Auxotrophic mutant of aureobasidium pullulans e1 and application thereof in coproduction of pullulan and beta-glucan

Abstract

The present application provides a strain of Aureobasidium pullulans E1 and its application in co-production of pullulan and beta-glucan, and belongs to the technical field of biological fermentation; specifically provides a strain of Aureobasidium pullulans E1, which is preserved in the China General Microbiological Culture Collection Center on March 17, 2025, the preservation address is No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.41836; the application of Aureobasidium pullulans E1 in co-production of pullulan and beta-glucan; the strain of Aureobasidium pullulans E1 provided by the present application can co-produce pullulan and beta-glucan, and the yield of both 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 ( Beta-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:

[0008] A strain of Aureobasidium pullulans (Aureobasidium pullulans ) E1, deposited on March 17, 2025 at China General Microbiological Culture Collection Center, located at No. 1, Michen West Road, Hua-yuan District, Beijing, with the accession number of CGMCC NO. 41836.

[0009] The application of the above-mentioned Aureobasidium pullulans E1 in co-production of pullulan and β-glucan.

[0010] According to the application, preferably, in the application, the molecular weight of the pullulan is 80 kD-120 kD.

[0011] The culture method of the above-mentioned Aureobasidium pullulans E1 for co-production of pullulan and β-glucan comprises 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°C, a pH of 4.0-8.0, 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 comprises 50 g / L of glucose, 5 g / L of yeast extract powder, 2 g / L of sodium chloride, and 3 g / L of potassium phosphate dibasic, with water as the solvent.

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

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

[0019] The nitrogen source comprises 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, monopotassium phosphate, zinc sulfate, and copper sulfate.

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

[0022] Formulation one: 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, monopotassium phosphate 1.5 g / L, magnesium sulfate 2 g / L, ammonium sulfate 3 g / L, and water as solvent;

[0023] Formulation two: 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, monopotassium phosphate 1 g / L, magnesium sulfate 2 g / L, ammonium sulfate 5 g / L, and water as solvent;

[0024] Formulation three: 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, monopotassium phosphate 1 g / L, magnesium sulfate 5 g / L, ammonium sulfate 5 g / L, and water as solvent.

[0025] The beneficial effects of the present application at least include the following:

[0026] Compared with the prior art, the strain Aureobasidium pullulans E1 provided by the present application can co-produce pullulan and beta-glucan, and the yield of both is high, the yield of pullulan reaches 153 g / L, and the yield of beta-glucan reaches 23 g / L. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Figure for detection of the molecular weight of pullulan in Example 2.

[0028] Figure 2 Figure for detection of the molecular weight of pullulan in Example 3.

[0029] Figure 3 Figure for detection of the molecular weight of pullulan in Example 4.

[0030] Figure 4 Figure for detection of the molecular weight of pullulan produced by the initial strain M1. DETAILED DESCRIPTION

[0031] The technical solutions of the present application are further described below in combination with examples, but the scope of protection of the present application is not limited thereto.

[0032] The experimental methods not specified in the following examples are usually carried out under conventional conditions.

[0033] Example 1

[0034] Mutation screening of bacterial strains

[0035] 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.

[0036] 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.

[0037]

[0038] 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:

[0039] 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.

[0040] Example 2

[0041] The method for culturing Aureobasidium pullulans E1 to co-produce pullulan and β-glucan comprises the following steps:

[0042] (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.

[0043] Solid 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;

[0044] (2) Primary shake flask seed culture: scrape 1 piece of strain, inoculate in 50 mL seed culture medium, 30℃, 200 rpm shaker culture to OD 600 10, as a primary seed;

[0045] (3) Secondary shake flask seed culture: take 30 mL of primary seed, inoculate in a secondary shake flask containing 300 mL of seed culture medium, 30℃, 200 rpm shaker culture to OD 600 12, as a secondary seed;

[0046] 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;

[0047] (4) Fermenter culture: 5L fermenter with 3L liquid volume for fermentation, take 300 mL of cultured secondary seed liquid, inoculate in the fermenter, aeration rate 9 L / min (ventilation ratio 1:3), rotation speed 300 rpm, pH 4.0, temperature 30℃, culture for 120h, to obtain the fermentation broth.

[0048] 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, potassium phosphate dibasic 2 g / L, potassium phosphate monobasic 1.5 g / L, magnesium sulfate 2 g / L, ammonium sulfate 3 g / L, solvent is water.

[0049] The content of pullulan and β-glucan in the fermentation broth and the molecular weight of pullulan were detected, and the specific method was as follows:

[0050] Pullulan detection: centrifuge the fermentation broth at 10000 rpm for 10 min, take the supernatant, add 1‰ β-glucanase, 55℃, pH 5.5, enzymolysis for 4h. Take 20 mL of enzyme solution and add 3 times of ethanol, centrifuge at 10000 rpm for 5 min, dry the precipitate and weigh, calculate the yield of pullulan.

