Aureobasidium pullulans E1 and application thereof in co-production of pullulan and beta-glucan

Abstract

The invention provides aureobasidium pullulans E1 and application of the aureobasidium pullulans E1 in co-production of pullulan and beta-glucan, and belongs to the technical field of biological fermentation. The aureobasidium pullulans E1 is preserved in the China General Microbiological Culture Collection Center on March 17, 2025, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.41836; the invention relates to an application of aureobasidium pullulans E1 in co-production of pullulan and beta-glucan. The strain aureobasidium pullulans E1 provided by the invention can co-produce pullulan and beta-glucan, and the yield of pullulan and beta-glucan is high.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and particularly relates to a budding Aureobasidium pullulans E1 strain and application thereof in the co-production of pullulan and beta-glucan. Background Art

[0002] Pullulan is a natural, water-soluble polysaccharide produced by microorganisms, primarily through fermentation metabolism by Aureobasidium pullulans. Its structure consists of repeating maltotriose units linked by α-1,6 glycosidic bonds. It exhibits excellent film-forming properties, adhesion, and biocompatibility, making it widely used in food, pharmaceuticals, cosmetics, and packaging.

[0003] β-glucan ( β-Glucan β-glucans are natural polysaccharides composed of glucose monomers linked by β-glycosidic bonds. They are widely found in the cell walls of yeast, cereals (such as oats and barley), fungi (such as shiitake mushrooms and Ganoderma lucidum), and some bacteria. Their molecular structure varies depending on their source, with common bond types including β-1,3, β-1,4, and β-1,6 glycosidic bonds. Due to its unique biological activity and physicochemical properties, β-glucans have important applications in food, medicine, cosmetics, and other fields.

[0004] Aureobasidium pullulans (scientific name: Aureobasidium pullulans ) is a polymorphic fungus widely distributed in nature, belonging to the phylum Ascomycota ( Ascomycota )、Saccharomyces( Saccharomycetes Its unique physiological properties and environmental adaptability make it valuable in fields such as biotechnology, the food industry, and environmental remediation, making it a key production strain in industrial microbiology. Currently, most literature mentions that Aureobasidium pullulans is primarily used to produce pullulan, polymalic acid, and melanin, with only a few studies suggesting that Aureobasidium pullulans can also secrete extracellular β-glucan.

[0005] Numerous studies have been conducted on strains producing either pullulan or β-glucan. For example, Chinese patent CN 117660204 A (Application Number: 202311240831.1) describes a method for producing β-glucan by knocking out genes involved in pullulan to produce β-glucan alone. However, studies on strains producing both polysaccharides are limited, and yields have been relatively low. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the present invention provides a strain of Aureobasidium pullulans E1 and its application in the co-production of pullulan and β-glucan.

[0007] The technical solutions adopted in the present invention are as follows: A strain of Aureobasidium pullulans ( Aureobasidium pullulans) E1, deposited on March 17, 2025 in the General Microbiology Center of China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.41836.

[0008] The application of the above-mentioned Aureobasidium pullulans E1 in the co-production of pullulan and β-glucan.

[0009] Preferably according to the present invention, in the application, the molecular weight of pullulan is 80kD~120kD.

[0010] The above-mentioned method for culturing Aureobasidium pullulans E1 to co-produce pullulan and β-glucan comprises the following steps: (1) Inoculate the bacteria into the seed culture medium and culture until OD 600 ≥5, as first-level seed; (2) Inoculate the first-level seeds into the seed culture medium and culture until OD 600 ≥5, as secondary seeds; (3) The secondary seeds were inoculated into the fermentation medium at a volume ratio of 1-10%, and the fermentation culture was carried out at a rotation speed of 50-500 rpm, a ventilation ratio of 1:(0.5-3), a temperature of 25-35°C, a pH of 4.0-8.0, and a fermentation time of 60-120 h to obtain a fermentation liquid.

[0011] Preferably, in step (1) or step (2), the culture is carried out until the OD 600 ≥10.

[0012] Preferably, in step (1) or step (2), the seed culture medium comprises: 50 g / L glucose, 5 g / L yeast extract powder, 2 g / L sodium chloride, 3 g / L potassium hydrogen phosphate, and the solvent is water.

[0013] Preferably, in step (3), the fermentation medium comprises: 80-200 g / L of carbon source, 10-100 g / L of nitrogen source and 0.1-20 g / L of inorganic salt.

[0014] Preferably, in step (3), the carbon source in the fermentation medium components includes: at least one of fructose, maltose, glucose, sucrose, lactose, galactose, and molasses; The nitrogen source comprises at least one of yeast extract powder, yeast extract, peptone, soybean cake powder, corn steep liquor, ammonium sulfate, ammonium chloride, and ammonium nitrate; The inorganic salt includes 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.

