Method for producing pullulan by aureobasidium pullulans

Abstract

The invention provides a method for producing pullulan by aureobasidium pullulans, which is characterized in that a microbial conversion method is adopted for the first time, and pullulan is produced by catalyzing conversion of fructooligosaccharide through an enzyme reaction liquid, and the pullulan is produced by taking an aureobasidium pullulans seed culture solution as a carrier, fructooligosaccharide as a substrate and the enzyme reaction liquid as a catalyst. In a 5.0 L biological reaction container, a mode of one-time substrate addition or multiple feeding of fructooligosaccharide and enzyme reaction liquid is adopted, catalytic reaction is performed for 60-72 hr, 61.43 g / L-71.43 g / L of fructooligosaccharide is consumed together, the highest yield of pullulan is 60.2 g / L-64.26 g / L, and the conversion rate of fructooligosaccharide is up to 87.5%-97.98%. Compared with the existing method for producing the pullulan by a fermentation method, the biotransformation method can solve the problem of insufficient enzyme amount of catalytic reaction depended in the pullulan synthesis process on one hand, and also can solve the problem of reverse regulation of the substrate and the product on pullulan synthesis in the pullulan synthesis process on the other hand; and the problems of low substrate carbon source utilization rate, low substrate conversion rate and the like caused thereby are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial pharmaceutical manufacturing, and in particular relates to a production process for producing pullulan 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, tasteless, heat-resistant, salt-resistant, acid-resistant, low viscosity, strong plasticity, strong film-forming ability, good film-forming ability and very good thickening effect. It has been widely used in the fields of medicine, food, light industry, chemical industry and petroleum.

[0003] On May 19, 2006, the Ministry of Health issued Announcement No. 8, and pullulan was one of the four new food additives. The Biochemical Research Institute of Hayashibara Company in Japan cooperated with Pfizer Company in the United States to successfully develop hard capsules made of pullulan as raw materials. For the first time, pullulan was applied to the medical field. This new hard capsule product has good disintegration, oxygen impermeability and stability. In 2020, my country also included pullulan hollow capsules in the pharmaceutical excipients section of Part IV of the 2020 Chinese Pharmacopoeia. Domestic pullulan has been used in candies, chocolate coatings, diaphragms, fruit and vegetable juice drinks, etc. Therefore, pullulan is a new multifunctional biomaterial with great economic value and development potential, and has broad market prospects.

[0004] Fructooligosaccharide (FOS), also known as oligofructose, is a type of small molecular weight polysaccharide formed by fructose residues and D-fructose molecules connected by β-1,2 glycosidic bonds (Sheng Dongfeng, 2005). Fructooligosaccharide is a colorless powdery solid, the solution is colorless and transparent, with high solubility, and thermal stability is greatly affected by acid and alkali. When the temperature is between 0 and 70 °C, the viscosity of fructooligosaccharide decreases with the increase of temperature. The fructose formed by the connection of sucrose molecules with 1 molecule of D-fructose, 2 molecules of D-fructose, and 3 molecules of D-fructose is called fructooligotrisaccharide (GF2), fructooligotetrasaccharide (GF3), and fructooligopentasaccharide (GF4) respectively (Wang Bo, 2015). FOS exists naturally in about 30,000 species of plants, such as Campanulaceae, Asteraceae, Gentianaceae, Lobelia, Malvaceae, Primulaceae, Boraginaceae, etc. (Wu Dongru, 1987).

