Method for producing pullulan using Aureobasidium pullulans

Abstract

The invention provides a method for producing pullulan by using Aureobasidium pullulans. The method adopts a microbial conversion method for the first time, wherein fructooligosaccharide is converted into pullulan by catalyzing an enzyme reaction liquid. A budding Aureobasidium pullulans seed culture liquid is used as a carrier, fructooligosaccharide is used as a substrate, and the enzyme reaction liquid is used as a catalyst. A one-time substrate addition or multiple flow addition of fructooligosaccharide and the enzyme reaction liquid is adopted in a 5.0L bioreactor. The catalytic reaction is carried out for 60hr-72hr, and a total consumption of fructooligosaccharide is 61.43g / L-71.43g / L. The pullulan yield reaches a maximum of 60.2g / L-64.26g / L, and the conversion rate of fructooligosaccharide is as high as 87.5%-97.98%. Compared with the existing fermentation method for producing pullulan, the biotransformation method can solve the problem of insufficient enzyme amount in the catalytic reaction relied on in the pullulan synthesis process on the one hand, and on the other hand, it can also solve the reverse regulation of pullulan synthesis by the substrate and product during the pullulan synthesis process, as well as the resulting low substrate carbon source utilization rate and low substrate conversion rate.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial pharmaceutical manufacturing, and particularly relates to a production process for producing pullulan polysaccharide by a budding Aureobasidium pullulans transformation method. Background Art

[0002] The production of pullulan by microbial fermentation was first discovered by German scientist Bender in 1959. Pullulan is a viscous extracellular polysaccharide produced by Aurebasidium pulluans using sugar compounds as a substrate through aerobic fermentation. It is non-toxic, harmless, colorless, and odorless, and is resistant to heat, salt, acids, and alkalis. It exhibits low viscosity, strong plasticity, excellent film-forming properties, and excellent thickening properties. It has been widely used in the pharmaceutical, food, light industry, chemical, and petroleum industries.

[0003] On May 19, 2006, the Ministry of Health issued Announcement No. 8, adding pullulan as one of four new food additives. The Biochemical Research Institute of Hayashibara Corporation in Japan, in collaboration with Pfizer in the United States, successfully developed hard capsules made from pullulan, marking the first application of pullulan in the pharmaceutical field. This new hard capsule product exhibits excellent disintegration, oxygen impermeability, and stability. In 2020, my country also included pullulan hollow capsules in the pharmaceutical excipient section of Part IV of the 2020 Chinese Pharmacopoeia. Domestically, pullulan has been used in candies, chocolate coatings, films, and fruit and vegetable juice beverages. Therefore, pullulan is a new multifunctional biomaterial with great economic value and development potential, and has broad market prospects.

[0004] Fructooligosaccharides (FOS), also known as oligofructose, are small molecular weight polysaccharides composed of fructose residues and D-fructose molecules linked by β-1,2-glycosidic bonds (Sheng Dongfeng, 2005). FOS are colorless powdered solids that are colorless and transparent in solution. They have high solubility, but their thermal stability is significantly affected by acids and bases. The viscosity of FOS decreases with increasing temperature between 0 and 70°C. Fructooligosaccharides formed by linking sucrose molecules with one D-fructose molecule, two D-fructose molecules, and three D-fructose molecules are called fructooligotrisaccharides (GF2), fructooligotetrasaccharides (GF3), and fructooligopentaaccharides (GF4), respectively (Wang Bo, 2015). FOS occur naturally in approximately 30,000 plant species, including those in the Campanulaceae, Asteraceae, Gentianaceae, Lobelia, Malvaceae, Primulaceae, and Boraginaceae families (Wu Dongru, 1987).