[0015] Preferably, in step (3), the fermentation medium components include any of the following formulas: Formula 1: Glucose 140g / L, sucrose 85g / L, peptone 8g / L, yeast extract powder 10g / L, sodium chloride 5g / L, dipotassium hydrogen phosphate 2g / L, potassium dihydrogen phosphate 1.5g / L, magnesium sulfate 2g / L, ammonium sulfate 3g / L, solvent is water; Formula 2: sucrose 130g / L, lactose 80g / L, peptone 9g / L, sodium chloride 2g / L, potassium carbonate 2g / L, dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 2g / L, ammonium sulfate 5g / L, solvent is water; Formula 3: sucrose 130g / L, glucose 80g / L, peptone 9g / L, yeast extract powder 3g / L, sodium chloride 3g / L, potassium carbonate 2g / L, dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 5g / L, ammonium sulfate 5g / L, solvent is water.

[0016] The beneficial effects of the present invention include at least the following: Compared with the prior art, the strain Aureobasidium pullulans E1 provided by the present invention can co-produce pullulan and β-glucan, and the yields of both are high, with the pullulan yield reaching 153 g / L and the β-glucan yield reaching 23 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a detection diagram of the pullulan molecular weight in Example 2.

[0018] Figure 2 This is a detection diagram of the pullulan molecular weight in Example 3.

[0019] Figure 3 This is a detection diagram of the pullulan molecular weight in Example 4.

[0020] Figure 4 This is a detection chart of the molecular weight of pullulan produced by the initial strain M1. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described below in conjunction with embodiments, but the scope of protection of the present invention is not limited thereto.

[0022] The experimental methods in the following examples without specific conditions are generally based on conventional conditions.

[0023] Example 1 Mutation screening of bacterial strains The initial strain M1 is a budding Aureobasidium strain preserved in the laboratory of Shandong Mimei Biotechnology Co., Ltd., and was isolated in an orchard in Linqu County, Weifang City.

[0024] Strain M1 was used as the initial mutagenic strain. The bacteria were cultured on a plate for 4 days and the cells or spores were washed off the plate with sterile saline. The cells were diluted to 10 6 Use 20W UV lamp, irradiate at a distance of 30cm for 0s, 10s, 20s, 30s, 40s, 50s, 60s, 80s, and perform gradient dilution under red light. 3 , 10 4 , 10 5 Three gradients were applied to solid culture medium plates (the solid culture medium: 50 g / L glucose, 5 g / L yeast extract powder, 2 g / L sodium chloride, 3 g / L potassium hydrogen phosphate, 15 g / L agar powder, and the solvent was water), wrapped in black plastic bags, and incubated at 30°C in the dark for 2 days. Appropriate gradients were selected for plate counting and calculation of the lethality.

[0025]

[0026] UV mutagenesis was performed at an irradiation time that resulted in an 80% lethality rate. Strains with white colonies and large growth were selected for fermentation verification in Erlenmeyer flasks containing fermentation medium. Pullulan and β-glucan production was measured. Mutagenesis was repeated until a strain, Aureobasidium pullulans E1, was identified as a high-yield strain of both. The selected strains were deposited with the China National Center for the Administration of Microbial Culture Collection. Detailed deposit information is as follows: A strain of Aureobasidium pullulans ( Aureobasidium pullulans ) E1, deposited on March 17, 2025 in the General Microbiology Center of China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.41836.

[0027] Example 2 The method for culturing Aureobasidium pullulans E1 to co-produce pullulan and β-glucan comprises the following steps: (1) Activation of bacterial strains: Use an inoculation loop to take one loop of glycerol bacterial solution and inoculate it onto a solid culture medium plate using the streak method. Place the plate upside down at 30°C incubator and culture for 3 days. Solid culture medium: glucose 50g / L, yeast extract powder 5g / L, sodium chloride 2g / L, potassium dihydrogen phosphate 3g / L, agar powder 15g / L, solvent is water; (2) Primary shake flask seed culture: scrape 1 piece of bacterial strain, inoculate it into 50mL seed culture medium, and culture it in a shaker at 30℃ and 200rpm until the OD 600 10, as a first-level seed; (3) Secondary shake flask seed culture: Take 30 mL of primary seeds and inoculate them into a secondary shake flask containing 300 mL of seed culture medium. Cultivate at 30°C, 200 rpm, and shake until the OD 60012, as a secondary seed; The seed culture medium in step (2) and step (3) includes 50 g / L glucose, 5 g / L yeast extract powder, 2 g / L sodium chloride, and 3 g / L potassium hydrogen phosphate, and the solvent is water; (4) Fermentation tank culture: A 5L fermentation tank was filled with 3L of liquid for fermentation. 300mL of the cultured secondary seed liquid was inoculated into the fermentation tank with an aeration rate of 9L / min (ventilation ratio 1:3), a rotation speed of 300rpm, a pH of 4.0, and a temperature of 30°C. The culture was carried out for 120h to obtain the fermentation liquid.

