Aureobasidium pullulans g1 and its application in co-production of pullulan and beta-glucan

Abstract

The application provides an Aureobasidium pullulans G1 and application thereof in co-production of pullulan and beta-glucan, and belongs to the technical field of biological fermentation; specifically provides an Aureobasidium pullulans G1, which is preserved in the China General Microbiological Culture Collection Center on March 17, 2025, and the address is No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.41837; application of the Aureobasidium pullulans G1 in co-production of pullulan and beta-glucan; the Aureobasidium pullulans G1 provided by the application can co-produce pullulan and beta-glucan, and the yield of the two is high.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, specifically relating to a budding short-stem mold G1 and its application in the co-production of pullulan and β-glucan. Background Technology

[0002] Pullulan is a natural water-soluble polysaccharide produced by microorganisms, primarily by *Brachystomata* through fermentation. Its structure consists of repeating maltotriose units linked by α-1,6 glycosidic bonds, exhibiting excellent film-forming, adhesive, and biocompatibility properties. It is widely used in food, pharmaceuticals, cosmetics, and packaging materials.

[0003] β-glucan ( Beta-Glucan β-glucan is a class of natural polysaccharides composed of glucose monomers linked by β-glycosidic bonds. It is widely found in the cell walls of yeast, grains (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. Due to its unique biological activity and physicochemical properties, β-glucan has important applications in food, medicine, cosmetics, and other fields.

[0004] Aureobasidium pullulans (scientific name: Aureobasidium pullulans It is a pleomorphic fungus widely distributed in nature, belonging to the phylum Ascomycota ( Ascomycota Yeast () Saccharomycetes Its unique physiological characteristics and environmental adaptability make it of great value in fields such as biotechnology, food industry, and environmental remediation, and it is an important production strain in industrial microbiology. Currently, most data mention that *Brachysporium buddingense* is mainly used to produce pullulan, polymalic acid, and melanin, while very few studies mention that *Brachysporium buddingense* can also secrete extracellular β-glucan.

[0005] There are numerous existing research reports on strains producing pullulan or β-glucan monopolysaccharides. For example, Chinese patent document CN 117660204 A (application number: 202311240831.1) discloses a melanin-producing *Brachystomata* strain and its usage method, which describes a method for producing β-glucan by knocking out the gene involved in pullulan. However, there are relatively few existing studies on strains that co-produce these two polysaccharides, and the yields are low. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a budding short-stem mold G1 and its application in the co-production of pullulan and β-glucan.

[0007] The technical solution adopted in this invention is as follows:

[0008] A strain of Aureobasidium pullulans (G1) Aureobasidium pullulans , which was preserved in the China General Microbiological Culture Collection Center on March 17, 2025, at an address of No. 1, Beichen West Road, Haidian District, Beijing, with a preservation number of CGMCC NO. 41837.

[0009] Application of the above-mentioned Aureobasidium pullulans G1 in co-production of pullulan and β-glucan.

[0010] According to the application, preferably, in the application, the molecular weight of the pullulan is 150 kD-200 kD.

[0011] The culture method of the above-mentioned Aureobasidium pullulans G1 for co-production of pullulan and β-glucan includes the following steps:

[0012] (1) inoculate the strain into a seed culture medium and culture until OD 600 ≥5 as a first-level seed;

[0013] (2) inoculate the first-level seed into a seed culture medium and culture until OD 600 ≥5 as a second-level seed;

[0014] (3) inoculate the second-level seed into a fermentation culture medium at a volume ratio of 1-10%, and perform fermentation culture at a rotation speed of 50-500 rpm, a ventilation ratio of 1:(0.5-3), a temperature of 25-35℃, a pH of 4.0-8.0, a glucose concentration controlled by feeding sugar liquid to be above 8 g / L during the fermentation process, and a fermentation time of 60-120 h to obtain a fermentation liquor.

[0015] Preferably, in step (1) or step (2), the culture is performed until OD 600 ≥10.

[0016] Preferably, in step (1) or step (2), the seed culture medium includes glucose 50 g / L, yeast extract powder 5 g / L, sodium chloride 2 g / L, and dipotassium hydrogen phosphate 3 g / L, with water as the solvent.