[0005] Currently, efforts to increase pullulan yields primarily rely on identifying high-yielding strains, constructing genetically engineered strains, performing mutagenesis screening, and optimizing fermentation medium components and fermentation control processes, often with limited success. Kim JH et al. believe that excessive carbon sources can actually reduce pullulan yield. Zheng Aiquan ("Introduction to Modern Biotechnology," p. 88) suggests that adding fresh feed containing one or more nutrients once or multiple times during fermentation can extend the product synthesis cycle and increase yield. This, compared to traditional batch fermentation, can alleviate the inhibition of nutrient substrates. However, he also notes that the feed amount or rate during fermentation is still determined empirically and is somewhat unpredictable. This makes it difficult to simultaneously meet the needs of microbial growth and product synthesis, and it is impossible to completely avoid substrate-mediated reactions. Therefore, simply adding fresh feed containing one or more nutrients during fermentation cannot address substrate-mediated reactions during pullulan synthesis, particularly the inhibition caused by insufficient enzyme production in the catalytic reactions required for substrate carbon source metabolism, resulting in low substrate carbon source utilization and conversion rates. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a method for producing pullulan by using Aureobasidium pullulans. The method adopts a microbial conversion method for the first time. The entire pullulan production process does not require the use of a fermentation medium for fermentation culture. Fructose oligosaccharides are directly converted into pullulan by an enzyme reaction liquid. The method uses Aureobasidium pullulans seed culture liquid as a carrier, fructooligosaccharides as a substrate, and an enzyme reaction liquid as a catalyst. A one-time substrate addition or multiple flow additions of fructooligosaccharides and enzyme reaction liquid are carried out in a 5.0L bioreactor. The catalytic reaction is carried out for 60hr-72hr, and a total consumption of fructooligosaccharides is 61.43g / L-71.43g / L. The pullulan yield is as high as 60.2g / L-64.26g / L, and the conversion rate of fructooligosaccharides is as high as 87.5%-97.98%. Compared with existing fermentation methods for producing pullulan, the bioconversion method can solve the problem of insufficient enzymes for the catalytic reaction relied upon during pullulan synthesis. It also addresses the negative regulatory effects of substrate and product on pullulan synthesis during pullulan synthesis, as well as the resulting low substrate carbon source utilization and conversion rate. The Aureobasidium pullulans strain utilized was deposited with the China General Microbiological Culture Collection (CGMCC) on April 19, 2022; the deposit number is CGMCC No. 40158, and the classification name is Aureobasidium pullulans. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The invention discloses a method for producing pullulan by using Aureobasidium pullulans, which mainly comprises the following steps.

[0008] Seed plate culture: The EP1001 strain, which is deposited in the China General Microbiological Culture Collection Center and has a deposit number of CGMCC No. 40158, was inoculated into a PDA plate culture medium by streaking with an inoculating needle for activation culture. The specific steps are as follows: the budding Aureobasidium pullulans strain EP1001 was taken out of a -80 refrigerator, thawed naturally on ice, and transferred to a PDA plate culture medium using an inoculating needle on a sterile laminar bench. The culture was then placed in a constant temperature incubator at 30°C in the dark for 72 hours ( Figure 1 、 Figure 2 ).

[0009] Seed liquid culture: The activated Aureobasidium pullulans strain EP1001 in the above step was transferred to liquid seed culture medium and cultured for 24 hours ( Figure 3 The specific steps are as follows: a single colony of the culture activated on a PDA plate medium for 72 hours was inoculated into a 500 ml Erlenmeyer flask containing 100 ml of liquid seed medium, cultured at 30°C, shaken at 220 rpm, and shaken for 24 hours to obtain a liquid culture of the Aureobasidium pullulans strain.

[0010] To prepare the enzyme reaction solution, remove the Aureobasidium pullulans strain EP1001 from a -80 freezer, thaw naturally on ice, and transfer the culture to a PDA plate using an inoculating needle on a sterile laminar flow hood. Incubate in a constant-temperature incubator at 30°C in the dark for 72 hours. A single colony from the activated culture on the PDA plate for 72 hours was inoculated into a 500ml Erlenmeyer flask containing 100ml of liquid seed medium. The culture was shaken at 30°C and 220 rpm for 24 hours. The resulting Aureobasidium pullulans liquid culture was centrifuged at 12,000 rpm for 15 minutes. The supernatant obtained was the enzyme reaction solution.

[0011] Furthermore, in the process of converting fructooligosaccharide to produce pullulan by Aureobasidium pullulans, the enzyme reaction solution is added to the conversion culture medium in batches during the microbial conversion process.

[0012] Furthermore, the enzyme reaction solution was added all at once at 20 hr, 40 hr, and 60 hr of microbial transformation.

[0013] Furthermore, the content of the enzyme reaction solution in the microbial transformation culture medium is 0.0 ml / -500 ml.

[0014] Furthermore, the enzyme reaction liquid is derived from the supernatant separated from the fungal fermentation liquid.

[0015] Furthermore, the fungal fermentation liquid includes all fungal microorganisms that can produce enzyme reaction liquid.

