Aureobasidium pullulans with high yield of high molecular weight pullulan and application thereof

Abstract

The application discloses an Aureobasidium pullulans strain with high yield of high-molecular-weight pullulan and application thereof, and belongs to the field of industrial biotechnology. The application provides the Aureobasidium pullulans, the strain number of which is BL80, and the preservation number of which in the China General Microbiological Culture Collection Center is CGMCC No. 23807. High-molecular-weight pullulan is produced by fermentation of the Aureobasidium pullulans BL80, the molecular weight of the high-molecular-weight pullulan is up to 3.3 MDa, the yield is 86 g / L, the fermentation rate is 1.43 g / L / h, and the substrate conversion rate is up to 75%, which are all significantly better than those of existing high-molecular-weight pullulan production strains, and the cost can be reduced. Moreover, the Aureobasidium pullulans strain does not produce melanin during fermentation, which is beneficial to simplifying a later-stage purification process and further reducing production cost.

Description

Technical Field

[0001] This invention belongs to the field of industrial biotechnology, specifically relating to a budding short-stem mold that produces high molecular weight pullulan polysaccharide and its applications. Background Technology

[0002] Pullulan is a polysaccharide produced by *Brachystomata*. (Aureobacidium pullulans) Pullulan is a secreted extracellular water-soluble mucopolysaccharide, a linear polysaccharide composed of repeating maltotriose units linked by α-1,4-glycosidic bonds and linked by α-1,6-glycosidic bonds. Its unique glycosidic bond linkage gives pullulan high elasticity and high water solubility. Pullulan possesses characteristics such as high viscosity, water solubility, safety, water retention, shaping properties, and low digestibility, and can be used as a food quality improver, thickener, and low-calorie food additive. Japan classifies pullulan and starch as additives with no usage restrictions, and my country approved its use as a food additive in 2006. Pullulan possesses plasticity and biodegradability, making it suitable as a non-polluting plastic. It also exhibits film-forming and oxygen-barrier properties, making it an excellent material for drug capsules, superior to commonly used capsule materials (gelatin and hydroxypropyl methylcellulose capsules). Furthermore, it demonstrates good biocompatibility, lack of immunogenicity, non-toxic degradation products, controllable degradation rate, and moldability, making it suitable as a drug carrier, tissue engineering scaffold, and 3D printing bio-ink. Finally, its water solubility, dispersibility, film-forming properties, hygroscopicity, and non-toxicity allow it to be used as a moisturizing and thickening agent in cosmetics, at a price far lower than hyaluronic acid, which has similar effects.

[0003] Pullulan, as a linear polymer, exhibits significant advantages in terms of molecular weight and distribution, which are crucial parameters affecting its properties. High molecular weight pullulan materials possess superior mechanical properties, slower degradation rates, and require less cross-linking agent during preparation. Therefore, high molecular weight pullulan offers substantial advantages in material applications, particularly in the development of high-value-added medical materials. Furthermore, high molecular weight pullulan can significantly increase solution viscosity, allowing for lower addition amounts in food and cosmetic applications. Thus, producing high molecular weight pullulan can improve product quality or reduce usage costs. In addition to its high molecular weight, the high substrate conversion rate, yield, and efficiency during fermentation, along with a simple purification process, greatly reduce the production cost of pullulan.

