation medium, the concentration of glucose can be 45 g / L, 50 g / L, or 60 g / L, etc.; the concentration of tryptone can be 8 g / L, 10 g / L, or 15 g / L, etc.; the concentration of ammonium sulfate can be 1.5 g / L, 1.8 g / L, or 2.0 g / L, etc.; the concentration of potassium dihydrogen phosphate can be 1.0 g / L, 1.2 g / L, or 1.5 g / L, etc.; the concentration of dipotassium hydrogen phosphate can be 1.0 g / L, 1.2 g / L, or 1.5 g / L, etc.; the concentration of magnesium sulfate can be 0.5 g / L, 0.6 g / L, or 0.7 g / L, etc.; the concentration of calcium carbonate can be 2 g / L, 2.5 g / L, or 3 g / L, etc.; and the amount of trace element solution added can be 1.0 mL / L, 1.2 mL / L, or 1.5 mL / L, etc.

[0064] More preferably, the trace element solution comprises the following raw material components at the following concentrations: ferrous sulfate heptahydrate 8-10 mg / L, manganese chloride tetrahydrate 8-10 mg / L, zinc sulfate heptahydrate 8-10 mg / L, and cobalt chloride hexahydrate 0.5-10 mg / L.

[0065] For example, in the trace element solution, the concentration of ferrous sulfate heptahydrate can be 8 mg / L, 9 mg / L, or 10 mg / L, etc.; the concentration of manganese chloride tetrahydrate can be 8 mg / L, 9 mg / L, or 10 mg / L, etc.; the concentration of zinc sulfate heptahydrate can be 8 mg / L, 9 mg / L, or 10 mg / L, etc.; and the concentration of cobalt chloride hexahydrate can be 0.5 mg / L, 5 mg / L, or 10 mg / L, etc.

[0066] For example, in the first stage of the culture conditions in step (2), the temperature can be 29℃, 29.5℃ or 30℃, etc.; the pH can be 6.8, 6.9 or 7.0, etc.; the aeration rate can be 0.6vvm, 0.7vvm or 0.8vvm, etc.; the stirring speed can be 250rpm, 260rpm or 280rpm, etc.; and the flow rate of the Astragalus fermentation broth can be 10mL / h, 12mL / h or 15mL / h, etc.

[0067] For example, in the second stage of the cultivation conditions in step (2), the temperature can be 26℃, 26.5℃ or 27℃, etc.; the pH can be 5.5, 5.8 or 6.0, etc.; the aeration rate can be 1.2vvm, 1.3vvm or 1.5vvm, etc.; the stirring speed can be 320rpm, 335rpm or 350rpm, etc.; the cultivation time can be 40h, 42h or 44h, etc.; the flow rate of the Astragalus fermentation broth can be 1.0mL / h, 1.5mL / h or 2.0mL / h, etc.; and the flow rate of the brown algae oligosaccharide solution can be 2mL / h, 3.5mL / h or 5mL / h, etc.

[0068] Preferably, in step (2), the preparation method of the Astragalus fermentation broth includes the following steps:

[0069] a) Preparation of Astragalus substrate: Astragalus powder and water were extracted twice in a water bath at a ratio of 1:10-15 (w / v), and the extracts were combined; glucose and yeast powder were added to the extract and mixed evenly to obtain the Astragalus substrate; the amount of glucose added was 4-6 g / L, and the amount of yeast powder added was 1-2 g / L.

[0070] b) Preparation of Bacillus hainanensis seed culture: Activated Bacillus hainanensis was inoculated into LB liquid medium and cultured to obtain Bacillus hainanensis seed culture;

[0071] c) Fermentation: The Bacillus hainanensis seed liquid was inoculated into the Astragalus membranaceus substrate at an inoculation rate of 4% to 5% (v / v), and fermented at 36 to 38°C and 180 to 190 rpm for 48 to 60 hours to obtain the fermented product;

[0072] d) Post-processing: The fermented product is heated to inactivate and then centrifuged. The supernatant is collected and filtered to obtain the Astragalus fermentation broth.

[0073] For example, in step a), the ratio (w / v) of the astragalus powder to water can be 1:10, 1:12, or 1:15, etc.; the amount of glucose added can be 4g / L, 5g / L, or 6g / L, etc.; and the amount of yeast powder added can be 1.0g / L, 1.5g / L, or 2.0g / L, etc.

[0074] For example, in step b), the LB liquid culture medium comprises the following raw material components at the following concentrations: tryptone 10 g / L,

[0075] Yeast extract 5 g / L, NaCl 10 g / L.