[0016] Furthermore, the strain deposit number of the fungal microorganism is CGMCC No. 40158, the classification name is Aureobasidium pullulans, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0017] Transformation culture medium: 1.0L-2.0L of liquid culture from seed liquid culture was transferred to a 5.0L bioreactor containing 0.0L-3.5L of 100g / L fructooligosaccharide solution sterilized at 121℃. Figure 5 ), control the stirring speed at 200-600 rpm, the aeration rate at 1.0:1.0-2.0 (V / V), the tank pressure at 0.01-0.02 MPa, the temperature at 25-28°C, and the pH at 2.5-3.5. During this time, add 0.0-100 g / L of fructooligosaccharide solution at a single dose or continuously at a flow rate of 1.0-3.0 g / L·h. Add 0.0-500 ml of the enzyme reaction solution at 20, 40, and 60 hours of conversion, respectively. The conversion incubation period ends after 60-72 hours.

[0018] Furthermore, the fructooligosaccharides in the transformation medium are mainly extracts obtained from plants of Campanulaceae, Asteraceae, Gentianaceae, Lobelia, Malpighiaceae, Primulaceae, and Boraginaceae.

[0019] Furthermore, the extract mainly consists of small molecular heteropolysaccharides formed by fructose residues and D-fructose molecules connected by β-1,2 glycosidic bonds.

[0020] Furthermore, the small molecule heteropolysaccharide is mainly a combination of one or more of kestose, kestotetraose and kestopentaose.

[0021] Furthermore, the concentration of fructooligosaccharide in the transformation medium is 0.0 g / L-100 g / L.

[0022] Furthermore, when fructooligosaccharide is added to the bioreactor vessel at one time, its concentration in the transformation medium is 100 g / L.

[0023] Furthermore, when fructooligosaccharide is added by continuous addition during the transformation process, the initial concentration of fructooligosaccharide in the transformation medium of the bioreactor vessel is 0.0 g / L and the final concentration is 100 g / L.

[0024] Furthermore, the addition rate of fructooligosaccharide during the conversion process is 1.0 g / L•h-3.0 g / L•h.

[0025] Furthermore, the final transformation medium volume was 3.5 L.

[0026] Furthermore, the final conversion cycle is 60hr-72hr.

[0027] Determination of pullulan yield: Take 10 ml of the transformation culture medium and centrifuge it at 12000 rpm for 15 minutes. Transfer the supernatant to a new centrifuge tube and add 1.8 times the volume of anhydrous ethanol as the supernatant. Mix by inversion 30 times. Centrifuge at 12000 rpm in a high-speed centrifuge for 20 minutes. Discard the supernatant and dry the precipitate at 120°C for 24 hours. Then weigh it to obtain the pullulan yield.

[0028] The advantage of the present invention is that the Aureobasidium pullulans strain EP1001 was deposited in the China General Microorganism Culture Collection Center (CGMCC for short) on April 19, 2022; the deposit number is CGMCC No. 40158, and the classification name is Aureobasidium pullulans, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The method adopts a microbial conversion method. The entire pullulan production process does not require the use of a fermentation medium for fermentation culture. The pullulan is produced by directly catalyzing the conversion of fructooligosaccharides through an enzyme reaction liquid. With Aureobasidium pullulans seed culture liquid as a carrier, fructooligosaccharides as a substrate, and an enzyme reaction liquid as a catalyst, a fructooligosaccharide solution 2.5 times the volume of the seed liquid is added once or continuously in a 5.0L bioreactor during the conversion process. The seed liquid is continuously diluted during the conversion process, thereby reducing the concentrations of the substrate carbon source and the product pullulan in the conversion liquid, effectively eliminating the inhibitory effects of the high concentration of the carbon source and the product pullulan in the substrate on the synthesis of pullulan. At the same time, the enzyme reaction liquid is introduced during the conversion process, thereby improving the decomposition efficiency of the substrate carbon source and the synthesis efficiency of the pullulan, and greatly improving the utilization efficiency of the substrate carbon source. In the present invention, a total of 61.43 g / L-71.43 g / L of fructooligosaccharides is consumed, while the maximum pullulan yield is 60.2 g / L-64.26 g / L, and the conversion rate of fructooligosaccharides is as high as 87.5%-97.98%. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 EP1001 strain grown on PDA plate medium for 72 hours;

[0030] Figure 2 Mycelial morphology of strain EP1001 grown on PDA plate medium for 72 hours;