[0004] Pullulan is mainly obtained through microbial fermentation. The microbial strain itself and the fermentation process are key factors affecting the molecular weight, yield, and substrate conversion rate of pullulan. There are some reports on the fermentation production of pullulan with different molecular weights. However, most reports show that the molecular weight of pullulan produced by microbial fermentation is low, below 600,000 Da, which greatly limits the production and application of pullulan. Currently, a few reports describe the microbial fermentation production of high molecular weight pullulan. Patent 20121055529.9 reports obtaining pullulan with a molecular weight of 500,000-2,000,000 Da by adjusting the phosphate concentration and initial pH in the culture medium, but no yield report has been found. Patent 201710426466.1 reports a strain suitable for producing high molecular weight, pigment-free pullulan under alkaline conditions, which can produce pullulan with a molecular weight of 814,000 Da, higher than the molecular weight of commercial pullulan (average 200,000 Da), but its molecular weight is still relatively low, and the yield is only 40.4 g / L. Patent 201610030180.7 reports a high-yield strain of pullulan using compound mutagenesis, with a molecular weight greater than 2.56 million Da, but the yield is only 35 g / L. An article (International Journal of Biological Macromolecules 117 (2018) 727–734) reports a strain with gene knockout of pullulan degradation-related enzymes, which can obtain pullulan with a molecular weight of 3.02 MDa after fermentation for 96 h in a 10 L fermenter, but its substrate conversion rate is only 48%, the yield is only 58.1 g / L, and the yield is only 0.62 g / L / h.

[0005] As can be seen from the aforementioned publicly available patents and published articles, current research mainly focuses on the production of low-molecular-weight pullulan using microbial fermentation. A few studies have used mutagenesis or genetic modification techniques to obtain strains capable of producing high-molecular-weight pullulan, but the yield, productivity, and substrate conversion rate are generally low, increasing production costs. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to prepare high molecular weight pullulan.

[0007] To solve the above-mentioned technical problems, in a first aspect, the present invention provides a budding short-stemmed mold, wherein the budding short-stemmed mold is a budding short-stemmed mold ( Aureobasidium pullulans The strain number is BL80, and its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 23807.

[0008] To address the aforementioned technical problems, in a second aspect, the present invention provides the application of the aforementioned budding short-stem mold in the preparation of high molecular weight pullulan polysaccharide.

[0009] Furthermore, in the above applications, the molecular weight of the high molecular weight pullulan polysaccharide can be 3.3 M Da.

[0010] To address the aforementioned technical problems, in a third aspect, the present invention provides a culture containing the aforementioned budding short-stem mold.

[0011] To address the aforementioned technical problems, in a fourth aspect, the present invention provides a method for preparing high molecular weight pralam polysaccharide, the method comprising culturing the aforementioned budding short-stem mold in a culture medium, collecting the culture, and obtaining high molecular weight pralam polysaccharide.

[0012] Furthermore, in the above method, the culture medium may be: 11.6% sucrose, 0.28% yeast extract, 0.07% ammonium sulfate, 0.45% dipotassium hydrogen phosphate, 0.02% magnesium sulfate heptahydrate, 0.09% sodium chloride, and the remainder being water, where % refers to the mass percentage.

[0013] Furthermore, the above method also includes a purification step: heating the collected culture at 100°C to boiling for 10 minutes, centrifuging (11000 rpm) for 30 minutes, and collecting the supernatant.

[0014] Furthermore, the above method further includes the step of precipitating the collected supernatant with ethanol and collecting the precipitate.

[0015] Furthermore, in the above method, the molecular weight of the high molecular weight pullulan polysaccharide can be 3.3 M Da.

[0016] To solve the above-mentioned technical problems, in a fifth aspect, the present invention provides a microbial agent containing the above-mentioned budding short-stem mold or the above-mentioned culture.

[0017] To address the aforementioned technical problems, in a sixth aspect, the present invention provides the application of the above-mentioned microbial agent in the preparation of high molecular weight pullulan polysaccharide.

[0018] Furthermore, in the above applications, the molecular weight of the high molecular weight pullulan polysaccharide can be 3.3 M Da.

[0019] In this invention, the molecular weight is the weight-average molecular weight, which can be determined using the high-performance gel filtration chromatography-multi-angle laser scattering (HPGFC-MALLS) method.

[0020] The term "culture" refers to a liquid or solid product (i.e., fermentation product) that has grown a microbial community after artificial inoculation and cultivation. It is a product obtained by growing and / or amplifying microorganisms; it can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites, or other components produced during the cultivation process.