[0076] For example, in step c), the inoculation amount of the Bacillus hymeni seed liquid can be 4%, 4.5%, or 5%, etc.; the fermentation temperature can be 36℃, 37℃, or 38℃, etc.; the fermentation speed can be 180 rpm, 185 rpm, or 190 rpm, etc.; and the fermentation time can be 48h, 54h, or 60h, etc.

[0077] Preferably, the Bacillus haynesii is Bacillus haynesii DXGZ2-6.

[0078] Preferably, in step (2), the mass concentration of the alginate oligosaccharide solution is 15–40 g / L. For example, the mass concentration of the alginate oligosaccharide solution can be 15 g / L, 30 g / L, or 40 g / L, etc.

[0079] Preferably, in step (3), the centrifugation is performed at 8000-9000 r / min at 4°C for 20-25 min. For example, the centrifugation speed can be 8000 r / min, 8500 r / min, or 9000 r / min, etc.; and the centrifugation time can be 20 min, 22 min, or 25 min, etc.

[0080] Preferably, in step (3), the purification is performed by purifying the supernatant using an integrated membrane system and / or a continuous ion exchange system; the concentration is one or more of nanofiltration concentration, membrane concentration or vacuum concentration.

[0081] For example, the purification operation of the integrated membrane system is as follows: the supernatant is filtered through a ceramic microfiltration membrane with a pore size of 0.22 μm at 0.2 MPa and 25°C, and the filtrate is collected; the filtrate is then passed into an ultrafiltration membrane system with a molecular weight cutoff of 30 kDa and ultrafiltration is performed at 0.3 MPa, and the ultrafiltration retentate is collected. The purification operation of the continuous ion exchange system is as follows: the ultrafiltration retentate is pumped into the continuous ion exchange system, the influent flow rate is controlled at 3 BV / h, the operating temperature is 30°C, the effluent is collected and monitored in real time, and collection is stopped when the conductivity of the effluent drops below 200 μS / cm and the pH is neutral, thus obtaining the purified solution.

[0082] For example, the concentration is as follows: the purified solution is passed into a nanofiltration membrane system with a molecular weight cutoff of 300 Da and concentrated to 1 / 4 of its original volume under conditions of 2.0 MPa and 25°C to obtain a nanofiltration concentrate; the nanofiltration concentrate is pumped into a scraped film evaporator, and the heating temperature is controlled at 65°C and the feed rate is 50 L / h for rapid evaporation to remove water, to obtain a secondary concentrate; finally, the secondary concentrate is transferred to a vacuum concentration tank and concentrated under low temperature vacuum conditions of -0.08 MPa and 55°C until the solid mass fraction of the concentrate reaches 35%, thus obtaining a high-purity β-glucan final product.

[0083] The present invention also provides an application of the above-mentioned β-glucan in the preparation of cosmetics.

[0084] Preferably, the cosmetic is a cosmetic with moisturizing and / or soothing effects.

[0085] Preferably, the content of β-glucan in the cosmetic is 0.1% to 10%. For example, the content of β-glucan in the cosmetic can be 0.1%, 0.5%, 3%, 8%, 10%, etc.

[0086] The present application will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present application.

[0087] Example 1

[0088] This embodiment provides a β-glucan, which is produced using Rhizobiumpus ense CC604-1. The production method includes the following steps:

[0089] 1) Seed liquid preparation

[0090] (1) Preparation of seed culture medium

[0091] Weigh the following raw material components according to the following mass concentrations: glucose 10 g / L, tryptone 4 g / L, dipotassium hydrogen phosphate 0.5 g / L, and magnesium sulfate heptahydrate 0.1 g / L. Prepare the seed culture medium according to the above formula and adjust its pH to 6.8. Incubate the prepared seeds at 121℃ for 15 min and then cool for later use.

[0092] (2) Seed culture

[0093] The activated Rhizobium pusense CC604-1 was inoculated into the seed culture medium at an inoculation rate of 5% (v / v) and cultured at 28℃ and 180 rpm for 48 h to obtain the seed liquid.

[0094] 2) Fermentation culture

[0095] (1) Preparation of trace element solution

[0096] Weigh the following raw material components according to the following mass concentrations: ferrous sulfate heptahydrate 10 mg / L, manganese chloride tetrahydrate 8 mg / L, zinc sulfate heptahydrate 10 mg / L, and cobalt chloride hexahydrate 0.5 mg / L. Prepare the trace element solution according to the above formula for later use.

