Method for producing soluble beta-glucan by using inert carrier

Abstract

The invention provides application of an inert immobilization carrier in preparation of soluble beta-glucan and a preparation method of the inert immobilization carrier. The inert immobilization carrier is at least one of rice bran, wheat straw, wheat bran, corncob, loofah sponge, bagasse, activated carbon, a polypropylene material and reticular polyurethane foam. The method comprises the steps of carrier pretreatment, strain activation, seed culture and fermentation sugar production. According to the method disclosed by the invention, the yield of beta-glucan (beta-1, 3-1, 6-LG) is greatly improved through optimization of an inert immobilized carrier and culture conditions. The inert immobilized carrier is beneficial to absorption of nutrient substances and synthesis and secretion of secondary metabolites, high fructose corn syrup is adopted as an alternative carbon source, the yield is increased, the cost is reduced, and the method has a good industrial application prospect. Compared with the prior art, the method has great breakthrough, has the advantages of low fermentation cost, high yield, strong technical economy, continuous and repeated production and the like, and can be applied to industrial production.

Description

Technical Field

[0001] This invention belongs to the field of fermentation engineering technology, specifically relating to a method for producing β-glucan by immobilizing cells using an inert carrier. Background Technology

[0002] β-Glucan is a naturally occurring polysaccharide with important biological activities, composed of glucose monomers linked by β-glycosidic bonds. It is widely found in the cell walls of yeast, cereals (such as oats and barley), fungi (such as shiitake mushrooms and Ganoderma lucidum), and certain bacteria. Its molecular structure varies depending on its source, with common bond types including β-1,3, β-1,4, or β-1,6 glycosidic bonds. It shows great promise for applications in the food, pharmaceutical, and cosmetic fields.

[0003] Aureobasidium pullulans is one of the commonly used fungal species for producing β-glucan. However, most current data mention that Aureobasidium pullulans is mainly used to produce pullulan, polymalic acid, and melanin. Only a few studies mention that Aureobasidium pullulans can also secrete extracellular β-glucan.

[0004] Currently, the main industrial production of β-glucan involves extraction from natural sources such as grains, edible fungi, and yeast. These methods typically require cell wall disruption, which is complex and costly. It is worth noting that the cell walls of *Brachystomia melanogaster* also contain small amounts of high-molecular-weight, low-water-soluble β-glucan (β-1,3-1,6-HG), requiring high-temperature hydrolysis to convert it into a water-soluble form, further increasing the complexity of the process. The current fermentation yield of β-glucan from *Brachystomia melanogaster* remains low, severely restricting its large-scale production and subsequent application development.

[0005] In existing patented technologies, agricultural processing byproducts such as rice bran, wheat bran, and wheat straw are commonly used as nutrients in the preparation of microbial culture media. For example, Chinese patent application number CN202110973243.3, filed on August 24, 2021, discloses a high-β-glucan-producing melanin-producing *Syngonium brevichorum* strain and its applications. The liquid fermentation medium used in this application contains 0.3%–1.0% (w / v) of agricultural byproducts, 3%–10% (w / v) of sucrose, and a pH of 6–7. This existing technology primarily utilizes these agricultural byproducts to provide carbon, nitrogen, and other nutrients for microbial growth.

[0006] To increase yield and control costs, existing technologies mainly focus on optimizing the composition of fermentation media. In media optimization, the selection of carbon source is a key factor. Existing patented technologies mostly use glucose or sucrose as the main carbon source. For example, Chinese patent application number CN201710142161.8, filed on March 7, 2017, discloses a method for producing β-glucan by fermentation of *Acer cinnamomea*, using glucose as the carbon source. Although the yield reaches 17 g / L, the culture period is as long as 8 days. In addition, existing technologies also disclose techniques to increase yield through gene knockout. For example, Chinese patent application number CN202510372757.1, filed on March 27, 2025, discloses a melanin-producing *Brachystomata* Kxk gene knockout mutant strain and its construction method and application. The Kxk knockout strain, cultured for 144 hours, achieved a β-glucan yield of 4.84 g / L.

[0007] Therefore, existing technologies rely on traditional carbon sources, making it difficult to achieve significant breakthroughs in yield while controlling costs. Thus, developing a β-glucan production process that can balance high yield, high stability, and low cost has become an urgent problem to be solved in this field. Summary of the Invention

[0008] The purpose of this invention is to prepare extracellular secretory β-glucan (*Brachystomia melanogaster* β-G) using *Brachystomia melanogaster* without disrupting the cell wall, obtaining soluble β-1,3-1,6-LG through direct centrifugation of the fermentation broth. This invention has the advantages of low production cost and simple separation and purification.

[0009] Another objective of this invention is to provide the application of agricultural by-products in the preparation of extracellular secretory soluble β-glucan, particularly the application of agricultural by-products in the preparation of extracellular secretory soluble β-1,3-1,6-LG. This invention is the first to propose the use of agricultural by-products as inert immobilization carriers in the fermentation process, rather than the carbon source, nitrogen source, and other nutrients used in traditional fermentation processes. The function of the agricultural by-products in the fermentation system of this invention is fundamentally different from that in the prior art.

[0010] The purpose of this invention is to provide a method for producing soluble β-glucan by immobilizing melanin-producing short-skinned sclerotium cells. This method, through carrier pretreatment, strain activation, seed culture, and fermentation sugar production, does not introduce toxic or harmful substances, thus achieving environmental friendliness. By introducing an immobilized carrier, the fermentation cycle is shortened, dissolved oxygen is increased, and the conversion rate of β-glucan is effectively improved.

[0011] To achieve the above objectives, the present invention provides the application of an inert immobilization carrier in the preparation of soluble β-glucan, wherein the inert immobilization carrier is at least one of rice bran, wheat straw, wheat bran, corn cob, loofah sponge, sugarcane bagasse, activated carbon, polypropylene material, and reticulated polyurethane foam.

[0012] Preferably, the present invention provides the application of an inert immobilized carrier in the preparation of soluble β-1,3-1,6-glucan (hereinafter referred to as β-1,3-1,6-LG).

[0013] In this invention, the inert immobilization carrier is used to adsorb and immobilize bacterial cells, and does not participate in metabolism as a carbon or nitrogen source, nor in the fermentation metabolic process.

[0014] This invention also provides a method for producing soluble β-glucan by cell immobilization using an inert carrier, with *Bretschneidera melanin-producing* as the fermentation strain. The method includes carrier pretreatment, strain activation, seed culture, and fermentation for sugar production. The fermentation for sugar production is a process in which *Bretschneidera melanin-producing* inoculated into a fermentation medium and co-fermented with an inert immobilization carrier. The inert immobilization carrier is selected from at least one of rice bran, wheat straw, wheat bran, corn cob, loofah sponge, sugarcane bagasse, activated carbon, polypropylene material, and reticulated polyurethane foam.

[0015] Furthermore, the fermentation for sugar production refers to the process of inoculating a strain into a shake flask for fermentation; the shake flask contains an inert immobilization carrier, and β-glucan, especially soluble β-1,3-1,6-glucan, is produced by culturing *Bacillus melaninus* in a culture medium containing carbon source, nitrogen source, inorganic salt, and trace elements.