[0005] At present, the increase in pullulan production is mainly through finding high-yield strains, constructing genetically engineered bacteria, mutagenesis screening, and optimizing fermentation medium components and fermentation control processes, etc., and the effect is often not obvious. Kim JH et al. believe that excessive carbon sources are believed to reduce the yield of pullulan. Zheng Aiquan ("Introduction to Modern Biotechnology", page 88) believes that fresh feed liquid containing one or more nutrients is added once or multiple times during the fermentation process to achieve the purpose of extending the product synthesis cycle and increasing the yield; compared with traditional batch fermentation, it can relieve the inhibition of nutrient matrix; at the same time, it is also mentioned that the feed amount or feed rate during the fermentation process is still determined by experience in production, with a certain degree of blindness, and it is difficult to meet the needs of microbial growth and product synthesis simultaneously, and it is impossible to completely avoid the regulation reaction of the matrix. Therefore, simply adding fresh feed liquid with one or more nutrients during the fermentation process cannot solve the regulation reaction of the matrix during the synthesis of pullulan, especially the inhibition of the substrate caused by insufficient enzyme production of the catalytic reaction on which the substrate carbon source metabolism depends, and the resulting low utilization rate of the substrate carbon source and low substrate conversion rate. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for producing pullulan by using Aureobasidium pullulans. The method adopts a microbial transformation method for the first time. The whole process of producing pullulan does not need to use a fermentation medium for fermentation culture. The method directly uses an enzyme reaction liquid to catalyze the conversion of fructooligosaccharides to produce pullulan. The method uses a budding Aureobasidium pullulans seed culture liquid as a carrier, fructooligosaccharides as a substrate, and an enzyme reaction liquid as a catalyst. In a 5.0L bioreactor, a one-time substrate addition or multiple flow additions of fructooligosaccharides and enzyme reaction liquid are used. The catalytic reaction is carried out for 60hr-72hr, and a total of fructooligosaccharides are consumed. 61.43g / L-71.43g / L, while 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 the existing fermentation method for producing pullulan, the bioconversion method can solve the problem of insufficient enzymes in the catalytic reaction relied on in the pullulan synthesis process, and can also solve the reverse regulatory reaction of the substrate and product on the pullulan synthesis in the pullulan synthesis process, as well as the resulting low substrate carbon source utilization rate and low substrate conversion rate. The Aureobasidium pullulans strain used was deposited in the China General Microbiological Culture Collection Center (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 No. 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, is inoculated into the PDA plate culture medium by streaking with an inoculation needle for activation culture; the specific steps are: the budding short bacillus strain EP1001 is taken out of the minus 80 refrigerator, naturally thawed on ice, transferred to the PDA plate culture medium on a sterile clean bench with an inoculation needle, and cultured in a constant temperature incubator at 30°C in the dark for 72 hours ( Figure 1 , Figure 2 ).

[0009] Seed liquid culture: The activated budding Aureobasidium 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 and cultured on the PDA plate medium for 72 hours is inoculated into a 500 ml Erlenmeyer flask containing 100 ml of liquid seed medium, the culture temperature is 30°C, the shaker speed is 220 rpm, and the shaking culture is carried out for 24 hours to obtain a liquid culture of the budding Aureobasidium strain.

[0010] Obtaining the enzyme reaction solution: Take the budding short-stalked mold strain EP1001 out of the negative 80 refrigerator, dissolve it naturally on ice, transfer it to the PDA flat culture medium on a sterile clean bench with an inoculation needle, and culture it in a constant temperature incubator at 30°C in the dark for 72 hours. Inoculate a single colony of the culture activated and cultured on the PDA flat culture medium for 72 hours in a 500ml triangular flask containing 100ml of liquid seed culture medium, culture at 30°C, shaker speed 220rpm, shake culture for 24 hours, obtain the budding short-stalked mold strain liquid culture, centrifuge it at 12000rpm for 15 minutes, and the obtained supernatant is the enzyme reaction solution.

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

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

[0013] Furthermore, the content of the enzyme reaction solution in the microbial transformation 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 No. 1, Beichen West Road, Chaoyang District, Beijing.

[0017] Transformation culture medium: 1.0L-2.0L of the liquid culture from the seed liquid culture was transferred to a 5.0L bioreactor containing 0.0L-3.5L of fructooligosaccharide solution at a concentration of 100g / L and sterilized at 121℃ ( Figure 5 ), control the stirring speed to 200rpm-600rpm, the ventilation volume to 1.0:1.0-2.0 (V / V), the tank pressure to 0.01Mpa-0.02Mpa, the temperature to 25℃-28℃, and the pH value to 2.5-3.5. During this period, add 0.0g / L-100g / L at one time or add 2.5L of 100g / L fructooligosaccharide solution continuously at a flow rate of 1.0g / L•h-3.0g / L•h. Add 0.0ml-500ml of enzyme reaction solution at 20hr, 40hr, and 60hr of conversion, respectively. The conversion culture ends after 60hr-72hr ( Figure 4 ).

