Aureobasidium pullulans NCPS-D9187 and application of aureobasidium pullulans NCPS-D9187 in fermentation preparation of high-molecular-weight pullulan

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to aureobasidium pullulans NCPS-D9187 and application of the aureobasidium pullulans NCPS-D9187 in preparation of high-molecular-weight pullulan through fermentation. The aureobasidium pullulans NCPS-D9187 provided by the invention is preserved in the China Center for Type Culture Collection on November 17, 2025, and the preservation number is CCTCC NO: M 20252577. After a carbon source of a fermentation culture medium of the aureobasidium pullulans NCPS-D9187 is optimized, when the concentration of the carbon source is 250g / L, the molecular weight of pullulan produced by fermentation of the strain is up to 2.49 MDa, and the pullulan can be used for producing pullulan. Meanwhile, the yield of the pullulan is up to 189.67 g / L, and the molecular weight and the yield of the pullulan are synchronously increased.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial fermentation, and particularly relates to Aureobasidium pullulans NCPS-D9187 and application thereof in fermentative preparation of high molecular weight pullulan. BACKGROUND

[0002] Pullulan is a linear polysaccharide synthesized by Aureobasidium pullulans, and the molecule is composed of maltotriose units connected by α-1, 6 glycosidic bonds. Due to its good film-forming property, excellent biocompatibility and degradability, pullulan is widely used in food packaging, medical sustained-release, biological materials and other fields. Among them, high molecular weight pullulan has higher mechanical strength and better film toughness, and is the core raw material for high-end application scenarios such as medical degradable film and high-barrier food packaging.

[0003] Aureobasidium pullulans is the main microbial strain for preparing pullulan by biological fermentation. Due to the differences in strains, culture medium composition and fermentation conditions, the molecular weight and yield of pullulan show significant differences. For example, patent CN105420127A uses a gas lifting type fermentation tank to produce high molecular weight pullulan, and obtains pullulan with a molecular weight of up to 3.33 million Da, but the yield is only 35 g / L. In addition, patent CN112094752A improves the yield of pullulan to 100-120 g / L through batch fermentation process, but the molecular weight of pullulan is only 2.5 x 10 3 ~9.0 x 10 3 Da; another article (International Journal of Biological Macromolecules 117 (2018) 727-734) reports that a strain with gene knockout of pullulan degradation related enzymes can obtain pullulan with a molecular weight of 3.02 MDa after 96 h of fermentation in a 10 L fermenter, but the yield is only 58.1 g / L, which is significantly lower than the level of high yield process.

[0004] At present, related researches focus more on the production of medium and low molecular weight pullulan, and a few studies on high molecular weight products such as mutagenesis breeding and genetically modified strains generally have the problem of low yield and substrate conversion rate, which directly leads to the increase of production cost of enterprises.

[0005] Therefore, the development of strains and corresponding production processes that can synergistically improve the yield and molecular weight of pullulan has important practical significance for improving product yield, reducing production cost, broadening the application scenarios of pullulan in high-end fields, and improving product added value. SUMMARY

[0006] The application provides an Aureobasidium pullulans dominant strain Aureobasidium pullulans NCPS-D9187 capable of high-yield high-molecular-weight pullulan, and application of the strain in fermentation production of high-molecular-weight pullulan.

[0007] The technical scheme provided by the application is as follows: The first aspect of the application is to provide an Aureobasidium pullulans NCPS-D9187 capable of high-yield high-molecular-weight pullulan, wherein the Aureobasidium pullulans NCPS-D9187 is preserved in the China Center for Type Culture Collection on November 17, 2025, the address is Wuhan University, Wuhan, China, the classification name is Aureobasidium pullulans (Aureobasidium pullulans) NCPS-D9187, and the preservation number is CCTCC NO: M 20252577. Aureobasidiu pullulans )NCPS-D9187, and the preservation number is CCTCC NO: M 20252577.

[0008] The second aspect of the application is to provide application of the Aureobasidium pullulans NCPS-D9187 in fermentation preparation of high-molecular-weight pullulan.

[0009] In the application, preferably, the weight average molecular weight of the high-molecular-weight pullulan is 2.4-2.5 MDa.

[0010] Further preferably, the weight average molecular weight of the high-molecular-weight pullulan is 2.49 MDa.

[0011] Preferably, the application specifically refers to obtaining the high-molecular-weight pullulan by activating the Aureobasidium pullulans NCPS-D9187 strain, seed culture and fermentation culture, wherein the fermentation culture medium used in the fermentation culture contains glucose, and the concentration of the glucose is 230-260 g / L.