[0016] The preferred inert immobilization carrier of this invention has a porous structure to enhance the adsorption and immobilization effect of the bacteria. The porous structure of the inert immobilization carrier provides an ideal surface area for easy attachment and a naturally favorable living environment, allowing *B. melanin-producing* to exhibit elongation, branching, and filamentous mycelium formation in the liquid culture medium of this invention. This morphological feature has a high surface area to volume ratio, increasing the effective contact area between the bacteria and the culture medium, thereby facilitating the absorption of nutrients and the synthesis and secretion of secondary metabolites.

[0017] Preferably, the carrier pretreatment includes:

[0018] Step 1: Cut or break the inert immobilized carrier into small pieces and boil them.

[0019] Step 2: Wash with deionized water.

[0020] Step 3: Dry and sterilize the inert immobilized carrier after step 2.

[0021] Preferably, in step 1, the boiling time is 10-20 minutes. More preferably, the boiling time is 10, 15, or 20 minutes or a range thereof.

[0022] Preferably, in step 2, the vehicle is washed 1-5 times with deionized water to remove any interference from the carrier-borne components that might affect the fermentation process. More preferably, the number of washes is 1, 2, 3, 4, or 5 times, or a range thereof.

[0023] Preferably, in step 3, drying is carried out at 60-70°C. More preferably, the drying temperature is 60, 65, 70°C or a range thereof.

[0024] In any of the above-mentioned preferred embodiments, in step 3, the sterilization conditions are high-pressure sterilization at 115-121°C for 15-30 minutes.

[0025] In a preferred embodiment of the present invention, the carrier pretreatment includes: cutting or breaking the inert immobilized carrier into small pieces, boiling for 10-20 minutes; washing with deionized water 1-5 times to remove the interference of carrier-carried components on the fermentation process; drying at 60-70°C, and autoclaving at 115-121°C for 15-30 minutes together with the fermentation medium before cell immobilization.

[0026] In a preferred embodiment of the present invention, the carrier pretreatment method is as follows: the loofah sponge and the mesh polyester fiber foam are cut into small pieces of 1 cm × 1 cm × 1 cm, the carrier is boiled for 15 min, and washed three times with deionized water. Then it is dried and sterilized.

[0027] In a preferred embodiment of the present invention, wheat bran is crushed using a plant tissue crusher, then sieved using sieves of different mesh sizes (preferably 10-80 mesh), boiled for 15 min, and washed three times with deionized water. Finally, it is dried overnight at 65°C to obtain an inert immobilization carrier. Before cell immobilization, the inert immobilization carrier and fermentation medium are autoclaved together at 121°C for 20 min.

[0028] Preferably, the inert immobilization carrier comprises wheat bran and / or activated carbon, with a mass ratio of wheat bran to activated carbon of 0.2 to 5. More preferably, the mass ratio of wheat bran to activated carbon is 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, or a range thereof.

[0029] Preferably, the fermentation medium used in the fermentation sugar production step includes a carbon source, a nitrogen source, inorganic salts, and trace elements.

[0030] In any of the above-mentioned preferred embodiments, the carbon source in the fermentation medium used in the fermentation sugar production step includes at least one of glucose, sucrose, fructose, lactose, xylose, arabinose, mannose, and high-fructose corn syrup.

[0031] Preferably, in any of the above-mentioned steps, the nitrogen source in the fermentation medium used in the fermentation sugar production step includes an organic nitrogen source and / or an inorganic nitrogen source.

[0032] In any of the above-mentioned preferred embodiments, the organic nitrogen source in the fermentation medium used in the fermentation sugar production step comprises yeast extract and / or yeast extract powder. The yeast extract powder is preferably Aoboxin yeast extract powder, trade number 01-012.

[0033] In any of the above-mentioned preferred embodiments, the inorganic nitrogen source in the fermentation medium used in the fermentation sugar production step includes ammonium sulfate.

[0034] In any of the above-mentioned preferred embodiments, the total sugar content of the carbon source in the fermentation medium used in the fermentation sugar production step is 25-150 g / L.

[0035] In any of the above-mentioned preferred embodiments, the carbon source in the fermentation medium used in the fermentation sugar production step comprises fructose with a concentration of 25-150 g / L and / or fructose syrup with a concentration of 40-50 g / L.

[0036] Preferably, in any of the above-mentioned steps, the concentration of fructose in the fermentation medium used in the fermentation sugar production step is 25, 50, 75, 100, 125, 150 g / L or a range thereof.

[0037] Preferably, the concentration of fructose syrup in the fermentation medium used in the fermentation sugar production step is 40, 45, 50 g / L or within the range thereto.

[0038] Preferably, the inorganic salt comprises K₂HPO₄ and / or NaCl; and the trace element comprises magnesium sulfate heptahydrate and / or zinc sulfate.

[0039] Preferably, the fermentation conditions for sugar production are: 25-30℃, 150-200 rpm shaking culture.

[0040] In any of the above-mentioned preferred embodiments, the temperature in the fermentation sugar production conditions is preferably 25, 26, 27, 28, 29, 30°C or a range thereof.

[0041] In any of the above-mentioned preferred embodiments, the shaking rate in the fermentation sugar production conditions is preferably 150, 160, 170, 180, 190, 200 rpm and the range thereof.

[0042] In a preferred embodiment of the present invention, the fermentation medium used in the fermentation sugar production step comprises the following components: a carbon source, a nitrogen source, inorganic salts, and trace elements. Preferably, the carbon source in the fermentation medium is selected from at least one of lactose, fructose, glucose, xylose, xylitol, arabinose, mannose, sucrose, and high-fructose corn syrup. Preferably, the total sugar content of the carbon source in the fermentation medium is 25–150 g / L. Preferably, the total sugar content of the carbon source in the fermentation medium is 25, 50, 75, 100, 125, 150 g / L or a range thereof. Preferably, the fermentation medium contains 5 g / L of yeast extract. Preferably, the fermentation medium contains 2 g / L of dipotassium hydrogen phosphate. Preferably, the fermentation medium contains 0.5 g / L of ammonium sulfate. Preferably, the fermentation medium contains 0.3 g / L of magnesium sulfate heptahydrate. Preferably, the fermentation medium contains 0.3 g / L of zinc sulfate. Preferably, the fermentation medium contains 0.5 g / L of sodium chloride.

[0043] In any of the above-mentioned preferred embodiments, the melanin-producing short-stem fungus is first propagated to a stable phase in a seed culture medium containing a carbon source and a nitrogen source before fermentation.

[0044] In any of the above-mentioned preferred embodiments, the carbon source of the seed culture medium during the seed culture process is glucose.

[0045] In any of the above-mentioned preferred embodiments, the nitrogen source of the seed culture medium during the seed culture process is potato starch.