[0051] Pullulan molecular weight detection: ethanol precipitated and dried pullulan sample, 2% sodium nitrite aqueous solution, prepared into 10 g / L sample solution. According to the molecular exclusion chromatography method (Chinese Pharmacopoeia 2020 edition general 0514) for detection. Water gel chromatography column, 2% sodium nitrite solution (water as solvent) as mobile phase, column temperature 30℃, flow rate is 0.5 mL per minute, differential refractive index detector detection.

[0052] Detection of β-glucan: centrifuge the fermentation broth at 10,000 rpm for 10 min, take the supernatant, add 1‰ pullulanase, and enzymatically hydrolyze at 55°C and pH 5 for 8 h. Take 20 mL of the enzymatically hydrolyzed solution, add 4 times of ethanol, centrifuge at 10,000 rpm for 5 min, dry the precipitate, and weigh to calculate the yield of β-glucan.

[0053] Detection results: the yield of pullulan in the fermentation broth is 153 g / L, the molecular weight of pullulan is 85 kD (see Figure 1 ), and the yield of β-glucan is 23 g / L.

[0054] Example 3

[0055] The culture method for coproducing pullulan and β-glucan by Aureobasidium pullulans E1 includes the following steps:

[0056] (1) Primary shake flask seed culture: take 0.5 mL of glycerol bacterial solution, inoculate into 50 mL of seed culture medium, and culture at 30°C and 200 rpm on a shaker until OD 600 11 is reached as a primary seed;

[0057] (2) Secondary shake flask seed culture: take 30 mL of the primary seed, inoculate into a secondary shake flask containing 300 mL of seed culture medium, and culture at 30°C and 200 rpm on a shaker until OD 600 15 is reached as a secondary seed;

[0058] The seed culture medium in step (1) and step (2) is the same as the seed culture medium in Example 2 above.

[0059] (3) Fermentor culture: use a 5 L fermentor, and ferment 3 L of fermentation medium. Take 300 mL of the cultured secondary seed solution, inoculate into the fermentor, set the aeration rate to 6 L / min (ventilation ratio 1:2), the rotation speed to 400 rpm, the pH to 7.0, and the temperature to 25°C, and culture for 100 h to obtain a fermentation broth.

[0060] Fermentation medium: sucrose 130 g / L, lactose 80 g / L, peptone 9 g / L, sodium chloride 2 g / L, potassium carbonate 2 g / L, potassium phosphate dibasic 3 g / L, potassium phosphate monobasic 1 g / L, magnesium sulfate 2 g / L, and ammonium sulfate 5 g / L, with water as the solvent.

[0061] Detect the contents of pullulan and β-glucan in the fermentation broth and the molecular weight of pullulan, and the specific method is the same as in Example 2.

[0062] Detection results: the yield of pullulan is 139 g / L, the molecular weight of pullulan is 110 kD (see Figure 2 ), and the yield of β-glucan is 17 g / L.

[0063] Example 4

[0064] The culture method for coproducing pullulan and beta-glucan by Ausdadia brevipes E1 comprises the following steps:

[0065] (1) Primary shake flask seed culture: 0.5 mL of glycerol bacterial solution is inoculated into 50 mL of seed culture medium, and cultured at 30°C and 200 rpm to OD 600 11 as a primary seed;

[0066] (2) Secondary shake flask seed culture: 30 mL of the primary seed is inoculated into a secondary shake flask containing 300 mL of seed culture medium, and cultured at 30°C and 200 rpm to OD 600 13 as a secondary seed;

[0067] The seed culture medium in step (1) and step (2) is the same as the seed culture medium in Example 2 above.

[0068] (3) Fermentor culture: 5 L of fermentation tank is used for fermentation with a liquid volume of 3 L, 300 mL of the cultured secondary seed solution is inoculated into the fermentation tank, the aeration volume is 7.5 L / min (ventilation ratio 1:2.5), the rotating speed is 200 rpm, the pH is 6.0, and the temperature is 29°C, and the culture is performed for 80 h to obtain a fermentation liquor.

[0069] The fermentation culture medium comprises 130 g / L of sucrose, 80 g / L of glucose, 9 g / L of proteose peptone, 3 g / L of yeast extract powder, 3 g / L of sodium chloride, 2 g / L of potassium carbonate, 3 g / L of potassium phosphate dibasic, 1 g / L of potassium phosphate monobasic, 5 g / L of magnesium sulfate, and 5 g / L of ammonium sulfate, and the solvent is water.

[0070] The content of pullulan and beta-glucan in the fermentation liquor and the molecular weight of pullulan are detected by the same method as in Example 2.

[0071] The detection result is that the yield of pullulan is 116 g / L, the molecular weight of pullulan is 115 kD (see Figure 3 ), and the yield of beta-glucan is 20 g / L.

[0072] According to the culture method in Example 4, the initial strain M1 is fermented, and the content of pullulan and beta-glucan in the fermentation liquor of the initial strain M1 and the molecular weight of pullulan are detected by the same detection method as in Example 2. The detection result is that the yield of pullulan is 40 g / L, the molecular weight of pullulan is 129 kD (see Figure 4 ), and no beta-glucan is detected.

[0073] The strain Ausdadia brevipes E1 provided by the application can coproduce pullulan and beta-glucan, and the yield of both is 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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