[0028] Fermentation medium: glucose 140 g / L, sucrose 85 g / L, peptone 8 g / L, yeast extract powder 10 g / L, sodium chloride 5 g / L, dipotassium hydrogen phosphate 2 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate 2 g / L, ammonium sulfate 3 g / L, solvent is water.

[0029] The content of pullulan and β-glucan in the fermentation broth and the molecular weight of pullulan were detected as follows: Pullulan assay: Centrifuge the fermentation broth at 10,000 rpm for 10 minutes, remove the supernatant, add 1‰ β-glucanase, and incubate at 55°C, pH 5.5, for 4 hours. Add 3x ethanol to 20 mL of the hydrolyzate, centrifuge at 10,000 rpm for 5 minutes, dry the precipitate, and weigh it to calculate the pullulan yield.

[0030] Pullulan molecular weight test: Prepare a 10 g / L sample of pullulan after ethanol precipitation and drying with a 2% aqueous sodium nitrite solution. Determination was performed using size exclusion chromatography (Chinese Pharmacopoeia 2020 General Chapter 0514). An aqueous gel chromatography column was used with a 2% sodium nitrite solution (water as the solvent) as the mobile phase, column temperature at 30°C, flow rate at 0.5 mL / min, and detection was performed using a differential refractive index detector.

[0031] β-glucan assay: Centrifuge the fermentation broth at 10,000 rpm for 10 minutes, remove the supernatant, add 1‰ pullulanase, and incubate at 55°C, pH 5, for 8 hours. Add 4x ethanol to 20 mL of the hydrolyzate, centrifuge at 10,000 rpm for 5 minutes, dry and weigh the precipitate, and calculate the β-glucan yield.

[0032] Test results: The pullulan yield in the fermentation broth reached 153g / L, and the molecular weight of pullulan was 85kD (see Figure 1 ); β-glucan production was 23 g / L.

[0033] Example 3 The method for culturing Aureobasidium pullulans E1 to co-produce pullulan and β-glucan comprises the following steps: (1) Primary shake flask seed culture: Take 0.5 mL of glycerol bacterial solution and inoculate it into 50 mL of seed culture medium. Incubate at 30°C and 200 rpm in a shaker until the OD 600 11, as a first-level seed; (2) Secondary shake flask seed culture: Take 30 mL of primary seeds and inoculate them into a secondary shake flask containing 300 mL of seed culture medium. Cultivate at 30°C, 200 rpm, and shake until the OD 600 15, as a secondary seed; The seed culture medium in step (1) and step (2) is the same as the seed culture medium in Example 2 above.

[0034] (3) Fermentation tank culture: A 5 L fermentation tank was used with a fermentation medium volume of 3 L for fermentation. 300 mL of the cultured secondary seed liquid was inoculated into the fermentation tank with an aeration volume of 6 L / min (ventilation ratio 1:2), a rotation speed of 400 rpm, a pH of 7.0, and a temperature of 25 °C. The culture was carried out for 100 h to obtain the fermentation liquid.

[0035] Fermentation medium: sucrose 130 g / L, lactose 80 g / L, peptone 9 g / L, sodium chloride 2 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 5 g / L, solvent is water.

[0036] The contents of pullulan and β-glucan in the fermentation broth and the molecular weight of pullulan were detected using the same method as in Example 2.

[0037] Test results: Pullulan yield reached 139g / L, and the molecular weight of pullulan was 110kD (see Figure 2 ), the β-glucan production reached 17g / L.

[0038] Example 4 The method for culturing Aureobasidium pullulans E1 to co-produce pullulan and β-glucan comprises the following steps: (1) First-stage shake flask seed culture: Take 0.5 mL of glycerol bacterial solution and inoculate it into 50 mL of seed culture medium. Incubate at 30°C and 200 rpm in a shaker until the OD 600 11, as a first-level seed; (2) Secondary shake flask seed culture: Take 30 mL of primary seeds and inoculate them into a secondary shake flask containing 300 mL of seed culture medium. Cultivate at 30°C, 200 rpm, and shake until the OD 600 13, as a secondary seed; The seed culture medium in step (1) and step (2) is the same as the seed culture medium in Example 2 above.