[0046] Preferably, the melanin-producing short-stem fungus is the strain Aureobasidium melanogenum with accession number CGMCC No. 18996, which was deposited on November 22, 2019, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0047] In the preliminary research of this invention, the ability of *Brieflychee melaninosa*, especially *Brieflychee melaninosa* CGMCC No. 18996, to produce β-glucan was verified:

[0048] In the verification experiment, the following experimental groups were set up: Experimental group: *Bacillus melanogaster* CGMCC No. 18996, using activated carbon as an inert immobilization carrier; Control group: *Bacillus melanogaster* CGMCC No. 18996, without an inert immobilization carrier. The culture media were as follows: Primary seed culture medium included the following components: glucose 20 g / L, potato extract 13 g / L; Secondary seed culture medium included the following components: glucose 20 g / L, potato extract 15 g / L; The components and contents of the fermentation medium used were: glucose 50 g / L, yeast extract 3 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium sulfate 0.6 g / L, magnesium sulfate heptahydrate 0.2 g / L, zinc sulfate 0.1 g / L, sodium chloride 1 g / L, natural pH.

[0049] After fermentation, the fermentation products were tested. The yields of β-glucan and polymalic acid in the control group were 2.55 g / L and 16.8 g / L, respectively; while the yields of β-glucan and polymalic acid in the experimental group were 6.43 g / L and 6.02 g / L, respectively.

[0050] Then, according to the relevant experimental methods in the reference (DOI: 10.1007 / s00253-021-11538-x), the activities of three enzymes closely related to glucan synthesis—β-glucan synthase (β-GS), UGP, and PGM—intracellularly in the experimental group and control group of the present invention were measured. Samples were taken at 48 h of fermentation, and the contents of β-GS, UGP, and PGM in the experimental group and control group were measured. The control group had β-GS 94.27 ng / ml, UGP 95.85 pg / ml, and PGM 94 pg / ml; the experimental group had β-GS 97.33 ng / ml, UGP 115.1 pg / ml, and PGM 141.43 pg / ml.

[0051] The data above indicate that in the experimental group with the addition of an inert immobilization carrier, the yields of several enzymes closely related to glucan synthesis were increased, especially the contents of UGP and PGM, which could meet the precursor material requirements for polysaccharide synthesis. This is consistent with the increased β-glucan yield in the experimental group. The inert immobilization carrier, by inhibiting the main pathway of polymalic acid synthesis, unlocked the central carbon metabolism, thereby directing carbon flow to the previously inhibited β-glucan synthesis pathway.

[0052] This invention provides a method for preparing soluble β-1,3-1,6-glucan using an inert immobilized carrier. This method is not limited to strains with accession number CGMCC No. 18996; any melanin-producing short-stem fungus capable of producing β-glucan is applicable to this invention.

[0053] The method provided by this invention produces β-1,3-1,6-glucan with a yield of not less than 10 g / L.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] The inert immobilization carriers screened in this invention effectively increased the yield of β-1,3-1,6-LG, especially when activated carbon was used as the immobilization carrier, resulting in the greatest yield increase. Fructose was found to be a preferred carbon source for promoting β-glucan synthesis, and a novel approach was proposed to use high-fructose corn syrup instead of pure fructose, effectively reducing raw material costs while maintaining high yields, demonstrating promising industrial application prospects. In this invention, cell immobilization with activated carbon increased the yield of β-glucan (β-1,3-1,6-LG) by more than 2 times; the use of fructose as the preferred carbon source further increased the yield of β-glucan (β-1,3-1,6-LG) by 23%; and the use of high-fructose corn syrup as a substitute carbon source further increased the yield of β-glucan (β-1,3-1,6-LG) by 17%. The inert immobilization carrier provided by this invention offers cells an ideal surface area for easy attachment and a favorable natural living environment, causing cells to elongate, branch, and form filamentous mycelia. This morphological feature has a high surface area to volume ratio, increasing the effective contact area between the cells and the culture medium, which is beneficial for the absorption of nutrients and the synthesis and secretion of secondary metabolites.

[0056] This invention represents a significant breakthrough compared to existing technologies, offering advantages such as low fermentation costs, high yields, strong technical and economic efficiency, and the ability to be continuously and repeatedly produced, making it suitable for industrial production. Attached Figure Description

[0057] Figure 1 The yield of β-1,3-1,6-LG and total sugar in the fermentation broth after fermentation using agricultural and industrial by-products as an inert immobilization carrier in the preferred embodiment of the present invention is shown.

[0058] Figure 2 The preferred embodiment of the present invention, in which synthetic materials are used as inert immobilization carriers, yields β-1,3-1,6-LG and total sugars in the fermentation broth after fermentation.

[0059] Figure 3 The yield of β-1,3-1,6-LG and total sugar in the fermentation broth after fermentation using three types of wheat bran as inert immobilization carriers in the preferred embodiment of the present invention is shown in Example 3.

[0060] Figure 4 The yield of β-1,3-1,6-LG and total sugar in the fermentation broth after fermentation using wheat bran of different mesh sizes as an inert immobilization carrier in the preferred embodiment 2 of the present invention.

[0061] Figure 5The yields of β-1,3-1,6-LG and total sugars in the fermentation broth after fermentation using different amounts of 40-mesh wheat bran as an inert immobilization carrier in the preferred embodiment of the present invention are shown.

[0062] Figure 6 The preferred embodiment of the present invention, 6, shows the yields of β-1,3-1,6-LG and total sugars in the fermentation broth after fermentation using different amounts of 40-mesh activated carbon as an inert immobilization carrier.

[0063] Figure 7 The preferred embodiment of the present invention, 7, shows the yield of β-1,3-1,6-LG and total sugar in the fermentation broth after fermentation using activated carbon of different mesh sizes (20 g / L) as an inert immobilization carrier.

[0064] Figure 8 The preferred embodiment of the present invention, 8, shows the yield of β-1,3-1,6-LG and total sugar in the fermentation broth after fermentation using wheat bran and activated carbon in different mass ratios as inert immobilization carriers.

[0065] Figure 9 The preferred embodiment of the present invention, in which different carbon sources are used to ferment the yield of β-glucan and total sugar in the fermentation broth, as well as the biomass of melanin-producing short-stem mold, is described.

[0066] Figure 10 The preferred embodiment of the present invention, 10, utilizes different concentrations of fructose to determine the yield of β-glucan and total sugar in the fermentation broth, as well as the biomass of *Syngonium brevicorum* that produces melanin.

[0067] Figure 11 The preferred embodiment of the present invention utilizes different types of fructose syrup to ferment the yield of β-glucan and total sugar in the fermentation broth, as well as the biomass of melanin-producing short-stem mold.

[0068] Figure 12 The electron microscopy results are shown for the inert immobilized carrier before and after fermentation and the bacterial cells after fermentation in the preferred embodiment 12 of the present invention.

[0069] Figure 13 The electron microscopy results are shown for activated carbon and the fermented bacteria after fermentation using activated carbon as an inert immobilization carrier in the preferred embodiment 12 of the present invention.