[0031] Figure 3Changes in hyphae morphology of EP1001 strain when cultured in liquid seed medium for 24 h;

[0032] Figure 4 Morphological changes of the EP1001 strain during the first 52 hours of mycelial growth under different transformation culture conditions, where PA-0017, PA-0018, and PA-0020 correspond to Examples 1, 2, and 3, respectively;

[0033] Figure 5 The structure of the 5.0L bioreactor and its liquid filling before transformation. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0035] A method for producing pullulan by using Aureobasidium pullulans comprises the following steps:

[0036] 1) Seed cultivation

[0037] 1.1) Prepare plate seed culture medium;

[0038] 1.1.1) Prepare potato dextrose agar: Weigh 200 g of peeled potatoes, cut into small pieces, add 1000 mL of water, and boil for 30 minutes. Filter the clear solution through six layers of gauze. Add water to the clear solution to make it 1000 mL. Then, add 20 g of glucose and dissolve it completely. Then, add 20 g of agar and stir until thoroughly mixed. This will produce potato dextrose agar.

[0039] 1.1.2) Prepare plate seeds. Remove strain EP1001, deposited with the China General Microbiological Culture Collection (CGMCC No. 40158), from a -80 freezer, thaw naturally on ice, and then transfer to a PDA slant medium using an inoculating needle on a sterile laminar bench. Incubate in a constant temperature incubator at 27°C in the dark for 72 hours. Figure 1 、 Figure 2 );

[0040] 1.1.3) Prepare liquid seeds by inoculating a single colony from a plate of A. pullulans into liquid seed culture medium for shake flask activation. Specifically, place 100 ml of seed culture medium into a 500 ml Erlenmeyer flask and sterilize it with high temperature and high pressure. After cooling, inoculate a single colony from a plate of A. pullulans into liquid seed culture medium for shake flask activation. Culture conditions are: rotation speed 220 rpm, temperature 30°C, and incubation for 24 hours ( Figure 3The liquid seed culture medium comprises the following components: yeast extract 4.0 g / L, glucose 20.0 g / L, sucrose 10.0 g / L, molasses 10.0 g / L, (NH4)2SO4 1.32 g / L, NaNO3 1.68 g / L, K2HPO4 10.0 g / L, NaCl 2.0 g / L, MgSO4·7H2O 0.4 g / L, pH 7.0, sterilized at 121°C for 20 min;

[0041] 2) Transformation culture medium

[0042] 2.1) Transfer 1.0L-2.0L of the activated liquid seed culture to a 5.0L bioreactor containing 0.0L-3.5L of fructooligosaccharide solution ( Figure 5 ) for pullulan production; wherein the conversion medium comprises the following components: 0.0g / L-100g / L fructooligosaccharide, 0.0ml-500ml enzyme reaction solution;

[0043] 2.2) Transformation culture control process: in a 5.0 L bioreactor ( Figure 5 ), control the stirring speed at 200-600 rpm, the ventilation volume at 1.0:1.0-2.0 (V / V), the tank pressure at 0.01-0.02 MPa, the temperature at 25-28°C, and the pH at 2.5-3.5. During this period, 0.0-100 g / L of fructooligosaccharide is added in a single dose or 1.0-3.0 g / L / h of fructooligosaccharide is added continuously. 0.0-500 ml / L of enzyme reaction solution is added at 20, 40, and 60 hours of conversion, respectively. The conversion and incubation are continued for 60-72 hours. The pH is controlled between 2.5-3.5 using 1.0 M hydrochloric acid solution and 1.0 M sodium hydroxide solution, respectively. The conversion and incubation are completed after 60-72 hours.

[0044] 2.3) Determination of pullulan yield: Centrifuge 10 ml of the conversion solution at 12,000 rpm for 15 minutes. Transfer the supernatant to a fresh centrifuge tube and add 1.8 times the volume of anhydrous ethanol to the supernatant. Mix by inversion 30 times. Centrifuge at 12,000 rpm for 20 minutes in a high-speed centrifuge. Discard the supernatant and dry the precipitate at 120°C for 24 hours. The weight of the precipitate represents the pullulan yield. Example 1

[0045] See 1) Seed culture steps for seed culture.

[0046] 1.0 L of the activated liquid seed culture was transferred to a 5.0 L bioreactor containing 2.5 L of 100 g / L fructooligosaccharide solution for pullulan production. The total reaction volume was 3.5 L ( Figure 5).