[0097] (2) Preparation of fermentation medium

[0098] Prepare the following raw material components at the following concentrations: glucose 50 g / L, tryptone 10 g / L, ammonium sulfate 1.5 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, calcium carbonate 3 g / L, and trace element solution 1.5 mL / L. Prepare the fermentation medium according to the above formula and adjust its pH to 7.0. Sterilize the prepared seed culture at 121℃ for 15 min, then cool for later use.

[0099] (3) Preparation of Astragalus fermentation broth

[0100] a) Preparation of Astragalus substrate: Astragalus powder and water were extracted twice in a water bath at a ratio of 1:15 (w / v), and the extracts were combined; glucose and yeast powder were added to the extract and mixed evenly to obtain the Astragalus substrate; the amount of glucose added was 5 g / L and the amount of yeast powder added was 1 g / L.

[0101] b) Preparation of Bacillus haynesii seed culture: Activated Bacillus haynesii DXGZ2-6 was inoculated into LB liquid medium at an inoculation rate of 5% (v / v) to obtain Bacillus haynesii seed culture.

[0102] c) Fermentation: The Bacillus hainanensis seed liquid was inoculated into the Astragalus membranaceus substrate at an inoculation rate of 5% (v / v) and fermented at 38°C and 180 rpm for 48 h to obtain the fermentation product;

[0103] d) Post-processing: The fermented product is heated to inactivate and then centrifuged. The supernatant is collected and filtered to obtain the Astragalus fermentation broth.

[0104] (4) Preparation of brown algae oligosaccharide solution

[0105] Prepare a solution of brown algae oligosaccharide to a concentration of 30 g / L using water, and set aside for later use.

[0106] (5) Fermentation

[0107] The seed culture was inoculated into a 5L fermenter containing fermentation medium at an inoculation rate of 8% (v / v), and cultured in two stages, wherein:

[0108] The first stage of cultivation conditions were: temperature 30℃, pH 6.8, aeration rate 0.6 vvm, stirring speed 280 rpm, and cultivation time 24 h; during the 8th to 20th h of cultivation, Astragalus fermentation broth was added at a flow rate of 15 mL / h.

[0109] The second stage of cultivation conditions were: temperature 27℃, pH 6.0, aeration rate 1.2 vvm, stirring speed 320 rpm, and cultivation time 42 h; during the 5th to 30th h of cultivation, Astragalus fermentation broth and brown algae oligosaccharide solution were added, with the Astragalus fermentation broth flowing at a rate of 1 mL / h and the brown algae oligosaccharide solution flowing at a rate of 3 mL / h.

[0110] Once fermentation is complete, the fermentation broth is obtained.

[0111] 3) Post-processing

[0112] (1) Centrifugation: Centrifuge at 8000 r / min and 4℃ for 25 min to remove bacterial precipitate and obtain supernatant;

[0113] (2) Purification: The supernatant was filtered through a ceramic microfiltration membrane with a pore size of 0.22 μm at 0.2 MPa and 25 °C, and the filtrate was collected. The filtrate was then passed into an ultrafiltration membrane system with a molecular weight cutoff of 30 kDa and ultrafiltration was performed at 0.3 MPa, and the ultrafiltration retentate was collected. The purification operation of the continuous ion exchange system was as follows: the ultrafiltration retentate was pumped into the continuous ion exchange system, the influent flow rate was controlled at 3 BV / h, the operating temperature was 30 °C, the effluent was collected and monitored in real time, and the collection was stopped when the conductivity of the effluent dropped below 200 μS / cm and the pH was neutral, and the purified solution was obtained.

[0114] (3) Concentration: The purified solution is passed into a nanofiltration membrane system with a molecular weight cutoff of 300 Da and concentrated to 1 / 4 of the original volume under the conditions of 2.0 MPa and 25°C to obtain nanofiltration concentrate; the nanofiltration concentrate is pumped into a scraped film evaporator, and the heating temperature is controlled at 65°C and the feed rate is 50 L / h for rapid evaporation to remove water, to obtain secondary concentrate; finally, the secondary concentrate is transferred into a vacuum concentration tank and concentrated under low temperature vacuum at a vacuum degree of -0.08 MPa and a temperature of 55°C to obtain β-glucan concentrate product with a solid content of 35%.

[0115] Comparative Example 1

[0116] Comparative Example 1 provides a β-glucan. The production method of the β-glucan in this comparative example differs from that in Example 1 only in step 2) fermentation culture. Step 2) does not involve two-stage fermentation or the addition of feed solution. Specifically, the seed culture is inoculated into the fermentation medium at an inoculation rate of 8% (v / v) and fermented for 64 h at a temperature of 30°C, pH 6.8, an aeration rate of 1.0 vvm, and a rotation speed of 300 rpm.