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

[0019] Furthermore, the extract is mainly composed 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 nitrosaccharide, nitrosaccharide tetraose and nitrosaccharide pentasaccharide.

[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 fructooligosaccharides were added by continuous addition during the transformation process, the initial concentration of fructooligosaccharides in the transformation medium of the bioreactor vessel was 0.0 g / L and the final concentration was 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 3.5L.

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

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

[0028] The advantage of the present invention is that the Aureobasidium pullulans strain EP1001 was deposited in the China General Microbiological 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 No. 1, Beichen West Road, Chaoyang District, Beijing. The method adopts a microbial transformation method. The entire pullulan production process does not require the use of a fermentation medium for fermentation culture. The method of directly catalyzing the conversion of fructooligosaccharides to produce pullulan is carried out by an enzyme reaction liquid, using a budding Aureobasidium pullulans seed culture liquid as a carrier, fructooligosaccharides as a substrate, and an enzyme reaction liquid as a catalyst. In a 5.0L bioreactor, a fructooligosaccharide solution 2.5 times the volume of the seed liquid is added once or continuously during the transformation process. The seed liquid is continuously diluted during the transformation process, thereby reducing the concentrations of the substrate carbon source and the product pullulan in the transformation liquid, effectively eliminating the inhibitory effect 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 transformation 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, 61.43 g / L-71.43 g / L of fructooligosaccharides are consumed in total, while the maximum yield of pullulan 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 The EP1001 strain was 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 3 Changes in hyphae morphology of strain EP1001 when cultured in liquid seed medium for 24 hours;

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

[0033] Figure 5 The structure of the 5.0L bioreactor and its liquid content 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: 1) Seed cultivation 1.1) Prepare plate seed culture medium; 1.1.1) Prepare potato dextrose agar medium: weigh 200 g of peeled potatoes, cut into small pieces, add 1000 mL of water and boil for 30 min, filter through six layers of gauze to obtain a clear liquid, add water to the clear liquid to make it 1000 mL, then add 20 g of glucose to completely dissolve, then add 20 g of agar, stir and mix well to obtain potato dextrose agar medium; 1.1.2) Prepare plate seeds. Take out the EP1001 strain, which is deposited in the China General Microbiological Culture Collection Center and has a deposit number of CGMCC No.40158, from the negative 80 refrigerator. After it is naturally thawed on ice, use an inoculation needle to transfer it to the PDA slant medium on a sterile clean bench. Incubate it in a constant temperature incubator at 27°C in the dark for 72 hours ( Figure 1 , Figure 2 ); 1.1.3) Prepare liquid seeds, inoculate a single colony of A. pulvinus on a plate into a liquid seed medium for shake flask activation, specifically: put 100 ml of seed medium into a 500 ml Erlenmeyer flask for high temperature and high pressure sterilization, after cooling, inoculate a single colony of A. pulvinus on a plate into a liquid seed medium for shake flask activation, the culture conditions are: rotation speed 220 rpm, temperature 30 ° C, culture 24 hours ( Figure 3); 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, sterilized at 121°C for 20 min; 2) Transformation culture medium 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; 2.2) Transformation culture control process: in a 5.0L bioreactor ( Figure 5 ), control the stirring speed to 200rpm-600rpm, the ventilation volume to 1.0:1.0-2.0 (V / V), the tank pressure to 0.01Mpa-0.02Mpa, the temperature to 25℃-28℃, and the pH value to 2.5-3.5. During this period, add 0.0g / L-100g / L fructooligosaccharide at one time or add 1.0g / L / h-3.0g / L / h fructooligosaccharide in a continuous flow, add 0.0ml / L-500ml / L of enzyme reaction solution at 20hr, 40hr, and 60hr of conversion, respectively, and culture for 60hr-72hr. The pH value is controlled between 2.5-3.5 with 1.0M hydrochloric acid solution and 1.0M sodium hydroxide solution, respectively, and the conversion culture ends after 60hr-72hr ( Figure 4 ).

[0036] 2.3) Determination of pullulan yield: Take 10 ml of the conversion solution and centrifuge 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 and 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, which is the pullulan yield. Example 1

[0037] See 1) Seed culture steps for seed culture.

[0038] 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 fructo-oligosaccharide solution for pullulan production. The total reaction volume was 3.5 L ( Figure 5 ).