[0047] Transformation culture control process: In a 5.0 L bioreactor, the stirring speed was controlled at 600 rpm, the ventilation volume was 1.0:2.0 (V / V), the tank pressure was 0.01 MPa, the temperature was 28°C, and the pH value was controlled at 2.8 using 1.0 M hydrochloric acid solution and 1.0 M sodium hydroxide solution, respectively. Transformation culture was completed for 70 hours.

[0048] Determination of pullulan yield: After the conversion is completed, 10 ml of the conversion solution was taken and centrifuged at 12000 rpm for 15 minutes. The supernatant was transferred to a new centrifuge tube, and 1.8 times the volume of the supernatant was added with anhydrous ethanol. After inversion 30 times to mix, the tube was centrifuged at 12000 rpm in a high-speed centrifuge for 20 minutes. The supernatant was discarded and the precipitate was dried at 120°C for 24 hours and weighed. The pullulan yield was 62.5 g / L, and the substrate conversion rate was as high as 87.5% (Table 1). Example 2

[0049] See 1) Seed culture steps for seed culture.

[0050] 1.0 L of the activated liquid seed culture was transferred to a 5.0 L bioreactor containing 0.0 L of a 100 g / L fructooligosaccharide solution for pullulan production. The total reaction volume was 3.5 L.

[0051] Transformation culture control process: In a 5.0-L bioreactor, the stirring speed was controlled at 600 rpm, the ventilation volume was 1.0:2.0 (V / V), the tank pressure was 0.01 MPa, the temperature was 28°C, and the pH was controlled at 2.8 using 1.0 M hydrochloric acid solution and 1.0 M sodium hydroxide solution, respectively. As the seed culture was transferred to the 5.0-L bioreactor, the transformation timer began. 2.5 L of a 100 g / L fructooligosaccharide solution was continuously added at a flow rate of 1.0 g / L / h. After 70 hours of transformation culture, the pullulan yield was 64.26 g / L, and the substrate conversion rate was as high as 89.96% (Table 1). Example 3

[0052] See 1) Seed culture steps for seed culture.

[0053] 1.0 L of the activated liquid seed culture was transferred to a 5.0 L bioreactor containing 0.0 L of a 100 g / L fructooligosaccharide solution for pullulan production. The total reaction volume was 3.5 L.

[0054] Transformation culture control process: In a 5.0-L bioreactor, the stirring speed was controlled at 600 rpm, the ventilation volume was 1.0:2.0 (V / V), the tank pressure was 0.01 MPa, the temperature was 28°C, and the pH was controlled at 2.8 with 1.0 M hydrochloric acid solution and 1.0 M sodium hydroxide solution, respectively. As the seed culture was transferred to the 5.0-L bioreactor, the conversion timer began. 2.15 L of a 100 g / L fructooligosaccharide solution was continuously added at a flow rate of 1.0 g / L / h. During this period, 50 mL, 100 mL, and 200 mL of the enzyme reaction solution were added at 20, 40, and 60 hr, respectively. The transformation culture ended after 70 hr. The pullulan yield was 60.2 g / L, and the substrate conversion rate was as high as 97.98% (Table 1).

[0055] Table 1 Statistics of conversion rates of fructooligosaccharides after 70 hours of conversion in different examples

[0056] Conversion cycle 70hr Example 1 Example 2 Example 3 Liquid seed volume (L) 1.0L 1.0L 1.0L Fructo-oligosaccharide solution volume (L) 2.5L 2.5L 2.15L (enzyme reaction solution volume 0.35L) Final conversion volume (L) 3.5L 3.5L 3.5L Total fructooligosaccharide consumption at 70 hours (g) 250 250 215 Concentration of consumed fructooligosaccharides in the conversion volume (3.5 L) (g / L) 71.43 71.43 61.43 Pullulan yield (g / L) in conversion volume (3.5 L) 62.5 64.26 60.2 Conversion efficiency of fructooligosaccharides (%) 87.5 89.96 97.98