[0021] In this invention, the culture is the culture of the budding short-stemmed mold ( Aureobasidium pullulans The substance obtained by culturing BL80 in a microbial culture medium (i.e., fermentation product).

[0022] In this invention, Pullulan polysaccharide with a molecular weight of 3.3 MDa is produced by fermentation of *Brachystomata buddingii* BL80, with a yield of 86 g / L, a fermentation rate of 1.43 g / L / h, and a substrate conversion rate as high as 75%.

[0023] In this invention, the "M" in the molecular weight 3.3M Da is a counting unit, referring to millions. That is, the weight-average molecular weight of the high molecular weight pullulan polysaccharide described in this invention is 3.3 million Da.

[0024] The beneficial technical effects achieved by this invention are as follows: (1) The *Brucea buddingis* strain BL80 provided by this invention can produce pullulan with a molecular weight as high as 3.3 M Da, which is much higher than that of existing commercial pullulan. High molecular weight pullulan has excellent mechanical properties and a low degradation rate, allowing for the use of no or reduced cross-linking agents. It can be used to prepare high-value-added materials such as fibers, hydrogels, and 3D printing inks, or in the field of tissue engineering, greatly expanding the application areas of pullulan. High molecular weight pullulan can significantly increase solution viscosity, and in food and cosmetic applications, it can significantly reduce the amount of pullulan added, thus reducing usage costs.

[0025] (2) The BL80 strain of *Brucea buddingis* provided by this invention produces pullulan polysaccharide at a yield of up to 86 g / L, a fermentation rate of 1.43 g / L / h, and a substrate conversion rate of up to 75%. The high yield, high production rate, and high substrate conversion rate can greatly reduce the cost of fermentation production.

[0026] (3) The BL80 strain of budding short-stem fungus provided by the present invention does not produce melanin. White polysaccharide can be obtained by ethanol precipitation, which simplifies the purification process, improves the purification yield, and reduces the purification cost.

[0027] Preservation Instructions Strain name: Budding short-stemmed mold; Latin name: Aureobasidium pullulans ; Strain number: BL80; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures; The abbreviation for the depository institution is CGMCC. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Date of deposit: November 11, 2021; Registered with the China National Collection Center (CGMCC) No. 23807. Attached Figure Description

[0028] Figure 1 This is a viscosity graph of the fermentation broth for the initial screening strains.

[0029] Figure 2 The colony morphology of *Brachystomata BL80*.

[0030] Figure 3 Phylogenetic analysis diagram of Brugia BL80.

[0031] Figure 4 The curve of high molecular weight polysaccharide production by fed-batch fermentation of *Brachystomata BL80* in a 5L fermenter.

[0032] Figure 5 Infrared spectra of polysaccharide products and pullulan polysaccharide standards fermented by *Brachystomata BL80*.

[0033] Figure 6 High-performance gel filtration chromatography-multi-angle laser scattering (HPGFC-MALLS) chromatogram of pullulan produced by fermentation of *Brachystomata BL80*. Detailed Implementation

[0034] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0036] Example 1: Isolation, screening and identification of *Brugia buddingis* BL80, which produces high molecular weight plurosaccharides 1.1 Isolation and screening of *Brucea buddingis* BL80 1.1.1 Sample Collection and Processing On September 12, 2020, the experimenter collected plant leaves from the Forest Park in Tanggu District, Tianjin. The leaves were classified as new leaves and old leaves. Every 20 old leaves were rinsed with 200 mL of 0.9% physiological saline, and every 100 new leaves were rinsed with 200 mL of 0.9% physiological saline.

[0037] 1.1.2 Gradual dilution of bacterial culture Take 1 mL of the rinsing solution and place it into a test tube containing 9 mL of sterile water; this makes 10 mL of solution. -1 The bacterial solution was diluted again from 10... -1 Take 1 mL of the diluted bacterial solution and place it into a test tube containing 9 mL of sterile water; this yields a dilution of 10. -2 Diluted bacterial solution.