[0017] Preferably, in step (3), the fermentation culture medium includes a carbon source 80-200 g / L, a nitrogen source 10-100 g / L, and inorganic salt 0.1-20 g / L.

[0018] Preferably, in step (3), the carbon source includes at least one of fructose, maltose, glucose, sucrose, lactose, galactose, and molasses.

[0019] The nitrogen source includes at least one of yeast extract powder, yeast paste, peptone, soybean cake powder, corn syrup, ammonium sulfate, ammonium chloride, and ammonium nitrate.

[0020] The inorganic salt comprises at least one of sodium chloride, potassium chloride, potassium carbonate, ferrous sulfate, magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, zinc sulfate and copper sulfate.

[0021] Preferably, in step (3), the fermentation medium comprises any one of the following formulations:

[0022] Formulation one: glucose 80 g / L, sucrose 100 g / L, yeast extract powder 10 g / L, proteose peptone 5 g / L, sodium chloride 3.5 g / L, dipotassium hydrogen phosphate 2 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate 2 g / L, ammonium sulfate 4 g / L, and water as solvent;

[0023] Formulation two: sucrose 100 g / L, lactose 60 g / L, proteose peptone 8 g / L, yeast extract powder 3 g / L, sodium chloride 6 g / L, potassium carbonate 2 g / L, dipotassium hydrogen phosphate 3 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 2 g / L, ammonium sulfate 3.5 g / L, and water as solvent;

[0024] Formulation three: sucrose 100 g / L, yeast extract powder 12 g / L, sodium chloride 3.5 g / L, potassium carbonate 2 g / L, dipotassium hydrogen phosphate 3 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 4 g / L, ammonium sulfate 5 g / L, and water as solvent.

[0025] Preferably, in step (3), the feeding sugar solution in the fermentation process comprises sucrose solution and glucose solution.

[0026] The present application has at least the following advantages:

[0027] Compared with the prior art, the strain Aureobasidium pullulans G1 provided by the present application can co-produce pullulan and beta-glucan, and the yield of both is high, the yield of pullulan reaches 181 g / L, and the yield of beta-glucan reaches 25 g / L. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Figure 1 is a graph for detecting the molecular weight of pullulan in Example 2.

[0029] Figure 2 Figure 2 is a graph for detecting the molecular weight of pullulan in Example 3.

[0030] Figure 3 Figure 3 is a graph for detecting the molecular weight of pullulan in Example 4.

[0031] Figure 4 Figure 4 is a graph for detecting the molecular weight of pullulan produced by the initial strain M1. DETAILED DESCRIPTION

[0032] The technical solutions of the present application are further described below in conjunction with examples, but the scope of protection of the present application is not limited thereto.

[0033] Experimental methods in the following examples that do not specify specific conditions are generally performed under conventional conditions.

[0034] Example 1

[0035] strain mutagenesis screening

[0036] The initial strain M1 was a budding short-stem mold strain preserved in the laboratory of Shandong Mimei Biotechnology Co., Ltd., which was isolated from an orchard in Linqu County, Weifang City.

[0037] Using strain M1 as the initial strain for mutagenesis, bacterial cells or spores were washed off the plates after 4 days of culture with sterile physiological saline and diluted to 10⁻⁶. 6 Suspensions with a concentration of [number] cells / mL were prepared. A 20W UV lamp was used, irradiated at a distance of 30cm for 0s, 10s, 20s, 30s, 40s, 50s, 60s, and 80s. Serial dilutions were then performed under red light, with 10 [units] being selected as the final concentration. 3 10 4 10 5 Three gradients of solid culture medium (the solid culture medium consisted of 50 g / L glucose, 5 g / L yeast extract, 2 g / L sodium chloride, 3 g / L dipotassium hydrogen phosphate, and 15 g / L agar powder, with water as the solvent) were spread on solid culture plates, wrapped in black plastic bags, and incubated at 30°C for 2 days in the dark. Plate counts were performed at the appropriate gradient to calculate the lethality rate.