[0039] Transformation culture control process: In a 5.0L bioreactor, the stirring speed was controlled at 600rpm, the ventilation volume was 1.0:2.0 (V / V), the tank pressure was 0.01Mpa, the temperature was 28°C, the pH value was controlled at 2.8 using 1.0M hydrochloric acid solution and 1.0M sodium hydroxide solution respectively, and the transformation culture was completed after 70 hours.

[0040] Determination of pullulan yield: After the conversion, take 10 ml of the conversion solution, centrifuge at 12000 rpm for 15 minutes, transfer the supernatant to a new centrifuge tube, add 1.8 times the volume of the supernatant anhydrous ethanol, mix by inversion 30 times, centrifuge at 12000 rpm in a high-speed centrifuge for 20 minutes, discard the supernatant, dry the precipitate at 120°C for 24 hours and weigh it, that is, the pullulan yield is 62.5 g / L, and the substrate conversion rate is as high as 87.5% (Table 1). Example 2

[0041] See 1) Seed culture steps for seed culture.

[0042] 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, with a total reaction volume of 3.5 L.

[0043] Transformation culture control process: In a 5.0L bioreactor, the stirring speed was controlled at 600rpm, the ventilation volume was 1.0:2.0 (V / V), the tank pressure was 0.01Mpa, the temperature was 28℃, and the pH value was controlled at 2.8 with 1.0M hydrochloric acid solution and 1.0M sodium hydroxide solution, respectively. As the seed culture solution was transferred to the 5.0L bioreactor, the transformation timing began. 2.5L of 100g / L fructooligosaccharide solution was continuously added at a flow rate of 1.0g / L / h. The transformation culture ended after 70hr. The yield of pullulan was 64.26g / L, and the substrate conversion rate was as high as 89.96% (Table 1). Example 3

[0044] See 1) Seed culture steps for seed culture.

[0045] 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, with a total reaction volume of 3.5 L.

[0046] Transformation culture control process: In a 5.0L bioreactor, the stirring speed was controlled at 600rpm, the ventilation volume was 1.0:2.0 (V / V), the tank pressure was 0.01Mpa, the temperature was 28℃, and the pH value was controlled at 2.8 with 1.0M hydrochloric acid solution and 1.0M sodium hydroxide solution, respectively. As the seed culture solution was transferred to the 5.0L bioreactor, the transformation timing began. 2.15L of 100g / L fructooligosaccharide solution was continuously added at a flow rate of 1.0g / L / h. During this period, 50ml, 100ml, and 200ml of enzyme reaction solution were added at 20hr, 40hr, and 60hr, respectively. The transformation culture ended after 70hr. The yield of pullulan was 60.2g / L, and the substrate conversion rate was as high as 97.98% (Table 1).

[0047] Table 1 Statistics of conversion rates of fructooligosaccharides after 70 hours of conversion in different examples 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 FOS in conversion volume (3.5 L) (g / L) 71.43 71.43 61.43 Pullulan yield in conversion volume (3.5 L) (g / L) 62.5 64.26 60.2 Conversion efficiency of fructooligosaccharides (%) 87.5 89.96 97.98

[0048] The invention provides a new method for producing pullulan by using budding Aureobasidium pullulans. The method adopts a microbial transformation method for the first time. The whole process of producing pullulan does not need to adopt a fermentation medium for fermentation culture. The method directly uses an enzyme reaction liquid to catalyze the transformation of fructooligosaccharides to produce pullulan. The method uses a budding Aureobasidium pullulans seed culture liquid as a carrier, fructooligosaccharides as a substrate, and an enzyme reaction liquid as a catalyst. In a 5.0L bioreactor, a one-time substrate addition or multiple flow addition of a fructooligosaccharide solution and an enzyme reaction liquid with a volume of 2.5 times that of the seed liquid are used. The concentrations of the substrate carbon source and the product pullulan in the transformation liquid are reduced by continuously diluting the seed liquid in the transformation process, and the inhibitory effect of the high-concentration carbon source and the product pullulan in the substrate on the synthesis of pullulan is effectively eliminated. At the same time, the enzyme reaction liquid is introduced in the transformation process, so that the decomposition efficiency of the substrate carbon source and the synthesis efficiency of the pullulan are improved, and the utilization efficiency of the substrate carbon source is greatly improved. Especially at 72 hours of catalytic reaction, a total of 61.43 g / L of fructooligosaccharides were consumed, while the maximum pullulan yield was 60.2 g / L, and the conversion rate of fructooligosaccharides was as high as 97.98%. Compared with the existing fermentation method for producing pullulan, the bioconversion method can solve the problem of insufficient enzymes in the catalytic reaction relied on in the pullulan synthesis process, and also solve the reverse regulation of pullulan synthesis by substrate and product during the pullulan synthesis process, as well as the resulting low substrate carbon source utilization rate and low substrate conversion rate.