[0038] 1.1.3. Culture medium preparation Prepare PDA solid culture medium with the following formula: 200 g / L potato, 20 g / L glucose, and 20 g / L agar. Peel and boil the potatoes until soft, filter through four layers of gauze, and dissolve them together with the glucose and agar in distilled water. Sterilize at 115℃ for 20 min. The concentrations of Amp and Cm antibiotics in the culture medium are 140 μg / mL and 200 μg / mL, respectively.

[0039] Prepare YPD liquid culture medium from the following ingredients: 1% yeast extract, 2% peptone, 2% glucose, and the remainder being distilled water; pH 6.5–7.5. Wherein, “%” represents the mass percentage.

[0040] 1.1.4 Streaking culture Spread each grade of dilution onto PDA plates. Incubate at 28°C for approximately 3 days and observe colony growth. Select strains with significant stringing effect based on colony morphology, color, and stringing characteristics. Use a sterile inoculation needle to pick up these strains and streak them onto new PDA solid plates for further cultivation.

[0041] 1.1.5 Seed liquid preservation The purified strain obtained in 1.1.4 was inoculated into a glass test tube containing 5 mL of YPD liquid medium and cultured at 28℃ and 180 rpm for 15 h to obtain YPD seed culture.

[0042] Use glycerol with a final concentration of 15% to preserve the seed for later use.

[0043] 1.1.6. Strain Screening Take 5 mL of the YPD seed culture prepared in 1.1.5 and inoculate it into 50 mL of fermentation medium. Ferment at 28±2℃ and 200 rpm for 5 days and measure the viscosity of the fermentation broth.

[0044] Sixteen polysaccharide-producing strains were screened, and the viscosity of the fermentation broth was measured using a viscometer. After centrifugation to remove the bacterial cells, 30 mL of the fermentation broth supernatant was collected, and the viscosity of the fermentation broth was measured using a viscometer (BROOKFIFLD model DV3T) with a No. 64 rotor at 30 rpm for 5 min.

[0045] The fermentation broth viscosity of strain BL80 was 6455±1248 MPa·s, which was several tens of times higher than that of other strains. See the fermentation broth viscosity data below. Figure 1 Viscosity is positively correlated with molecular weight, indicating that strain BL80 can produce high molecular weight polysaccharides.

[0046] The fermentation medium formula is as follows: sucrose 11.6%, yeast extract 0.28%, ammonium sulfate 0.07%, dipotassium hydrogen phosphate 0.45%, magnesium sulfate heptahydrate 0.02%, sodium chloride 0.09%, dissolved and diluted with distilled water; pH 6.5~7.5.

[0047] 1.2 Identification of *Breastfungiformis* BL80 1.2.1 Morphological characteristics of strain BL80 On YPD medium, BL80 colonies were white, initially round, smooth, homogeneous, and viscous. After 48 hours of incubation, the colony edges showed a mycelial appearance. Microscopic observation of a portion of the cells revealed that BL80 cells had a morphology similar to yeast, exhibiting both yeast-like cells and mycelial cells. The colony morphology of strain BL80 is shown below. Figure 2 As shown.

[0048] 1.2.2 Molecular biological characteristics of strain BL80 Strain BL80 has the ITS sequence shown in SEQ ID No.1, which is 559 bp in size.

[0049] Blast comparisons were performed on NCBI databases, and a phylogenetic tree was constructed using the Neighbor-Joining algorithm with MEGA7 software. BL80 and Aureobasidium pullulans In the same branch (such as) Figure 3 Therefore, strain BL80 was identified as a budding short-stemmed fungus ( ). Aureobasidium pullulans ).

[0050] Strain BL80 has been deposited at the China General Microbiological Culture Collection Center (CGMCC). Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China. Deposit date: November 11, 2021; accession number: CGMCC No. 23807. Strain name: *Bratrichia coli*; Latin name: Aureobasidium pullulans The biological material (strain) of ginseng: BL80. Hereinafter referred to as BL80.