[0070] The preferred strain of this invention is *Aureobasidium melanogenum*, with accession number CGMCC No. 18996. This strain was deposited on November 22, 2019, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0071] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0072] In a preferred embodiment of the present invention, a preferred method for producing β-glucan using *Bacillus thuringiensis* cells immobilized on an inert carrier, wherein the preferred technical solutions for immobilization carrier pretreatment, strain activation, seed culture, and fermentation for sugar production are as follows:

[0073] In the step of activating the bacterial strain:

[0074] Preferably, the slant culture medium used for activating the strain is potato dextrose agar medium;

[0075] In any of the above-mentioned preferred embodiments, the activation of the bacterial strain is to inoculate the bacterial strain into a slant culture medium and incubate it at 28°C for 48-72 hours.

[0076] In the seed culture steps:

[0077] In any of the above-mentioned preferred embodiments, the seed culture medium used in the seed culture process comprises a primary seed culture medium and a secondary seed culture medium, wherein the primary culture medium comprises the following components: 20 g / L glucose and 13 g / L potato extract powder; and the secondary culture medium comprises the following components: 20 g / L glucose and 15 g / L potato extract powder.

[0078] Preferably, the seed culture process is as follows: well-grown slant seeds are selected and inoculated into a primary seed culture medium, and cultured at 28°C and 180 rpm for 24 hours; then, bacterial culture is obtained from the primary seed culture medium and inoculated into a secondary seed culture medium, and cultured at 28°C and 180 rpm for 24 hours.

[0079] In the fermentation process for sugar production:

[0080] In any of the above-mentioned preferred embodiments, the fermentation medium includes a fermentation medium and an immobilization carrier, wherein the inert immobilization carrier and the fermentation medium are sterilized together.

[0081] Preferably, the inert immobilization carrier comprises agricultural and industrial by-products and / or synthetic materials.

[0082] Preferably, the agricultural industrial by-product includes any one of rice bran, wheat straw, wheat bran, corn cob, loofah sponge, and sugarcane bagasse; preferably, it is wheat bran.

[0083] Preferably, the synthetic material comprises any one of activated carbon, polypropylene, and polyester fiber; activated carbon is preferred.

[0084] Preferably, the fermentation medium comprises the following components: total sugar content of carbon source is 25-150 g / L, yeast extract 5 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium sulfate 0.6 g / L, magnesium sulfate heptahydrate 0.2 g / L, zinc sulfate 0.1 g / L, and sodium chloride 1 g / L.

[0085] Preferably, the carbon source comprises any one or a combination of at least two of fructose, glucose, and high-fructose corn syrup.

[0086] In any of the above-mentioned preferred embodiments, the carbon source is fructose with a sugar content of 50 g / L.

[0087] Preferably, the fermentation medium comprises the following components: fructose 50 g / L, yeast extract 5 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium sulfate 0.6 g / L, magnesium sulfate heptahydrate 0.2 g / L, zinc sulfate 0.1 g / L, and sodium chloride 1 g / L.

[0088] Preferably, in any of the above embodiments, the carbon source is fructose syrup with a sugar content of 40-50 g / L. The present invention also provides a culture medium for cell immobilization and β-glucan production using fructose syrup as a raw material, for use in the method described in any of the above embodiments.

[0089] Preferably, the fermentation medium comprises the following components: a total sugar content of 45 g / L, yeast extract F617 5.3 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium sulfate 0.7 g / L, magnesium sulfate heptahydrate 0.5 g / L, zinc sulfate 0.18 g / L, and sodium chloride 0.3 g / L.

[0090] Preferably, the inoculum size during fermentation is 10% of the volume of the fermentation medium.

[0091] Preferably, the fermentation temperature is 28°C.

[0092] In a preferred embodiment of the present invention, the fermentation for sugar production refers to the process of inoculating a microbial strain in a shake flask for fermentation; the shake flask contains an inert immobilization carrier, and the selected inert immobilization carrier includes agricultural and industrial by-products and / or synthetic materials.

[0093] Preferably, the agricultural industrial by-product is selected from at least one of rice bran, wheat straw, wheat bran, corn cob, loofah sponge, and sugarcane bagasse.

[0094] Preferably, the wheat bran is at least one selected from oat bran, wheat bran, and rye bran. Preferably, the wheat bran is of mesh size 10, 20, 40, 60, 80, or within a range thereof. Preferably, the amount of wheat bran added is 10g, 20g, 30g, 40g / L or within a range thereof.

[0095] Preferably, the synthetic material is selected from activated carbon, polypropylene (PP) material, or polyurethane material. The synthetic material used as the inert immobilization carrier in this invention is activated carbon, polypropylene material, or polyurethane material with a porous structure. Preferably, the activated carbon is 6 mesh, 10 mesh, 20 mesh, 40 mesh, 100 mesh, or a range thereof. Preferably, the amount of activated carbon added is 5g, 10g, 20g, 30g, 40g / L, or a range thereof. Preferably, the polypropylene material is PP material with a thickness of 1.5 mm or 2 mm or a range thereof. Preferably, the polyurethane material is polyurethane fiber. Preferably, the polyurethane material is a mesh polyurethane foam. Preferably, the polyurethane material is 25ppi polyurethane, 45ppi polyurethane, or 25ppi-45ppi polyurethane (different ppi represent the pore size of polyurethane; the larger the ppi, the more pores per unit area). Preferably, the polyurethane material is cut into small pieces of 1cm × 1cm.

[0096] Preferably, the inert immobilization carrier is composed of wheat bran and activated carbon, with the mass ratio of wheat bran to activated carbon being 1:5, 2:4, 3:3, 4:2, 5:1, or a range thereof. Preferably, the total amount of wheat bran and activated carbon added is uniformly 20 g / L.

[0097] Example 1

[0098] In this embodiment, the specific process of fermenting β-glucan using agricultural industrial by-products as inert immobilization carriers is as follows: *Briestroemia melanin-producing* is transferred to an agar slant culture medium and cultured at 28°C for 72-96 hours. Well-grown agar slant seeds are then inoculated into a primary seed culture medium using an inoculation loop and cultured at 28°C and 180 rpm for 24 hours. Then, bacterial culture is obtained from the primary seed culture medium and inoculated into a secondary seed culture medium under the same conditions as the primary seed culture medium. The secondary seed culture medium is then inoculated into the fermentation medium at 10% of its volume. In Example 1, seven experimental groups were set up. The blank control group did not add an inert immobilization carrier to the fermentation culture system. The other groups added rice bran, wheat straw, wheat bran, corn cob, loofah sponge, and sugarcane bagasse, respectively, to the fermentation culture system. The amount of the inert immobilization carrier added to the fermentation culture system was 10 g / L, with the loofah sponge cut into 1 cm pieces. 3Small pieces of inert immobilized carrier were added to the fermentation medium after pretreatment and sterilized together. The fermentation volume was 100 ml. The mixture was then cultured at 28℃ and 180 rpm for 72 h. The yields of β-1,3-1,6-LG and total sugar in the fermentation broth were then measured.