[0117] Comparative Example 2

[0118] Comparative Example 2 provides a β-glucan. The production method of the β-glucan in this comparative example differs from that in Example 1 only in step 2) of fermentation culture. Step 2) does not involve two-stage fermentation. Specifically, the seed culture is inoculated into the fermentation medium at an inoculation rate of 8% (v / v) and fermented for 64 h at a temperature of 30°C, pH 6.8, aeration rate of 1.0 vvm, and rotation speed of 300 rpm. During the 8th to 20th h of culture, Astragalus fermentation broth is added at a flow rate of 15 mL / h. During the 29th to 54th h, Astragalus fermentation broth and a fucoidan solution are added at a flow rate of 1 mL / h and 3 mL / h, respectively.

[0119] Comparative Example 3

[0120] Comparative Example 3 provides a β-glucan. The production method of the β-glucan in this comparative example differs from that in Example 1 only in step 2) of fermentation culture. In step 2), the Astragalus fermentation broth and the brown algae oligosaccharide solution are not added. Specifically, the seed culture is inoculated into the fermentation medium at an inoculation rate of 8% (v / v) and cultured in two stages. The culture conditions for the first stage are: temperature 30℃, pH 6.8, aeration rate 0.6 vvm, stirring speed 280 rpm, and culture time 24 h. The culture conditions for the second stage are: temperature 27℃, pH 6.0, aeration rate 1.2 vvm, stirring speed 320 rpm, and culture time 42 h.

[0121] Comparative Example 4

[0122] Comparative Example 4 provides a β-glucan. The production method of the β-glucan in this comparative example differs from that in Example 1 only in step 2) of fermentation culture. In step 2), Astragalus fermentation broth is not added. Specifically, the seed culture is inoculated into the fermentation medium at an inoculation rate of 8% (v / v) and cultured in two stages. The culture conditions for the first stage are: temperature 30℃, pH 6.8, aeration rate 0.6 vvm, stirring speed 280 rpm, and culture time 24 h. The culture conditions for the second stage are: temperature 27℃, pH 6.0, aeration rate 1.2 vvm, stirring speed 320 rpm, and culture time 42 h. During the 5th to 30th h of culture, a fucoidan solution is added at a flow rate of 3 mL / h.

[0123] Comparative Example 5

[0124] Comparative Example 5 provides a β-glucan. The production method of the β-glucan in this comparative example is different from that in Example 1, except for step 2) fermentation culture. Specifically, the preparation method of Astragalus fermentation broth in steps 2) and 3) is different. The preparation method of Astragalus fermentation broth is as follows: Bacillus hainanensis is replaced with Bacillus subtilis. That is, the Astragalus fermentation broth of Comparative Example 3 is the Astragalus fermentation broth obtained by fermentation with Bacillus subtilis.

[0125] Comparative Example 6

[0126] Comparative Example 6 provides a β-glucan. The production method of the β-glucan in this comparative example differs from that in Example 1 only in step 2) of fermentation culture. Specifically, step 2) does not involve the addition of alginate solution. Instead, the seed culture is inoculated into the fermentation medium at an inoculation rate of 8% (v / v) and cultured in two stages. The first stage culture conditions are: temperature 30℃, pH 6.8, aeration rate 0.6 vvm, stirring speed 280 rpm, and culture time 24 h. During the 8th to 20th h of culture, Astragalus fermentation broth is added at a flow rate of 15 mL / h. The second stage culture conditions are: temperature 27℃, pH 6.0, aeration rate 1.2 vvm, stirring speed 320 rpm, and culture time 42 h. During the 5th to 30th h of culture, Astragalus fermentation broth is added at a flow rate of 1 mL / h.

[0127] Comparative Example 7

[0128] Comparative Example 7 provides a β-glucan. The production method of the β-glucan in this comparative example differs from that of Example 1 only in step 2) of fermentation culture. Specifically, the timing of adding the fucoidan solution in step 2) is different. Specifically, the seed culture is inoculated into the fermentation medium at an inoculation rate of 8% (v / v) and cultured in two stages. The first stage culture conditions are: temperature 30℃, pH 6.8, aeration rate 0.6 vvm, stirring speed 280 rpm, and culture time 24 h. During the 8th to 20th h of culture, Astragalus fermentation broth and fucoidan solution are added, with the Astragalus fermentation broth flowing at a rate of 15 mL / h and the fucoidan solution flowing at a rate of 3 mL / h. The second stage culture conditions are: temperature 27℃, pH 6.0, aeration rate 1.2 vvm, stirring speed 320 rpm, and culture time 42 h. During the 5th to 30th h of culture, Astragalus fermentation broth is added, with the Astragalus fermentation broth flowing at a rate of 1 mL / h.