[0038]

[0039] UV mutagenesis was induced by irradiation time with a lethality of 80%. Strains with white colonies and large growth were selected and fermented in Erlenmeyer flasks containing fermentation medium for verification. The yields of pullulan and β-glucan were measured. Mutagenesis was repeated until a high-yielding strain of *Bacillus buddingus* G1 was selected. The selected strain was deposited with the China General Microbiological Culture Collection Center. Specific deposit information is as follows:

[0040] A strain of Aureobasidium pullulans ( Aureobasidium pullulans G1 was deposited on March 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.41837.

[0041] Example 2

[0042] The cultivation method for the co-production of pullulan and β-glucan by *Bacillus buddingus* G1 includes the following steps:

[0043] (1) Strain activation: 1 ring of glycerol bacteria liquid was taken with a inoculation ring and inoculated on a solid culture medium plate by streaking method, and inverted in a 30°C incubator for culture for 3 days;

[0044] Solid culture medium: glucose 50 g / L, yeast extract powder 5 g / L, sodium chloride 2 g / L, potassium phosphate dibasic 3 g / L, agar powder 15 g / L, solvent is water;

[0045] (2) Primary seed culture in a shake flask: 1 piece of strain was scraped and inoculated in 50 mL of seed culture medium, and cultured at 30°C in a 200 rpm shaker until OD 600 10, as a primary seed;

[0046] (3) Secondary seed culture in a shake flask: 30 mL of the primary seed was inoculated in a secondary shake flask containing 300 mL of seed culture medium, and cultured at 30°C in a 200 rpm shaker until OD 600 11, as a secondary seed;

[0047] Seed culture medium in step (2) and step (3): glucose 50 g / L, yeast extract powder 5 g / L, sodium chloride 2 g / L, potassium phosphate dibasic 3 g / L, solvent is water;

[0048] (4) Fermenter culture: 5 L fermenter, fermentation culture medium liquid volume 3 L for fermentation, 300 mL of the cultured secondary seed liquid was inoculated in the fermenter, aeration rate 7.5 L / min (ventilation ratio 1:2.5), rotation speed 300 rpm, pH 4.0, temperature 30°C. The fermentation process was controlled by supplementing sucrose to control the glucose concentration to be more than 8 g / L, and the fermentation liquid was obtained after 110 h of culture.

[0049] Fermentation culture medium: glucose 80 g / L, sucrose 100 g / L, yeast extract powder 10 g / L, peptone 5 g / L, sodium chloride 3.5 g / L, potassium phosphate dibasic 2 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate 2 g / L, ammonium sulfate 4 g / L, solvent is water.

[0050] Supplement: sucrose solution with a mass concentration of 50%.

[0051] The content of pullulan and β-glucan in the fermentation liquid and the molecular weight of pullulan were detected by the following specific methods:

[0052] Pullulan detection: the fermentation liquid was centrifuged at 10,000 rpm for 10 min, and the supernatant was taken, 1‰ β-glucanase was added, and the enzyme hydrolysis was carried out at 55°C and pH 5.5 for 4 h. 20 mL of the enzyme hydrolysate was added with 3 times of ethanol, centrifuged at 10,000 rpm for 5 min, and the precipitate was dried and weighed to calculate the yield of pullulan.

[0053] Molecular weight detection of pullulan: The pullulan sample precipitated by ethanol and dried was dissolved in 2% sodium nitrite aqueous solution to prepare a sample solution with a concentration of 10 g / L. The sample solution was detected according to the molecular exclusion chromatography (Chinese Pharmacopoeia 2020 Edition General Rule 0514). The water phase gel chromatography column was used, 2% sodium nitrite solution (water as solvent) was used as the mobile phase, the column temperature was 30°C, the flow rate was 0.5 mL per minute, and the differential refractive index detector was used for detection.

[0054] Detection of β-glucan: The fermentation broth was centrifuged at 10,000 rpm for 10 min, and the supernatant was taken. 1‰ pullulanase was added, and the enzyme hydrolysis was carried out at 55°C and pH 5 for 8 h. 20 mL of the enzyme hydrolysis solution was taken, 4 times of ethanol was added, and centrifugation was carried out at 10,000 rpm for 5 min. The precipitate was dried and weighed, and the β-glucan yield was calculated.