[0099] Figure 1 The figure shows the yields of β-1,3-1,6-LG and total sugar in the fermentation broth after fermentation using agricultural industrial by-products as an inert immobilization carrier in Example 1. The results show that wheat bran was used as an inert immobilization carrier in Example 1, with the highest yields of β-1,3-1,6-LG and total sugar, which were 5.62 g / L and 13.84 g / L, respectively.

[0100] In Example 1, the components and contents of each culture medium are as follows: the primary seed culture medium includes the following components: glucose 20 g / L, potato extract powder 13 g / L; the secondary seed culture medium includes the following components: glucose 20 g / L, potato extract powder 15 g / L; the components and contents of the fermentation culture medium used are: glucose 50 g / L, yeast extract powder 3 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium sulfate 0.6 g / L, magnesium sulfate heptahydrate 0.2 g / L, zinc sulfate 0.1 g / L, sodium chloride 1 g / L, and natural pH.

[0101] Conclusion: By comparing the effects of different organic materials as immobilization carriers on β-1,3-1,6-LG yield, the results showed that the yields of β-1,3-1,6-LG in all experimental groups with added inert immobilization carriers were significantly higher than those in the blank control group. Wheat bran showed the best effect as an inert immobilization carrier, achieving a β-1,3-1,6-LG yield of 5.62 g / L. These results preliminarily indicate that the porous structure of the inert immobilization carrier provides a better attachment interface for bacterial cells, thereby promoting bacterial growth and metabolic activity.

[0102] Example 2

[0103] In this embodiment, the specific process of fermenting β-glucan production using synthetic materials as inert immobilization carriers is as follows: *Bifidobacterium melanogaster*, a melanin-producing fungus, is transferred to an agar slant culture medium and cultured at 28°C for 48-72 hours. Well-grown agar slant culture seeds are selected and inoculated into a primary seed culture medium, cultured at 28°C and 180 rpm for 24 hours. The bacterial culture from the primary seed culture medium is then inoculated into a secondary seed culture medium under the same conditions as the primary seed culture medium. Finally, 10% of the secondary seed culture is inoculated into the fermentation medium. In this example, seven experimental groups were set up. The blank control group did not add an inert immobilization carrier to the fermentation culture system. The other groups added 40-mesh activated carbon, 10-mesh activated carbon, 1.5 mm PP material, and 2 mm [other inert immobilization carriers] to the fermentation culture system, respectively. PP material, 25ppi polyurethane, 45ppi polyurethane (different ppi represent the pore size of polyurethane, the larger the ppi, the more pores per unit area), wherein the polyurethane is cut into small pieces of 1cm×1cm, the amount of the inert immobilization carrier added to the fermentation culture system is 10g / L, the inert immobilization carrier is pretreated and then added to the fermentation culture medium for sterilization, the fermentation volume is 100ml, and then cultured at 28℃ and 180rpm for 72h, and the content of β-glucan in the fermentation broth is detected.

[0104] The results are as follows Figure 2 The results show that the highest yield of β-1,3-1,6-LG was achieved when 40-mesh activated carbon was used as the immobilization carrier in this example, reaching 6.24 g / L.

[0105] The seed culture medium and fermentation culture medium used in Example 2 are the same as those in Example 1 above.

[0106] Conclusion: Example 2 evaluated the effects of various inorganic materials as inert immobilization supports on the yield of β-1,3-1,6-LG and total sugars. The results showed that porous activated carbon, PP materials, or polyurethane all significantly increased the yield of β-1,3-1,6-LG, with activated carbon showing the most significant effect as a support, simultaneously promoting the synthesis of both β-1,3-1,6-LG and total sugars. Specifically, 10-mesh and 40-mesh activated carbon increased the yield of β-1,3-1,6-LG by 109% and 138%, respectively.

[0107] Based on the above results, subsequent experiments selected activated carbon as the inert immobilization carrier and further screened its optimal mesh size.

[0108] Example 3

[0109] In this embodiment, the specific process of fermenting β-glucan using three types of wheat bran as immobilization carriers is as follows: *Briefiophytes brevichorae*, a melanin-producing fungus, is transferred to an agar slant culture medium and cultured at 28°C for 48-72 hours. Well-grown agar slant seeds are selected and inoculated into a primary seed culture medium using an inoculation loop, and cultured at 28°C and 180 rpm for 24 hours. The primary seed culture medium is then inoculated into a secondary seed culture medium under the same conditions as the primary seed culture medium. Finally, the secondary seed culture medium is inoculated at 10% of its volume. In this example, four groups of experiments were set up in the fermentation medium. The blank control group did not add an inert immobilization carrier to the fermentation culture system. The other groups added oat bran, wheat bran and rye bran to the fermentation culture system, respectively. The amount of inert immobilization carrier added to the fermentation culture system was 10 g / L. After pretreatment, the inert immobilization carrier was added to the fermentation culture medium and sterilized together. The fermentation volume was 100 ml. Then, it was cultured at 28℃ and 180 rpm for 72 h. The content of β-glucan in the fermentation broth was detected.

[0110] The results are as follows Figure 3 As shown, in Example 3, the yields of β-1,3-1,6-LG were similar when oat bran and wheat bran were used as inert immobilization carriers, at 4.79 g / L and 4.69 g / L, respectively.

[0111] The seed culture medium and fermentation culture medium used in Example 3 are the same as those used in Example 1.

[0112] Conclusion: When comparing the effects of three types of wheat bran as immobilization carriers, it was found that the yields of β-1,3-1,6-glucan and total sugars in each experimental group were superior to those in the blank control group. Wheat bran and oat bran showed comparable promoting effects on β-1,3-1,6-glucan, increasing yields by 87% and 90%, respectively. Given the similar yield increases of the two, and considering the issues of oat bran's high water absorption and cost, wheat bran will be the preferred immobilization carrier in future studies.

[0113] Example 4

[0114] In this embodiment, the specific process of fermenting β-glucan using wheat bran of different mesh sizes as an immobilization carrier is as follows: *Briefiophytes brevis*, a melanin-producing fungus, is transferred to an agar slant culture medium and cultured at 28°C for 48-72 hours. Well-grown agar slant seeds are selected and inoculated into a primary seed culture medium using an inoculation loop. The medium is then cultured at 28°C and 180 rpm for 24 hours. The primary seed culture is then added to a secondary seed culture medium under the same conditions as the primary seed culture medium. Finally, the secondary seed culture is inoculated into the fermentation medium at 10% of its volume, and six experimental groups are set up. In the experiment, the blank control group did not add an inert immobilization carrier to the fermentation culture system, while the other 5 groups used wheat bran with inert immobilization carriers of 10 mesh, 20 mesh, 40 mesh, 60 mesh, and 80 mesh, respectively. The wheat bran of different mesh sizes was obtained by crushing it using a plant tissue pulverizer and then screening it through sieves of different mesh sizes. The addition amount was 10 g / L. After pretreatment, the inert immobilization carrier was added to the fermentation culture medium and sterilized together. The fermentation volume was 100 ml, and then the mixture was cultured at 28℃ and 180 rpm for 72 h. The content of β-glucan in the fermentation broth was then detected.