[0129] Test 1: β-glucan production detection

[0130] The β-glucan content in the supernatants prepared in Example 1 and Comparative Examples 1-6 was detected by the alcohol precipitation-phenol sulfuric acid method. The detection results are shown in Table 1 below.

[0131] Table 1. Effects of different production processes on β-glucan yield

[0132]

[0133] As shown in Table 1, the preparation process used in Example 1 of this invention significantly increased the yield of β-glucan, reaching 50.6 g / L, demonstrating a significant increase in yield. Compared to Example 1, the yield of Comparative Example 1 decreased significantly because fermentation with a single environmental parameter resulted in most of the carbon source being consumed in the later stages of cell growth to maintain cell life activities, preventing the metabolic flux from efficiently shifting to β-glucan synthesis. Compared to Comparative Example 1, Comparative Example 3 achieved a slight increase in yield through two-stage regulation, but it was still far lower than that of Example 1. This is most likely because, under the environmental stress of the second stage, although the cells initiated a highly efficient sugar production mode, the lack of external addition of specific nutrients caused the cells to prematurely enter the death phase, leading to the premature termination of polysaccharide synthesis.

[0134] Although feedstock was added in Comparative Example 2, the lack of a two-stage culture condition resulted in the added substance being preferentially used by the bacteria for cell division and proliferation, rather than for the synthesis of protective extracellular polysaccharides, thus no significant increase in yield. Comparative Examples 4 and 6, with only a single substance added, resulted in cells either lacking precursor synthesis or lacking synthetic signal stimulation, thus limiting yield. Comparative Example 5, with the addition of Astragalus membranaceus fermentation broth, showed a reduced yield. This may be because the Bacillus haynesii DXGZ2-6 strain specifically screened in this invention can precisely release characteristic active peptides and oligosaccharides most suitable for absorption by Rhizobium pusa, while other strains may lead to incomplete substrate degradation or the production of unsuitable metabolic byproducts, thereby limiting the nutrient absorption and utilization rate of Rhizobium pusa, and consequently limiting yield. In Comparative Example 7, adding fucoidan in the first stage in advance resulted in a significant decrease in yield. This may be because introducing fucoidan too early interferes with normal cell division, leading to insufficient accumulation of total viable cells (biomass) at the end of the first stage, resulting in a lower final total sugar production.

[0135] In summary, this invention achieves rapid accumulation of bacterial biomass by switching temperature, pH, and dissolved oxygen in two stages (the growth phase and the sugar production phase), combined with the first stage of high-dose fed-in Astragalus fermentation broth specifically degraded by Bacillus thuringiensis. Secondly, under the superimposed environmental stress of the second stage, fucoidan is precisely introduced as an exogenous signal inducer, while a very low flow rate of Astragalus fermentation broth is used to maintain precursor supply. The synergistic effect of these two methods specifically activates the extracellular polysaccharide synthesis pathway of Rhizobium pusa, effectively increasing β-glucan production.

[0136] Test 2: β-glucan repair efficacy test

[0137] To further investigate the effect of fed-batch processing on the efficacy of β-glucan, the repair effects of the β-glucan prepared in Example 1 and Comparative Example 3 were tested. The specific testing methods are as follows:

[0138] 1. Experimental Principle

[0139] Repair refers to actions that help maintain the skin's normal state, keep it hydrated, and reduce the irritation and damage caused by external factors. Damaged skin barriers increase the skin's sensitivity to external stimuli, a major cause of erythema, dryness, peeling, and itching. In daily life, various factors such as sunlight and chemical irritants can damage the skin barrier, leading to increased skin permeability. Therefore, repairing the skin barrier and reducing external irritation is a crucial way to maintain normal skin condition. Using non-specific fluorescent staining reagents, changes in zebrafish skin barrier permeability can be efficiently observed, showing a positive correlation between the fluorescent area of ​​zebrafish embryonic skin and skin barrier permeability. Therefore, this experiment aims to construct a zebrafish embryonic skin barrier damage model, use fluorescent staining reagents to label skin permeability, and evaluate the activity of the test samples in inhibiting skin permeability by testing the red fluorescent area of ​​zebrafish skin, thereby reflecting the skin barrier repair efficacy of the test samples.

[0140] 2. Reagent preparation

[0141] (1) Fish embryo culture medium

[0142] This solution was prepared by dissolving 2940 mg of anhydrous calcium chloride, 1233 mg of magnesium sulfate heptahydrate, 630 mg of sodium bicarbonate, and 55 mg of potassium chloride in 10 L of water. The pH value is 6.5–8.5. All chemicals are of analytical grade.