[0051] Example 2: Production of polysaccharides using BL80 The production of polysaccharides using BL80 mainly includes the following steps: 2.1 Activation of microbial strains The BL80 strain preserved in 1.1.5 was streaked on YPD solid medium plates and incubated at 28°C for 2-3 days to obtain the activated strain.

[0052] The YPD solid culture medium is made from the following raw materials: 1% yeast extract, 2% peptone, 2% glucose, 1.5% agar, and the balance being distilled water; pH 6.5~7.5. "%" refers to the mass percentage.

[0053] 2.2 Seed Culture Take the activated bacterial strain obtained in 2.1, pick a single colony and inoculate it into 5 mL of YPD liquid medium, and incubate at 28℃ and 200 rpm for 20 h. Then transfer it to 300 mL of shake flask culture medium (YPD liquid medium) and incubate at 28℃ and 200 rpm for 16 h to obtain the seed culture.

[0054] 2.3 Fermentation Culture The seed culture prepared in 2.2 was inoculated at 10% (V / V) into a 5L fermenter with a liquid volume of 60% for fermentation. The fermentation temperature was 28±2℃, the stirring speed was 500 rpm / min, and then increased to 800 rpm / min when the dissolved oxygen dropped to 20%. The aeration rate was 3 vvm. After fermentation for 36 h, 800 mL of 140 g / L sucrose solution was added to the fermentation broth at a rate of 100 mL / h. After fermentation for 72 h, a fermentation broth containing a high concentration of pullulan was obtained. The fermentation medium consisted of: 11.6% sucrose, 0.28% yeast extract, 0.07% ammonium sulfate, 0.45% dipotassium hydrogen phosphate, 0.02% magnesium sulfate heptahydrate, and 0.09% sodium chloride, dissolved and brought to volume with distilled water; the pH was adjusted to 6.5-7.5, and the medium was sterilized at 121℃ for 15 minutes. The percentages (%) represent the mass percentage.

[0055] Samples were taken at 12, 24, 36, 48, 60, and 72 hours of fermentation to determine the pullulan content, sugar content, and viscosity of the fermentation broth. The determination methods are described below: The pullulan content in the fermentation broth was determined by the drying and weighing method. 50 mL of fermentation broth sample was centrifuged at high speed (11000 rpm, 10 min) at regular intervals to remove the bacterial cells. 10 mL of the fermentation broth supernatant was taken and precipitated with 20 mL of anhydrous ethanol. The resulting white precipitate was placed in an 80℃ oven for 48 h until dried. The pullulan yield at that time was obtained by weighing and converting the precipitate.

[0056] The calculation formula is shown in equation (I): Pullulan yield = M / 10 × 1000 (I) Where M is the pullulan yield in 10 mL of fermentation broth supernatant, in g; 10 represents the sampling volume of the fermentation broth supernatant, in mL; 1000 is the unit conversion factor, and the unit is mL / L.

[0057] Sugar content in the fermentation broth was measured by high-performance liquid chromatography (HPLC). 1 mL of fermentation broth was collected periodically, centrifuged at high speed (11000 rpm, 10 min) to remove bacterial cells, the supernatant was diluted 100-fold with pure water, and insoluble matter was removed by membrane filtration. The chromatogram was determined using an Agilent 1260 Infinity HPLC system equipped with a quaternary pump, an autosampler, a column oven, and a RID detector. The selected column was a Bio-Rad Aminex HPX-87P column (300 × 78 mm; 9 µm). The mobile phase was chromatographic grade pure water; the column temperature was 65℃. The injection time was 20 min, and the injection volume was 20 µL. Standard curves were constructed using sucrose, fructose, and glucose standards. The content of the three sugars in the fermentation broth was calculated based on the peak area and the standard curves, and the sum of these values ​​yielded the total residual sugar in the fermentation broth.