[0115] The results are as follows Figure 4 As shown in Example 4, the highest yield of β-1,3-1,6-LG was achieved when 40-mesh wheat bran was used as the immobilization carrier, at 5.48 g / L. The highest total sugar yield was achieved when 20-mesh wheat bran was used as the immobilization carrier, at 15.23 g / L.

[0116] The seed culture medium and fermentation culture medium used in Example 4 are the same as those used in Example 1.

[0117] Conclusion: The effects of wheat bran of different mesh sizes as immobilization carriers on the yield of β-1,3-1,6-LG and total sugars were investigated. The results showed that, compared with the blank control group without carriers, the addition of wheat bran effectively promoted the synthesis of β-1,3-1,6-LG and total sugars, with 40-mesh wheat bran showing the most significant effect, increasing the yield of β-1,3-1,6-LG by 110%.

[0118] Example 5

[0119] In this embodiment, the specific process of fermenting β-glucan using 40-mesh wheat bran (preferably wheat bran) as an inert immobilization carrier with different amounts of added material is as follows: *Briefiophyte brevicornu*, a melanin-producing fungus, is transferred to an agar slant culture medium and cultured at 28°C for 48-72 hours. Well-grown agar slant seeds are selected and inoculated into a primary seed culture medium using an inoculation loop, and cultured at 28°C and 180 rpm for 24 hours. The primary seed culture is then inoculated into a secondary seed culture medium under the same conditions as the primary seed culture medium. Finally, 10% of the secondary seed culture is inoculated into the fermentation medium. Six experimental groups are set up, with the amount of inert immobilization carrier added to the fermentation culture system being 0g, 5g, 10g, 20g, 30g, and 40g / L (preferably 40-mesh wheat bran). The inert immobilization carrier is pretreated and then added to the fermentation medium for sterilization. The fermentation volume is 100ml, and the mixture is then cultured at 28°C and 180 rpm for 72 hours. The β-glucan content in the fermentation broth is then measured.

[0120] The COD of the culture medium after sterilization was measured in both the blank group and each experimental group. The results are shown in Table 1 below:

[0121] Table 1:

[0122]

[0123] There was no significant difference in COD between the control group and the experimental group, indirectly indicating that no usable sugars and nutrients were dissolved after sterilization. Therefore, this demonstrates that the pretreated inert immobilized carrier in this invention does not participate in the sugar production metabolism process as a carbon or nitrogen source.

[0124] The results are as follows Figure 5 As shown, in Example 5, the highest yield of β-1,3-1,6-LG was achieved when 10 g / L wheat bran was used as the immobilization carrier, at 5.02 g / L. The highest yield of total sugar was achieved when 30 g / L wheat bran was used as the immobilization carrier, at 13.76 g / L.

[0125] The seed culture medium and fermentation culture medium used in Example 5 are the same as those used in Example 1.

[0126] Conclusion: Based on the experimental results, within the range of wheat bran addition of 10 g / L to 40 g / L, the yields of β-1,3-1,6-LG and total sugars were both increased compared to the control group. The effect was most significant at an addition of 10 g / L, where the yield of β-1,3-1,6-LG reached its peak, increasing by 91% compared to the control group.

[0127] Example 6

[0128] In this embodiment, the specific process of fermenting β-glucan using activated carbon (40 mesh) with different addition amounts as immobilization carriers is as follows: *Briefiophytes brevicornu*, a melanin-producing fungus, is transferred to an agar slant culture medium and cultured at 28°C for 48-72 hours. Well-grown agar slant seeds are selected and inoculated into a primary seed culture medium using an inoculation loop, and cultured at 28°C and 180 rpm for 24 hours. The primary seed culture is then inoculated into a secondary seed culture medium under the same conditions as the primary seed culture medium. Finally, 10% of the secondary seed culture is inoculated into the fermentation medium. In this example, six experimental groups are set up, using 0g, 5g, 10g, 20g, 30g, and 40g / L of activated carbon (40 mesh) as an inert immobilization carrier. After pretreatment, the inert immobilization carrier is added to the fermentation medium and sterilized together. The fermentation volume is 100ml, and the mixture is then cultured at 28°C and 180 rpm for 72 hours. The β-glucan content in the fermentation broth is then measured.

[0129] The results are as follows Figure 6 As shown, in this example, the highest yield of β-1,3-1,6-LG was achieved when the activated carbon addition was 20 g / L, at 6.69 g / L, while the highest yield of total sugar was achieved when the activated carbon addition was 30 g / L, at 14.38 g / L.

[0130] The seed culture medium and fermentation culture medium used in Example 6 are the same as those used in Example 1.

[0131] Conclusion: When the activated carbon addition amount was 5–20 g / L, the yield of β-1,3-1,6-LG increased significantly with increasing addition amount, reaching a maximum yield of 6.69 g / L at 20 g / L, an increase of 140%. However, further increasing the addition amount led to a decrease in yield, indicating that excessive activated carbon (>20 g / L) may inhibit the overall fermentation process by aggravating mass transfer resistance, increasing the viscosity of the fermentation broth, and promoting excessive cell adhesion.

[0132] Example 7

[0133] In this embodiment, the specific process of fermenting β-glucan production using activated carbon (20 g / L) of different mesh sizes as an immobilization carrier is as follows: *Briefiophytes brevicornu*, a melanin-producing fungus, is transferred to an agar slant culture medium and cultured at 28°C for 48-72 hours. Well-grown agar slant seeds are selected and inoculated into a primary seed culture medium using an inoculation loop, and cultured at 28°C and 180 rpm for 24 hours. The primary seed culture is then inoculated into a secondary seed culture medium under the same conditions as the primary seed culture medium. Finally, the secondary seed culture is inoculated... 10% by volume was inoculated into the fermentation medium. In this example, 6 groups of experiments were set up. The blank control group did not add inert immobilization carrier to the fermentation culture system. The other 5 groups used 6-mesh, 10-mesh, 20-mesh, 40-mesh and 100-mesh activated carbon as inert immobilization carriers. The addition amount was uniformly 20 g / L. The inert immobilization carrier was added to the fermentation culture medium after pretreatment and sterilized together. The fermentation volume was 100 ml. Then, it was cultured at 28℃ and 180 rpm for 72 h. The content of β-glucan in the fermentation broth was detected.

[0134] The results are as follows Figure 7 As shown, in this example, the yields of β-1,3-1,6-LG were not significantly different when 20-mesh and 40-mesh activated carbon were used as immobilization supports, at 6.8 g / L and 7.01 g / L, respectively. Considering that the 20-mesh activated carbon particles are larger and more convenient for subsequent experiments, 20-mesh activated carbon was selected.

[0135] The seed culture medium and fermentation culture medium used in Example 7 are the same as those used in Example 1.

[0136] Conclusion: Within the activated carbon mesh range of 6 to 40 mesh, the yield of β-1,3-1,6-LG gradually increased with increasing mesh size, reaching a maximum of 7.01 g / L at 40 mesh, representing a 143% increase compared to the control group. However, when the activated carbon mesh size increased to 100 mesh, the yield of β-1,3-1,6-LG decreased. The speculated reason is that smaller mesh sizes (e.g., 6–40 mesh) correspond to larger pore sizes, which facilitate the diffusion of the reaction substrate; while larger mesh sizes (e.g., 100 mesh), although possessing a higher specific surface area, may have limited mass transfer efficiency due to the reduced pore size, thus affecting product synthesis.