[0143] (2) Ectoin solution

[0144] Prepare a 1.5 g / L stock solution and store it under refrigeration. The working concentration is 0.15 g / L; prepare fresh before use.

[0145] (3) Gentian Violet Solution

[0146] Weigh 30 mg of gentian violet and dissolve it in 15 mL of embryo buffer to obtain a 2 mg / mL stock solution. Working solution: Dilute the stock solution 100 times and prepare fresh before use.

[0147] (4) β-glucan solution

[0148] Weigh 25g of the β-glucan described in Example 1 and Comparative Example 3 respectively, and dissolve it in 1L of distilled water to obtain 25g / L β-glucan solutions of the examples and the comparative examples.

[0149] 3. Test organism

[0150] A healthy zebrafish, 2 days after fertilization.

[0151] 4. Experimental Procedure

[0152] (1) Test group

[0153] The following groups need to be set up for the test:

[0154] Blank control group: 24 zebrafish were randomly selected, transferred to 6-well plates, and 6 mL of fish embryo culture medium was added.

[0155] Model control group setup: 24 zebrafish were randomly selected, transferred to 6-well plates, and 6 mL of fish embryo culture medium was added.

[0156] Positive control group setup: 24 zebrafish were randomly selected, transferred to a 6-well plate, and 6 mL of ectoine working solution was added.

[0157] Test group 1: 24 zebrafish were randomly selected, transferred to a 6-well plate, and 6 mL of the β-glucan solution from the example were added.

[0158] Group 2: 24 zebrafish were randomly selected, transferred to a 6-well plate, and 6 mL of comparative β-glucan solution was added.

[0159] (2) Test group processing

[0160] After culturing the 6-well plates of each test group in a constant temperature shaker at 28±1℃ for 4 hours, they were washed twice with fish embryo culture medium. The blank control group was given 6 mL of gentian violet working solution, and each well of the other groups was given 6 mL of a mixed solution of gentian violet and lactic acid (LA) (modeling agent) (the concentration of gentian violet was the same as that of the blank group, and the final concentration of lactic acid was 1.1 g / L). The plates were then placed in a constant temperature shaker at 28℃±1℃ for 20 min.

[0161] (3) Microscopic analysis of the sample

[0162] Embryos from each test group were aspirated and placed in 6-well plates, and washed with embryo buffer until the solution was colorless. After processing, the fish were placed on their sides. The fluorescent area of ​​the zebrafish was photographed under a fluorescence microscope using uniform parameters.

[0163] (4) Data and Result Calculation

[0164] Use analysis software such as ImageJ to mark the tail of each zebrafish, then select "Measure Area" in the "Measure" column, and select the "Area" in the test results as the degree of light damage.

[0165] (5) Calculation of results

[0166] Calculate the skin barrier permeability inhibition rate:

[0167] Skin barrier permeability inhibition rate = (fluorescent area of ​​model group - fluorescent area of ​​test group) / fluorescent area of ​​model group.

[0168] 5. Experimental Results

[0169] The repair efficacy of each group of zebrafish can be found in the table below. Figure 3 And Table 2.

[0170] Figure 3 The image shows representative areas of fluorescent staining in zebrafish embryos from each group. In the image, lesions are stained with red fluorescence.

[0171] Table 2. Statistical results of relative fluorescence area of ​​zebrafish in each group.

[0172]

[0173] In the table: **** (P < 0.0001) indicates a highly significant difference between the test group or positive group and the model group; *** (P < 0.001) indicates a significant difference between the test group or positive group and the model group; #### (P < 0.0001) indicates a highly significant difference between the model group and the blank group.

[0174] Combination Figure 3 As shown in Table 2, under the experimental conditions, when the β-glucan concentration in both groups was 25 g / L, the relative fluorescence area compared to the model group decreased (P < 0.05), indicating that the test substance at this concentration had a beneficial effect on lactic acid-induced skin barrier damage and was superior to the positive control group. This means that the β-glucan prepared in Example 1 and Comparative Example 3 of this invention both have good repair effects, demonstrating that the β-glucan produced by fermentation using specific Rhizobium pusense CC604-1 in this invention has a good repair effect. Meanwhile, the repair effect of test group 1 was 7 percentage points higher than that of test group 2, showing a significantly better effect. This may be because the fed-batch material, especially the specific Astragalus fermentation broth, contains a large number of small active Astragalus molecules (such as oligosaccharides, polypeptides, and astragaloside A precursors) released by Bacillus thuringiensis transformation. During the secretion of β-glucan by Rhizobium pusa, these active small molecules are tightly embedded or encapsulated within the three-dimensional gel network of β-glucan, forming a microscopic "polysaccharide-Astragalus micro-active ingredient complex". When applied to damaged sites, the three-dimensional network of β-glucan not only provides a microscaffold for cell migration but also continuously and slowly releases the internally embedded Astragalus active ingredients, thereby enhancing its repair effect.