[0039] (3) Fermentation tank culture: A 5 L fermentation tank was filled with 3 L of liquid for fermentation. 300 mL of the cultured secondary seed liquid was inoculated into the fermentation tank with an aeration volume of 7.5 L / min (ventilation ratio 1:2.5), a rotation speed of 200 rpm, a pH of 6.0, and a temperature of 29 °C. The culture was carried out for 80 h to obtain the fermentation liquid.

[0040] Fermentation medium: sucrose 130 g / L, glucose 80 g / L, peptone 9 g / L, yeast extract powder 3 g / L, sodium chloride 3 g / L, potassium carbonate 2 g / L, dipotassium hydrogen phosphate 3 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 5 g / L, ammonium sulfate 5 g / L, solvent is water.

[0041] The contents of pullulan and β-glucan in the fermentation broth and the molecular weight of pullulan were detected using the same method as in Example 2.

[0042] Test results: Pullulan yield reached 116g / L, and the molecular weight of pullulan was 115kD (see Figure 3 ), the β-glucan production reached 20g / L.

[0043] The inventors fermented the initial strain M1 according to the cultivation method of Example 4, and detected the contents of pullulan and β-glucan in the fermentation broth of the initial strain M1 and the molecular weight of pullulan according to the same detection method as in Example 2. The test results showed that the pullulan yield was 40 g / L and the molecular weight of pullulan was 129 kD (see Figure 4 ), no β-glucan was detected.

[0044] The strain Aureobasidium pullulans E1 provided by the present invention can co-produce pullulan and beta-glucan, and the yields of both are high.

Claims

1. A strain of Aureobasidium pullulans E1, characterized in that: Aureobasidium pullulans ( Aureobasidium pullulans ) E1 was deposited in the General Microbiology Center of China Culture Collection Administration on March 17, 2025. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.41836.

2. Use of the Aureobasidium pullulans E1 according to claim 1 in the co-production of pullulan and β-glucan.

3. The use according to claim 2, characterized in that The molecular weight of pullulan is 80kD~120kD.

4. The method for culturing Aureobasidium pullulans E1 for co-producing pullulan and β-glucan according to claim 1, characterized in that: The steps include: (1) Inoculate the bacteria into the seed culture medium and culture until OD 600 ≥5, as first-level seed; (2) Inoculate the first-level seeds into the seed culture medium and culture until OD 600 ≥5, as secondary seeds; (3) The secondary seeds were inoculated into the fermentation medium at a volume ratio of 1-10%, and the fermentation culture was carried out at a rotation speed of 50-500 rpm, a ventilation ratio of 1:(0.5-3), a temperature of 25-35°C, a pH of 4.0-8.0, and a fermentation time of 60-120 h to obtain a fermentation liquid.

5. The method according to claim 4, wherein In step (1) or step (2), culture until OD 600 ≥10.

6. The method according to claim 4, wherein In step (1) or step (2), the seed culture medium comprises: 50 g / L glucose, 5 g / L yeast extract powder, 2 g / L sodium chloride, 3 g / L potassium hydrogen phosphate, and the solvent is water.

7. The method according to claim 4, wherein In step (3), the fermentation medium components include: 80-200 g / L of carbon source, 10-100 g / L of nitrogen source and 0.1-20 g / L of inorganic salt.

8. The method according to claim 7, wherein In step (3), the carbon source in the fermentation medium components includes at least one of fructose, maltose, glucose, sucrose, lactose, galactose, and molasses; The nitrogen source comprises at least one of yeast extract powder, yeast extract, peptone, soybean cake powder, corn steep liquor, ammonium sulfate, ammonium chloride, and ammonium nitrate; The inorganic salt includes 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.

9. The method according to claim 8, wherein In step (3), the fermentation medium composition includes any of the following formulas: Formula 1: Glucose 140g / L, sucrose 85g / L, peptone 8g / L, yeast extract powder 10g / L, sodium chloride 5g / L, dipotassium hydrogen phosphate 2g / L, potassium dihydrogen phosphate 1.5g / L, magnesium sulfate 2g / L, ammonium sulfate 3g / L, solvent is water; Formula 2: sucrose 130g / L, lactose 80g / L, peptone 9g / L, sodium chloride 2g / L, potassium carbonate 2g / L, dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 2g / L, ammonium sulfate 5g / L, solvent is water; Formula 3: sucrose 130g / L, glucose 80g / L, peptone 9g / L, yeast extract powder 3g / L, sodium chloride 3g / L, potassium carbonate 2g / L, dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 5g / L, ammonium sulfate 5g / L, solvent is water.

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