[0137] Example 8

[0138] In this embodiment, the specific process of fermenting β-glucan using 40-mesh wheat bran and 20-mesh activated carbon in different mass ratios as immobilization carriers is as follows: *Briefomus brevis*, a melanin-producing fungus, is transferred to an agar slant culture medium and cultured at 28°C for 48-72 hours. Well-grown agar slant seeds are selected and inoculated into a primary seed culture medium using an inoculation loop, and cultured at 28°C and 180 rpm for 24 hours. The primary seed culture is then inoculated into a secondary seed culture medium under the same conditions as the primary seed culture medium. Finally, the secondary seed culture is... The culture medium was inoculated at 10% of the inoculum volume into the fermentation medium. In this example, 5 groups of experiments were set up. Wheat bran and activated carbon were added as inert immobilization carriers at mass ratios of 1:5, 2:4, 3:3, 4:2, and 5:1, respectively. The total amount of wheat bran and activated carbon added was uniformly 20 g / L. After pretreatment, the inert immobilization carrier was added to the fermentation medium and sterilized together. The fermentation volume was 100 ml. Then, the culture was carried out at 28℃ and 180 rpm for 72 h. The content of β-glucan in the fermentation broth was detected. Example 8 also included a control group with 20 g / L wheat bran as an inert immobilization carrier and a control group with 20 g / L activated carbon as an inert immobilization carrier. The yield of β-1,3-1,6-LG in the 20 g / L wheat bran as an inert immobilization carrier group was 3.90 g / L, and the total sugar was 13.49 g / L; the yield of β-1,3-1,6-LG in the 20 g / L activated carbon as an inert immobilization carrier group was 6.68 g / L, and the total sugar was 12.07 g / L.

[0139] The results are as follows Figure 8 As shown, in this example, the yield of β-1,3-1,6-LG was the highest at a wheat bran:activated carbon mass ratio of 1:5, reaching 7.05 g / L, while the total sugar yield showed no significant difference.

[0140] The seed culture medium and fermentation culture medium used were the same as in Example 1.

[0141] Conclusion: Based on the experimental results, wheat bran and activated carbon of different mass ratios are more effective as immobilization carriers than wheat bran alone, and are comparable to activated carbon alone.

[0142] Example 9

[0143] The specific process for producing β-glucan using different carbon sources in this embodiment is as follows: *Briestroemia melanin-producing* strains were transferred to slant culture medium and cultured at 28°C for 48-72 hours. Well-grown slant seeds were selected and inoculated into primary seed culture medium using an inoculation loop, and cultured at 28°C and 180 rpm for 24 hours. The primary seed culture was then inoculated into secondary seed culture medium under the same conditions as the primary seed culture medium. Then, 10% of the secondary seed culture was inoculated into the fermentation medium. In this example, seven experimental groups were set up, using glucose, mannose, fructose, xylose, lactose, sucrose, and arabinose as carbon sources, and 20-mesh activated carbon as an inert immobilization carrier, with a uniform addition amount of 20 g / L. The inert immobilization carrier was pretreated and then added to the fermentation medium for sterilization. The fermentation volume was 100 ml, and the mixture was cultured at 28°C and 180 rpm for 72 hours. The β-glucan content in the fermentation broth was then measured.

[0144] The results are as follows Figure 9 As shown, in this example, the yield of β-1,3-1,6-LG was the highest when fructose was used as the carbon source, at 7.50 g / L; the yield of total sugar was the highest when sucrose was used as the carbon source, at 20.92 g / L; and the yield of cellular biomass was the highest when xylose was used as the carbon source, at 20.35 g / L.

[0145] The seed culture medium used in Example 9 was the same as that used in Example 1; the components and contents of the fermentation culture medium were as follows: sugar 50 g / L, yeast extract 3 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium sulfate 0.6 g / L, magnesium sulfate heptahydrate 0.2 g / L, zinc sulfate 0.1 g / L, sodium chloride 1 g / L, and natural pH.

[0146] Conclusion: Based on the experimental results, different carbon sources significantly affected the yield of β-1,3-1,6-glucan (β-1,3-1,6-LG) and total sugars. Fructose showed the best effect as a carbon source, increasing β-1,3-1,6-LG yield by 23% compared to glucose. Sucrose yielded the highest total sugar yield (20.92 g / L), suggesting that different carbon sources may affect total sugar yield by forming different byproducts. Furthermore, xylose resulted in a cell biomass of 20.35 g / L when used as a carbon source, indicating that xylose is more conducive to promoting cell growth.

[0147] Example 10

[0148] The specific process for producing β-glucan by fermentation using different concentrations of fructose in this embodiment is as follows: *Briefiophyte brevicornu*, a melanin-producing fungus, is transferred to an agar slant culture medium and cultured at 28°C for 48-72 hours. Well-grown agar slant seeds are selected and inoculated into a primary seed culture medium using an inoculation loop, and cultured at 28°C and 180 rpm for 24 hours. The primary seed culture is then inoculated into a secondary seed culture medium under the same conditions as the primary seed culture. Finally, 10% of the secondary seed culture is inoculated into the fermentation medium. In this example, six experimental groups are set up, with fructose added at concentrations of 25, 50, 75, 100, 125, and 150 g / L as the carbon source, and 20-mesh activated carbon as the inert immobilization carrier. The amount of 20-mesh activated carbon added is uniformly 20 g / L. The inert immobilization carrier is pretreated and then added to the fermentation medium for sterilization. The fermentation volume is 100 ml, and the mixture is cultured at 28°C and 180 rpm for 72 hours. The β-glucan content in the fermentation broth is then measured.

[0149] The results are as follows Figure 10 As shown, in this example, the highest yield of β-1,3-1,6-LG was achieved when the fructose concentration was 50 g / L, at 7.47 g / L; the highest total sugar yield was achieved when the fructose addition was 150 g / L, at 29.21 g / L; and the highest cell biomass of *Bacillus melaninus* was achieved when the fructose addition was 125 g / L, at 25.45 g / L.

[0150] The components and contents of the seed culture media used are as follows: The primary seed culture medium includes the following components: glucose 20 g / L, potato extract powder 13 g / L; the secondary seed culture medium includes the following components: glucose 20 g / L, potato extract powder 15 g / L; the components and contents of the fermentation culture medium used are: fructose 25-150 g / L, yeast extract powder 3 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium sulfate 0.6 g / L, magnesium sulfate heptahydrate 0.2 g / L, zinc sulfate 0.1 g / L, sodium chloride 1 g / L, natural pH.