[0175] Test 3: Detection of the moisturizing and soothing effects of β-glucan

[0176] To further investigate the efficacy of the β-glucan of this invention, the moisturizing and soothing effects of the β-glucan prepared in Example 1 were tested. The specific testing methods are as follows:

[0177] 1. Preparation of β-glucan essence

[0178] The β-glucan prepared in Example 1 above was applied to a serum to obtain the serum. The specific serum formula is shown in Table 3 below:

[0179] Table 3. Serum Formula (%, w / w)

[0180]

[0181] The preparation method of the above-mentioned essence is as follows: According to the formula table, accurately weigh the deionized water of phase A, add it to the emulsification pot, start stirring, and heat to 80°C; premix phases B and C evenly, slowly add them to the water of phase A, and stir homogenously for 2 minutes until completely hydrated and dissolved to form a transparent gel matrix, then cool to below 45°C; add phase D to the above system and stir evenly; add each component of phase E in sequence, stir at medium speed for 10 minutes until the system is uniform and clear, discharge the material and let it stand to remove bubbles, thus obtaining the β-glucan essence.

[0182] 1. Subjects

[0183] Subjects were selected based on their facial condition (those who reported having one of the following symptoms of impaired skin barrier function: dryness, peeling, redness, etc.) and other individual health conditions. A total of 30 subjects were selected and completed the study, with 0 dropouts. Among them, there were 28 females and 2 males, with an average age of 37.4 ± 6.0 years, meeting the subject voluntary inclusion criteria.

[0184] 2. Experimental Design

[0185] After cleansing, participants should apply an appropriate amount of the product evenly to their face and gently massage until absorbed. This should be done twice a day for 28 days. The testing period is from July 8, 2025 to August 25, 2025.

[0186] 3. Testing

[0187] The effectiveness was evaluated using a self-comparison principle before and after the event.

[0188] (1) Testing basis

[0189] "China Industry-University-Research Collaboration Promotion Association Group Standard: Test Methods for Seven Efficacy Items in Cosmetics (Anti-wrinkle, Firming, Moisturizing, Oil Control, Repairing, Nourishing, and Soothing)" T / CAB 0152-2022

[0190] (2) Instruments and equipment

[0191] The Corneometer CM825 skin moisture test probe and the VISIA facial image analyzer both meet the performance requirements of the testing specifications.

[0192] (3) Testing environment

[0193] The instrument testing was conducted in an environment with a temperature of 21±1 °C and a relative humidity of 50±10% RH. Subjects were required to acclimatize to this environment for 30 minutes before the assessment and testing could proceed.

[0194] (4) Testing process

[0195] ① Cleaning: Before the test, cleanse your face with a cleansing product (Cetaphil facial cleanser) and dry it with a lint-free and fluorescent agent-free tissue; repeat the cleansing process for each follow-up visit.

[0196] ② Technicians sequentially performed facial image acquisition and measured baseline skin parameters such as VISIA and skin stratum corneum moisture content. During data acquisition, the operator needed to ensure the subject was in a comfortable position to measure skin parameters in the test area. Each test probe was in complete contact with the skin and kept perpendicular during testing, and data was recorded. The same tests were repeated after 2 weeks (D14) and 4 weeks (D28) of sample use, and all values ​​were averaged.

[0197] (5) Statistical methods

[0198] SPSS software was used for statistical analysis of the data. Quantitative data were expressed as mean ± standard deviation and subjected to a normality test. If the data met the requirements of a normal distribution, a paired t-test was used for comparisons of the data before and after the same data point; otherwise, a rank-sum test of two related samples was used.

[0199] 4. Test Results

[0200] (1) Results of skin stratum corneum moisture content and skin redness index before and after the subjects used β-glucan essence. The statistical analysis of the test results is shown in Tables 4 and 5, respectively.

[0201] Table 4 Statistical analysis of skin stratum corneum moisture content test results

[0202]

[0203] Table 5 Skin Redness Index Statistical analysis of value test results

[0204]

[0205] (2) Comparison of skin stratum corneum moisture content and skin redness index before and after the subjects used β-glucan essence. The values ​​are as follows: Figure 4 and Figure 5 As shown in the figure. The significance level is indicated by the following methods: "ns" indicates no statistical difference, P>0.05; " "Indicates a significant difference, 0.01 ≤ P < 0.05;" "P" indicates a significant difference, 0.001 ≤ P < 0.01; "" indicates a significant difference, P<0.001.