[0058] The calculation formula is shown in equation (II): Total residual sugar content (g / L) = sucrose concentration + fructose concentration + glucose concentration (II); The monosaccharide concentration was calculated based on the standard curve. sucrose standard curve: y = 330208x, R 2 =1.00; Glucose standard curve: y = 307599.03x, R0 2 =1.00; Fructose standard curve: y = 325155.82x, R 2 =1.00; Where y is the peak area and x is the sugar concentration, with x in g / L; Monosaccharide concentration = x (measured sugar concentration) × 100; Monosaccharides are selected from sucrose, glucose, or fructose; 100 is the dilution factor.

[0059] The viscosity of the fermentation broth was measured using a viscometer. After centrifuging 50 mL of fermentation broth to remove the bacterial cells, 30 mL of the supernatant was taken and the viscosity of the fermentation broth was measured using a viscometer (BROOKFIFLD model DV3T) with a No. 64 rotor at 30 rpm for 5 min.

[0060] The fermentation results of BL80 in a 5 L fermenter are shown in Table 1 and Figure 4 As shown.

[0061] Table 1: Fermentation results of BL80 in a 5 L fermenter

[0062] Example 3: Purification, identification and molecular weight determination of polysaccharides in fermentation broth 3.1 Purification of polysaccharides Fermentation was carried out for 60 hours according to the method described in 2.1-2.3 of Example 2. The resulting fermentation broth containing high concentrations of polysaccharides was then purified, identified, and its molecular weight was determined, including the following steps: Step 1: Heat the fermentation broth to 100℃ and boil for 10 minutes to obtain the boiled fermentation broth; Step 2: Centrifuge the fermentation broth boiled in Step 1 at 11,000 rpm for 30 minutes to remove bacterial cells and denatured proteins, and collect the supernatant. Step 3: Dilute the supernatant obtained in Step 2 with 2.5 times its volume of sterile water to obtain the diluted solution; Step 4: The diluted solution obtained in Step 3 is precipitated with twice the volume of anhydrous ethanol, centrifuged, and the precipitate is collected; Step 5: Dissolve the precipitate obtained in Step 4 in water to obtain a solution; Step 6: Centrifuge the solution obtained in Step 5 at 11000 rpm for 30 minutes and collect the polysaccharide aqueous solution; Repeat steps 4-6 above, and freeze-dry the polysaccharide aqueous solution to obtain a white product.

[0063] 3.2 Identification of white products The freeze-dried product obtained in Example 3.1 was subjected to infrared spectroscopy by the tablet pressing method. 1~2 mg of sample and 200 mg of pure potassium bromide were ground evenly until the particle size was less than 2 µm. The sample was placed in a mold and pressed into a transparent sheet under a pressure of 15 MPa using a hydraulic press. The sheet was then ready for measurement.

[0064] The polysaccharide chromatograms of the pure polysaccharide were determined using an infrared spectroscopy instrument. The results showed that the polysaccharide chromatograms in section 3.1 were completely consistent with those of the pullulan polysaccharide standard. Figure 5 ).

[0065] The pullulan standard product was purchased from FEIYUBIO (item number: FY33196, CAS number: 9057-02-7).

[0066] 3.3 Determination of molecular weight of the product The molecular weight of the white product was determined using high-performance gel filtration chromatography-multi-angle laser scattering (HPGFC-MALLS). The procedure was as follows: 50 mg of the lyophilized product obtained in Example 3.1 was weighed and dissolved in 0.1 N sodium nitrate and diluted to 10 mL. The molecular weight was determined using an Agilent 1260 high-performance liquid chromatograph (equipped with a Wyatt multi-angle light scattering detector, an Empower workstation, and a Shodex OHpak SB-805 HQ column). The mobile phase was 0.1 N sodium nitrate, the flow rate was 0.5 mL / min, and the column temperature was 40 °C.

[0067] HPGFC-MALLS measurement results are as follows: Figure 6 As shown, Figure 6 In the diagram, LS represents the light scattering signal, and DRI represents the differential signal. The results show that the molecular weight (MW) of pullulan polysaccharide samples fermented by BL80 is 3.3 MDa.