[0049] 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 scope of ordinary technicians in this field without departing from the purpose of the present invention.

Claims

1. A method for producing pullulan by using Aureobasidium pullulans, comprising Aureobasidium pullulans strain, PDA plate medium, and seed liquid medium, characterized in that: The production method consists of a transformation medium and a control process, wherein the transformation medium consists of two components: fructooligosaccharide and enzyme reaction solution; the control process consists of a 5.0L bioreactor, 0.0L-3.5L fructooligosaccharide solution, 10ml-500ml enzyme reaction solution, 1.0L-2.0L cultured liquid seeds, a controlled stirring speed of 200rpm-600rpm, a ventilation volume of 1.0:1.0-2.0 (V / V), a tank pressure of 0.01Mpa-0.02Mpa, a temperature of 25℃-28℃, and a pH value of 2.5-3.

5.

2. The method for producing pullulan by Aureobasidium pullulans according to claim 1, characterized in that: The fructooligosaccharide in the transformation medium is mainly an extract obtained from plants of Campanulaceae, Asteraceae, Gentianaceae, Lobelia, Malvaceae, Primulaceae and Boraginaceae.

3. The method for producing pullulan by Aureobasidium pullulans according to claim 2, characterized in that: The extract is a small molecular heteropolysaccharide formed by fructose residues and D-fructose molecules connected by β-1,2 glycosidic bonds.

4. The method for producing pullulan by Aureobasidium pullulans according to claim 3, characterized in that: The small molecule heteropolysaccharide is mainly a combination of one or more of kestose triose, kestose tetraose and kestose pentose.

5. The method for producing pullulan by Aureobasidium pullulans according to claim 1, characterized in that: The concentration of fructooligosaccharide in the transformation medium is 0.0g / L-100g / L.

6. The method for producing pullulan by Aureobasidium pullulans according to claim 5, characterized in that: The concentration of fructooligosaccharide in the transformation medium was 100 g / L, which was added to the bioreactor vessel at one time.

7. The method for producing pullulan by Aureobasidium pullulans according to claim 5, characterized in that: The concentration of fructo-oligosaccharide in the transformation medium was 0.0 g / L, and it was added to the bioreactor vessel by continuous flow addition during the transformation process.

8. The method for producing pullulan by Aureobasidium pullulans according to claim 7, characterized in that: The flow rate of fructooligosaccharide in the transformation medium is 1.0g / L•h-3.0g / L•h.

9. The method for producing pullulan by Aureobasidium pullulans according to claim 1, wherein: The enzyme reaction solution is added to the transformation medium in batches during the transformation process.

10. The method for producing pullulan by Aureobasidium pullulans according to claim 9, wherein: The enzyme reaction solution was added all at once at 20 hr, 40 hr, and 60 hr of the conversion.

11. The method for producing pullulan by Aureobasidium pullulans according to claim 10, wherein: The content of the enzyme reaction solution in the transformation medium is 0.0ml-500ml.

12. The method for producing pullulan by Aureobasidium pullulans according to claim 1, wherein: The enzyme reaction liquid comes from the supernatant separated from the fungal fermentation liquid.

13. The method for producing pullulan by Aureobasidium pullulans according to claim 12, wherein: The fungal fermentation liquid includes all fungal microorganisms that can produce the enzyme reaction liquid and are used for the metabolism of Aureobasidium pullulans to synthesize pullulan.

14. The method for producing pullulan by Aureobasidium pullulans according to claim 13, wherein: The strain preservation number of the fungal microorganism is CGMCC No.40158, and the classification name is Aureobasidium pullulans.

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

Images