[0055] Detection results: The yield of pullulan in the fermentation broth was 181 g / L, the molecular weight of pullulan was 177 kD, Figure 1 and the yield of β-glucan was 25 g / L.

[0056] Example 3

[0057] The culture method for producing pullulan and β-glucan by Aureobasidium pullulans G1 includes the following steps:

[0058] (1) Primary seed culture in a shake flask: 0.5 mL of glycerol bacterial solution was inoculated into 50 mL of seed culture medium, and the culture was carried out at 30°C and 200 rpm on a shaker until the OD 600 11 was reached, serving as the primary seed;

[0059] (2) Secondary seed culture in a shake flask: 30 mL of the primary seed was inoculated into a secondary shake flask containing 300 mL of seed culture medium, and the culture was carried out at 30°C and 200 rpm on a shaker until the OD 600 15 was reached, serving as the secondary seed;

[0060] The seed culture medium in step (1) and step (2) was the same as the seed culture medium in Example 2 described above.

[0061] (3) Fermenter culture: A 5 L fermenter was used for fermentation with 3 L of fermentation medium. 300 mL of the cultured secondary seed solution was inoculated into the fermenter, the aeration rate was 9 L / min (ventilation ratio 1:3), the rotation speed was 150 rpm, the pH was 7.0, the temperature was 25°C, and the glucose concentration was controlled to be above 8 g / L by supplementing glucose. The culture was carried out for 100 h to obtain the fermentation broth.

[0062] Fermentation medium: sucrose 100 g / L, lactose 60 g / L, peptone 8 g / L, yeast extract powder 3 g / L, sodium chloride 6 g / L, potassium carbonate 2 g / L, dipotassium hydrogen phosphate 3 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 2 g / L, ammonium sulfate 3.5 g / L, and water as the solvent.

[0063] Supplement: glucose solution with a mass concentration of 50%.

[0064] The content of pullulan and β-glucan in the fermentation broth and the molecular weight of pullulan were detected, and the specific method was the same as that in Example 2.

[0065] The detection results were as follows: the yield of pullulan reached 163 g / L, the molecular weight of pullulan was 197 kD, Figure 2 , and the yield of β-glucan reached 23 g / L.

[0066] Example 4

[0067] The culture method of Aureobasidium pullulans G1 for co-producing pullulan and β-glucan included the following steps:

[0068] (1) Primary seed culture in a shake flask: 0.5 mL of glycerol bacterial solution was inoculated into 50 mL of seed culture medium, and cultured at 30°C and 200 rpm on a shaking table until OD 600 11 was obtained as a primary seed;

[0069] (2) Secondary seed culture in a shake flask: 30 mL of the primary seed was inoculated into a secondary shake flask containing 300 mL of seed culture medium, and cultured at 30°C and 200 rpm on a shaking table until OD 600 15 was obtained as a secondary seed;

[0070] The seed culture medium in step (1) and step (2) was the same as the seed culture medium in Example 2 described above.

[0071] (3) Fermentation tank culture: a 5L fermentation tank was used for fermentation with 3L of fermentation medium, 300 mL of the cultured secondary seed solution was inoculated into the fermentation tank, the aeration rate was 4.5 L / min (ventilation ratio 1:1.5), the rotation speed was 400 rpm, the pH was 6.0, the temperature was 29°C, glucose was supplemented to control the glucose concentration to be more than 8 g / L, and the fermentation broth was obtained after 80 h of culture.

[0072] Fermentation medium: sucrose 100 g / L, yeast extract powder 12 g / L, sodium chloride 3.5 g / L, potassium carbonate 2 g / L, dipotassium hydrogen phosphate 3 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 4 g / L, ammonium sulfate 5 g / L, and water as the solvent.

[0073] Supplement: glucose solution with a mass concentration of 50%.

[0074] The content of pullulan and β-glucan in the fermentation broth and the molecular weight of pullulan were detected, and the specific method was the same as that in Example 2.

[0075] The detection results were as follows: the yield of pullulan reached 136 g / L, the molecular weight of pullulan was 193 kD,Figure 3 The yield of β-glucan was 20 g / L.