[0151] Conclusion: Based on the experimental data, the yield of β-1,3-1,6-glucan (β-1,3-1,6-LG) reached its highest level (7.47 g / L) when the fructose concentration was 50 g / L. However, further increasing the fructose concentration led to the accumulation of byproducts, thereby reducing the conversion efficiency of the target product. Biomass remained at a high level when the fructose concentration was increased to 125 g / L; however, when the concentration increased to 150 g / L, the biomass decreased slightly, indicating that excessively high fructose concentrations inhibited bacterial growth and interfered with its normal metabolism and growth processes.

[0152] Example 11

[0153] In this embodiment, the specific process for producing β-glucan by fermentation using different types of fructose syrup is as follows: *Bifidobacterium melanogaster*, a melanin-producing fungus, is transferred to an agar slant culture medium and cultured at 28°C for 48-72 hours. Well-grown agar slant seeds are then inoculated into a primary seed culture medium using an inoculation loop and cultured at 28°C and 180 rpm for 24 hours. The primary seed culture is then inoculated into a secondary seed culture medium under the same conditions as the primary seed culture medium. Finally, 10% of the secondary seed culture is inoculated into the fermentation medium. In this example, four groups are set up. In the experiment, fructose, F42 type fructose syrup, F55 type fructose syrup, and F60 type fructose syrup were added as carbon sources, respectively. Fructose, F42 type fructose syrup, F55 type fructose syrup, and F60 type fructose syrup were added at a total sugar concentration of 45 g / L. 20-mesh activated carbon was used as an inert immobilization carrier, and the amount of inert immobilization carrier added was uniformly 20 g / L. After pretreatment, the immobilization carrier was added to the fermentation medium and sterilized together. The fermentation volume was 100 ml, and then the mixture was cultured at 28℃ and 180 rpm for 72 h. The content of β-glucan in the fermentation broth was then detected.

[0154] The results are as follows Figure 11 As shown, in this example, when F55 type fructose syrup was used as the carbon source, the yield of β-1,3-1,6-LG was the highest at 10 g / L, and the cell biomass was 19.3 g / L. When F42 type fructose syrup was used as the carbon source, the yield of total sugar was the highest at 11.77 g / L.

[0155] The components and contents of the seed culture media used are as follows: The primary seed culture medium includes the following components: glucose 20 g / L, potato extract 13 g / L; the secondary seed culture medium includes the following components: glucose 20 g / L, potato extract 15 g / L; the components and contents of the fermentation culture medium used are as follows: total sugar content 45 g / L, yeast extract 5.3 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium sulfate 0.7 g / L, magnesium sulfate heptahydrate 0.5 g / L, zinc sulfate 0.18 g / L, sodium chloride 0.3 g / L, natural pH.

[0156] Conclusion: The experimental results show that the three types of fructose syrups are superior to fructose alone in promoting the synthesis of β-1,3-1,6-LG. While the yields of the products obtained from the different fructose syrups did not differ significantly, considering both performance and cost, F55 fructose syrup exhibited the best cost-effectiveness and has the potential to replace fructose as a carbon source for fermentation. Its application in subsequent processes is recommended.

[0157] Example 12

[0158] Example 12 showed electron microscopy examination of the unfermented inert immobilized carrier and the bacterial cells attached to the inert immobilized carrier after fermentation. The results are as follows: Figure 12 As shown.

[0159] Figure 12 In the figures, A to I represent the electron microscopy results of the unfermented inert immobilized carrier materials, where A is rye bran, B is oat bran, C is wheat straw, D is loofah sponge, E is rice bran, F is corn cob, G is sugarcane bagasse, H is polyurethane, and I is PP material; J is the control group, consisting of free bacteria without immobilization on the carrier; K and L represent the electron microscopy results before and after fermentation in Example 5, where K is wheat bran without bacterial attachment, and L is bacterial cells attached to the wheat bran after fermentation; M and N represent the electron microscopy results before and after fermentation in Example 7, where M is activated carbon without bacterial attachment, and N is bacterial cells attached to the activated carbon after fermentation. Observations show that the fermented bacteria after immobilization undergo significant morphological changes, exhibiting elongation and the formation of filamentous mycelia. This morphological feature provides a high surface area to volume ratio, increasing the effective contact area between the bacteria and the culture medium, thus facilitating nutrient absorption and the synthesis and secretion of secondary metabolites.

[0160] Figure 13 The image shows the electron microscopy results of activated carbon and the bacterial cells after fermentation using activated carbon as an inert immobilization carrier. The upper image shows the activated carbon, and the lower image shows the bacterial cells attached to the activated carbon after fermentation. The red circle in the image shows the "short string" mycelium with a multicellular structure formed by budding. The remaining uncircled bacterial cells also clearly have budding structures consisting of at least two cells.

[0161] The above embodiments are merely illustrative examples and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of an inert immobilized carrier in the preparation of soluble β-glucan, characterized in that, The inert immobilization carrier is at least one of rice bran, wheat straw, wheat bran, corn cob, loofah sponge, sugarcane bagasse, activated carbon, polypropylene material, and reticulated polyurethane foam.

2. The application as described in claim 1, characterized in that, The soluble β-glucan is β-1,3-1,6-glucan.

3. A method for producing soluble β-glucan by cell immobilization using an inert carrier, using *Brachystomiae mellea* as the fermentation strain, characterized in that... The method includes carrier pretreatment, strain activation, seed culture, and fermentation for sugar production. The fermentation for sugar production is a process in which a melanin-producing short-stem mold inoculated into a fermentation medium and an inert immobilized carrier are fermented together. The inert immobilized carrier is selected from at least one of rice bran, wheat straw, wheat bran, corn cob, loofah sponge, sugarcane bagasse, activated carbon, polypropylene material, and reticulated polyurethane foam.

4. The method as described in claim 3, characterized in that, The carrier pretreatment includes: cutting or breaking the inert immobilized carrier into small pieces and boiling it; washing it with deionized water; and drying and sterilizing the treated inert immobilized carrier.

5. The method as described in claim 4, characterized in that, The inert immobilization carrier includes wheat bran and / or activated carbon, with a mass ratio of wheat bran to activated carbon of 0.2 to 5.

6. The method as described in claim 4, characterized in that, The fermentation medium used in the sugar production step includes a carbon source, a nitrogen source, inorganic salts, and trace elements; the carbon source includes at least one of glucose, sucrose, fructose, lactose, xylose, arabinose, mannose, and high-fructose corn syrup; the nitrogen source includes an organic nitrogen source and / or an inorganic nitrogen source, the organic nitrogen source includes yeast extract and / or yeast extract powder, and the inorganic nitrogen source includes ammonium sulfate.

7. The method as described in claim 6, characterized in that, The total sugar content of the carbon source is 25-150 g / L.

8. The method as described in claim 6, characterized in that, The inorganic salts include K2HPO4 and / or NaCl; the trace elements include magnesium sulfate heptahydrate and / or zinc sulfate.

9. The method as described in claim 6, characterized in that, The fermentation conditions for sugar production are: 25-30℃, shaking culture at 150-200 rpm.

10. The method as described in claim 6, characterized in that, During the seed culture process, the carbon source of the seed culture medium is glucose, and the nitrogen source is potato starch.

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