[0057] The invention provides a novel method for producing pullulan by using Aureobasidium pullulans. The method adopts a microbial conversion method for the first time. The entire pullulan production process does not require fermentation culture using a fermentation medium. Fructooligosaccharide is directly converted into pullulan by catalysis of an enzyme reaction liquid. Aureobasidium pullulans seed culture liquid is used as a carrier, fructooligosaccharide is used as a substrate, and an enzyme reaction liquid is used as a catalyst. In a 5.0L bioreactor, a one-time substrate addition or multiple flow additions of a fructooligosaccharide solution and an enzyme reaction liquid with a volume 2.5 times that of the seed liquid are used. The seed liquid is continuously diluted during the conversion process, thereby reducing the concentrations of the substrate carbon source and the product pullulan in the conversion liquid, effectively eliminating the inhibitory effects of the high-concentration carbon source and the product pullulan in the substrate on the synthesis of pullulan. Simultaneously, the enzyme reaction liquid is introduced during the conversion process, thereby improving the decomposition efficiency of the substrate carbon source and the synthesis efficiency of the pullulan, and greatly improving the utilization efficiency of the substrate carbon source. In particular, after 72 hours of catalytic reaction, a total of 61.43 g / L of fructooligosaccharides was consumed, while the pullulan yield reached a maximum of 60.2 g / L, with a fructooligosaccharide conversion rate of 97.98%. Compared with existing fermentation methods for producing pullulan, the bioconversion method can solve the problem of insufficient enzyme for the catalytic reaction relied upon during pullulan synthesis. It also solves the problem of reverse regulation of pullulan synthesis by substrate and product during pullulan synthesis, and the resulting problems of low substrate carbon source utilization and low substrate conversion rate.

[0058] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in this field without departing from the purpose of the present invention.

Claims

1. A method for producing pullulan by Aureobasidium pullulans, comprising the following steps: S1. Activation culture of Aureobasidium pullulans, characterized in that the EP1001 strain is inoculated into a PDA plate culture medium by streaking with an inoculating needle and cultured in a constant temperature incubator at 30°C in the dark for 72 hours; the EP1001 strain was deposited with the China General Microbiological Culture Collection on April 19, 2022, and is classified as Aureobasidium pullulans with a deposit number of CGMCC No. 40158. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; S2. Liquid seed culture of Aureobasidium pullulans, characterized in that a single colony activated and cultured for 72 hours in step S1 is inoculated into a 500 ml Erlenmeyer flask containing 100 ml of liquid seed culture medium, cultured at 30°C, a shaker speed of 220 rpm, and shaken for 24 hours to obtain a liquid culture of Aureobasidium pullulans; S3, culturing in a conversion medium, transferring the liquid seed culture to a bioreactor containing a fructooligosaccharide solution for pullulan production; characterized in that 100 g / L of the fructooligosaccharide solution is added to the bioreactor all at once or continuously added to the bioreactor as the seed culture is transferred to the bioreactor and the conversion begins; S4. Transformation culture control process: In a 5.0 L bioreactor, control the stirring speed to 200-600 rpm, the ventilation volume to 1.0:1.0-2.0 (V / V), the tank pressure to 0.01-0.02 MPa, the temperature to 25°C-28°C, and the pH to 2.5-3.

5. Add the enzyme reaction solution at 20, 40, and 60 hours of transformation culture, respectively. The enzyme reaction solution is the supernatant obtained by centrifuging the liquid culture of Aureobasidium pullulans obtained in step S2. S5. The obtained transformation culture fluid is centrifuged to separate the supernatant, which is then precipitated with ethanol to obtain pullulan.

2. The method for producing pullulan by Aureobasidium pullulans according to claim 1, wherein: The liquid seed culture medium comprises the following components: yeast extract 4.0 g / L, glucose 20.0 g / L, sucrose 10.0 g / L, molasses 10.0 g / L, (NH4)2SO4 1.32 g / L, NaNO3 1.68 g / L, K2HPO4 10.0 g / L, NaCl 2.0 g / L, MgSO4·7H2O 0.4 g / L, pH 7.

0.

3. The method for producing pullulan by Aureobasidium pullulans according to claim 1, wherein: The 100 g / L fructooligosaccharide solution in the transformation medium is continuously fed as the seed culture solution is transferred to the bioreactor vessel and the transformation time is started, and the flow rate is 1.0 g / L·h-3.0 g / L·h.

4. The method for producing pullulan by Aureobasidium pullulans according to claim 1, wherein: The amount of the added enzyme reaction solution is 50ml-500ml.

5. The method for producing pullulan by Aureobasidium pullulans according to claim 1, wherein: The final transformation medium volume was 3.5 L, and the final transformation cycle was 60 hr-72 hr.

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