[0206] (3) The changes in facial redness in subjects 2 and 8 after 28 days of using β-glucan essence are as follows: Figure 6 and Figure 7 As shown.

[0207] The above charts show that after 28 days of use, the β-glucan serum significantly improved the moisture content of the stratum corneum in the test areas of the subjects (P < 0.05), indicating that the sample has moisturizing effects. Simultaneously, the skin redness index in the test areas of the subjects... The value showed a significant improvement (P < 0.05), indicating that the sample has a soothing effect. That is, under the conditions of this experiment, the β-glucan essence has moisturizing and soothing effects.

Claims

1. A method for producing β-glucan using Rhizobium pusense CC604-1, characterized in that, The method includes the following steps: (1) Seed liquid preparation: The activated Rhizobium pusense CC604-1 was inoculated into the seed culture medium for culture to obtain the seed liquid; the preservation number of Rhizobium pusense CC604-1 is GDMCC No.68720. (2) Fermentation culture: The seed culture is inoculated into the fermentation medium and cultured in two stages to obtain the fermentation broth; wherein: The first stage of cultivation conditions were: temperature 29–30℃, pH 6.8–7.0, aeration rate 0.6–0.8 vvm, stirring speed 250–280 rpm, and cultivation time 22–24 h; during the 8th–20th h of cultivation, Astragalus fermentation broth was added at a flow rate of 10–15 mL / h. The second stage of cultivation conditions were as follows: temperature 26–27℃, pH 5.5–6.0, aeration rate 1.2–1.5 vvm, stirring speed 320–350 rpm, and cultivation time 40–44 h. During the 5th–30th h of cultivation, Astragalus fermentation broth and alginate oligosaccharide solution were added, with the Astragalus fermentation broth flowing at a rate of 1–2 mL / h and the alginate oligosaccharide solution flowing at a rate of 2–5 mL / h. (3) Post-processing: Centrifuge the fermentation broth to obtain supernatant; purify and concentrate the supernatant sequentially to obtain the β-glucan.

2. The method as described in claim 1, characterized in that, In step (2), the preparation method of the Astragalus fermentation broth includes the following steps: a) Preparation of Astragalus substrate: Astragalus powder and water were extracted twice in a water bath at a ratio of 1:10-15 (w / v), and the extracts were combined; glucose and yeast powder were added to the extract and mixed evenly to obtain the Astragalus substrate; the amount of glucose added was 4-6 g / L, and the amount of yeast powder added was 1-2 g / L. b) Preparation of Bacillus hainanensis seed culture: Activated Bacillus hainanensis was inoculated into LB liquid medium and cultured to obtain Bacillus hainanensis seed culture; c) Fermentation: The Bacillus hainanensis seed liquid was inoculated into the Astragalus membranaceus substrate at an inoculation rate of 4% to 5% (v / v), and fermented at 36 to 38°C and 180 to 190 rpm for 48 to 60 hours to obtain the fermented product; d) Post-processing: The fermented product is heated to inactivate and then centrifuged. The supernatant is collected and filtered to obtain the Astragalus fermentation broth.

3. The method as described in claim 2, characterized in that, The Bacillus haynesii species mentioned is Bacillus haynesii DXGZ2-6, with accession number CGMCC NO.33710.

4. The method as described in claim 1, characterized in that, In step (2), the fermentation medium comprises the following raw material components at the following concentrations: glucose 45-60 g / L, tryptone 8-15 g / L, ammonium sulfate 1.5-2.0 g / L, potassium dihydrogen phosphate 1-1.5 g / L, dipotassium hydrogen phosphate 1-1.5 g / L, magnesium sulfate 0.5-0.7 g / L, calcium carbonate 2-3 g / L, and trace element solution 1-1.5 mL / L.

5. The method as described in claim 4, characterized in that, The trace element solution comprises the following raw material components at the following concentrations: ferrous sulfate heptahydrate 8-10 mg / L, manganese chloride tetrahydrate 8-10 mg / L, zinc sulfate heptahydrate 8-10 mg / L, and cobalt chloride hexahydrate 0.5-10 mg / L.

6. The method as described in claim 1, characterized in that, In step (2), the mass concentration of the brown algae oligosaccharide solution is 15-40 g / L.

7. The β-glucan produced by the method according to any one of claims 1-6.

8. The use of the β-glucan as described in claim 7 in the preparation of cosmetics with moisturizing, soothing and repairing effects.

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