[0076] The inventors carried out fermentation culture of the initial strain M1 according to the culture method of Example 4, and detected the contents of pullulan and β-glucan in the fermentation liquor of the initial strain M1 and the molecular weight of pullulan according to the same detection method as that of Example 2. The detection results were as follows: the yield of pullulan was 62 g / L, the molecular weight of pullulan was 115 kD (Mw), and no β-glucan was detected. Figure 4

[0077] The strain Aureobasidium pullulans G1 provided by the present application can co-produce pullulan and β-glucan, and the yield of both is high.​

Claims

1. A budding short-stalked mold G1, characterized in that, Aureobasidium pullulans ( Aureobasidium pullulans G1 was deposited on March 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.41837.

2. The application of the budding short-stem mold G1 as described in claim 1 in the co-production of pullulan and β-glucan.

3. The application as described in claim 2, characterized in that, Pullulan has a molecular weight of 150kD~200kD.

4. The cultivation method for co-producing pullulan and β-glucan by *Bacillus buddingus* G1 as described in claim 1, characterized in that... Includes the following steps: (1) Inoculate the bacterial strain into the seed culture medium and culture until OD. 600 ≥5, as a first-level seed; (2) Inoculate the primary seeds into the seed culture medium and culture until OD. 600 ≥5, as a secondary seed; (3) Inoculate the secondary seeds into the fermentation medium at a volume ratio of 1~10%, ferment and culture at a speed of 50~500 rpm, an aeration ratio of 1:(0.5~3), a temperature of 25~35℃, and a pH of 4.0~8.

0. During the fermentation process, the glucose concentration is controlled above 8g / L by feeding sugar solution. The fermentation time is 60~120h to obtain the fermentation broth.

5. The method as described in claim 4, characterized in that, In step (1) or step (2), culture until OD 600 ≥10.

6. The method as described in claim 4, characterized in that, In step (1) or step (2), the seed culture medium consists of 50 g / L glucose, 5 g / L yeast extract, 2 g / L sodium chloride, and 3 g / L dipotassium hydrogen phosphate, with water as the solvent.

7. The method as described in claim 4, characterized in that, In step (3), the fermentation medium consists of: 80-200 g / L carbon source, 10-100 g / L nitrogen source and 0.1-20 g / L inorganic salt.

8. The method as described in claim 7, characterized in that, In step (3), the carbon source in the fermentation medium includes at least one of fructose, maltose, glucose, sucrose, lactose, galactose, and molasses; The nitrogen source includes at least one of the following: yeast extract powder, yeast paste, peptone, soybean meal powder, corn steep liquor, ammonium sulfate, ammonium chloride, and ammonium nitrate; The inorganic salts include at least one of the following: sodium chloride, potassium chloride, potassium carbonate, ferrous sulfate, magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, zinc sulfate, and copper sulfate.

9. The method as described in claim 8, characterized in that, In step (3), the fermentation medium composition includes any of the following formulations: Formula 1: Glucose 80g / L, sucrose 100g / L, yeast extract powder 10g / L, peptone 5g / L, sodium chloride 3.5g / L, dipotassium hydrogen phosphate 2g / L, potassium dihydrogen phosphate 1.5g / L, magnesium sulfate 2g / L, ammonium sulfate 4g / L, solvent is water; Formula 2: 100g / L sucrose, 60g / L lactose, 8g / L peptone, 3g / L yeast extract, 6g / L sodium chloride, 2g / L potassium carbonate, 3g / L dipotassium hydrogen phosphate, 1g / L potassium dihydrogen phosphate, 2g / L magnesium sulfate, 3.5g / L ammonium sulfate, with water as the solvent; Formula 3: 100g / L sucrose, 12g / L yeast extract, 3.5g / L sodium chloride, 2g / L potassium carbonate, 3g / L dipotassium hydrogen phosphate, 1g / L potassium dihydrogen phosphate, 4g / L magnesium sulfate, 5g / L ammonium sulfate, with water as the solvent.

10. The method as described in claim 4, characterized in that, In step (3), the feed sugar solution during fermentation includes sucrose solution and glucose solution.

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