[0068] This invention relates to *Broodia buddingis* (… Aureobasidium pullulans BL80 fermentation produces high molecular weight pullulan. Through three fermentation experiments, we found that the weight-average molecular weight of pullulan produced by BL80 fermentation is 3.3 MDa, the yield of pullulan after 60 hours of fermentation is 86 g / L, and the production rate is 1.43 g / L / h. Based on the initial sucrose content of 116 g / L in the fermentation broth and the remaining sugar content of 39 g / L after 60 hours of fermentation, and the addition of 112 g of sucrose during fermentation, we can conclude that the fermentation consumed a total of 343 g of sugar and produced 258 g of pullulan, thus the substrate conversion rate is 75%. The fermentation of high molecular weight pullulan by *Brachystomata buddingii* strain BL80 significantly outperforms existing high molecular weight pullulan producing strains in terms of yield, substrate conversion rate, and fermentation rate, which is beneficial for reducing the production cost of high molecular weight pullulan. Moreover, this *Brachystomata buddingii* strain does not produce melanin during fermentation, which simplifies the subsequent purification process and further reduces production costs. This invention uses microbial strains to achieve low-cost production of high molecular weight pullulan.

[0069] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. sequence list <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> A budding short-stem mold that produces high molecular weight pullulan polysaccharide and its applications <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 559 <212> DNA <213> Aureobasidium pullulans <400> 1 atgtttgcgg gtatgatgct cagcgcccga cctccaccct ttgttgttaa aactaccttg 60 ttgctttggc gggaccgctc ggtctcgagc cgctggggat tcgtcccagg cgagcgcccg 120 ccagagttaa accaaactct tgttatttaa ccggtcgtct gagtaaaatt tttgaataaa 180 tcaaaacttt caacaacgga tctcttggtt ctcgcatcga tgaagaacgc agcgaaatgc 240 gataagtaat gtgaattgca gaattcagtg aatcatcgaa tctttgaacg cacattgcgc 300 cccttggtat tccgaggggc atgcctgttc gagcgtcatt acaccactca agctatgctt 360 ggtattgggc gtcgtcctta gttgggcgcg ccttaaagac ctcggcgagg ccactccggc 420 tttaggcgta gtagaattta ttcgaacgtc tgtcaaagga gaggaactct gccgactgaa 480 acctttattt ttctaggttg acctcggatc aggtagggat acccgctgaa cttaagcata 540 tcaaaagggc ggggaggaa 559

Claims

1. A budding short-stemmed mold, characterized by: The budding short-stalked fungus is budding short-stalked fungus ( Aureobasidium pullulans The strain number is BL80, and its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 23807.

2. The application of the budding short-stem mold according to claim 1 in the preparation of high molecular weight pullulan polysaccharide, wherein the high molecular weight pullulan polysaccharide has a molecular weight of 3.3 M Da.

3. A culture, characterized in that: The culture contains the budding short-stemmed fungus as described in claim 1.

4. A method for preparing high molecular weight pullulan, characterized in that: The method includes the steps of culturing the budding short-stem mold of claim 1 in a culture medium, collecting the culture, and obtaining high molecular weight pullulan polysaccharide, wherein the high molecular weight pullulan polysaccharide has a molecular weight of 3.3 M Da.

5. The method according to claim 4, characterized in that: The culture medium consists of: 11.6% sucrose, 0.28% yeast extract, 0.07% ammonium sulfate, 0.45% dipotassium hydrogen phosphate, 0.02% magnesium sulfate heptahydrate, 0.09% sodium chloride, and the remainder is distilled water. The percentages are by mass.

6. A microbial agent, characterized in that: The inoculum contains the budding short-stem fungus as described in claim 1 or the culture as described in claim 3.

7. The application of the bacterial agent according to claim 6 in the preparation of high molecular weight pullulan, wherein the high molecular weight pullulan has a molecular weight of 3.3 M Da.

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