[0012] Preferably, the concentration of the glucose in the fermentation culture medium used in the fermentation culture is 250 g / L.

[0013] Preferably, the fermentation culture medium comprises 250 g / L of glucose, 8-12 g / L of acid-hydrolyzed soybean meal, 7-8 g / L of potassium chloride, 0.5-1 g / L of ammonium sulfate, 0.5-1.5 g / L of sodium chloride, 0.1-0.3 g / L of magnesium sulfate and 1-3 g / L of light calcium carbonate.

[0014] Further preferably, the fermentation culture medium comprises 250 g / L of glucose, 10 g / L of acid-hydrolyzed soybean meal, 7.5 g / L of potassium chloride, 0.6 g / L of ammonium sulfate, 1 g / L of sodium chloride, 0.2 g / L of magnesium sulfate and 2 g / L of light calcium carbonate.

[0015] As preferred, the fermentation culture conditions are: culture temperature 25~30℃, culture duration 6~8 days.

[0016] As further preferred, the fermentation culture conditions are: culture temperature 28℃, culture duration 7 days.

[0017] The present application has the following advantages and effects relative to the prior art: The present application provides a strain of Aureobasidium pullulans NCPS-D9187 capable of producing high-molecular-weight pullulan, and experimental results show that when the carbon source concentration is 250g / L, the molecular weight of the pullulan produced by the strain through fermentation is as high as 2.49MDa, and the yield of the pullulan is as high as 189.67g / L, realizing the simultaneous improvement of the molecular weight and yield of the pullulan. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The yield, biomass, reducing sugar and pH value of the Aureobasidium pullulans NCPS-D9187 in the fermentation production of pullulan under different carbon source concentrations in Examples 1~7 of the present application are shown in the following table: Figure 2 The gel chromatogram for determining the molecular weight of the pullulan prepared in Example 6 of the present application is shown in the following figure. DETAILED DESCRIPTION

[0019] In order to enable those skilled in the art to better understand the present application, the present application will be further described in conjunction with specific embodiments.

[0020] Examples 1~7 The application of the Aureobasidium pullulans NCPS-D9187 in the fermentation preparation of high-molecular-weight pullulan, the Aureobasidium pullulans NCPS-D9187 was preserved in the China Center for Type Culture Collection on November 17, 2025, the preservation address is: China. Wuhan. Wuhan University, the classification name is: Aureobasidium pullulans (Aureobasidium pullulans) Aureobasidiu pullulans NCPS-D9187, and the preservation number is: CCTCC NO: M 20252577.

[0021] The fermentation medium used in the present example is prepared by the following method: Preparation of acid-hydrolyzed soybean meal (80 mL bottle): Add 6.4 g soybean meal, 1.08 mL concentrated hydrochloric acid, and 48.84 mL water to an Erlenmeyer flask. Sterilize at 121℃ for 30 min. After sterilization, adjust the pH of the soybean meal to 5.0-5.5 with high-concentration sodium hydroxide. Add a rotor to the acid-hydrolyzed soybean meal and stir. Add 6 g potassium chloride, 0.144 g ammonium sulfate, 2.752 g sodium chloride, and 0.16 g magnesium sulfate. Adjust the pH to 7.0, bring the volume to 80 mL, and sterilize at 115℃ for 30 min.

[0022] Prepare the sugar solution (500 mL bottle): Weigh 184~208g of glucose, dissolve in water, and bring the volume to 690 mL. Divide the solution into 10 bottles, each containing 69 mL. Add 1.2 g of light calcium carbonate to each bottle. Sterilize at 115℃ for 30 min, seal with gauze and cover with newspaper.

[0023] The *Brucea buddingis* NCPS-D9187 provided in this invention is based on *Brucea buddingis* NCPS2022-M disclosed in patent CN115637278B, with the isopullanase gene A9187 knocked out in its genome. Further induction and screening using ARTP were then performed. The ARTP induction and screening process was conducted using an ARTP-IIIS instrument. Specifically, the A9187 gene-deleted strain was cultured on a YPD plate at 28°C for 48 hours. Single colonies were then transferred to a 500mL shake flask containing 80mL of screening medium and cultured at 28°C and 220rpm for 4 days. Finally, the yield of pullulan produced by the mutant was tested. The only difference between the screening medium and the fermentation medium used in the screening process is that the glucose concentration in the screening medium is 200 g / L. The application of *Brucea buddingis* NCPS-D9187 in the fermentation preparation of high molecular weight pullulan is as follows: Remove the cryopreservation tubes of the strain, thaw them, streak them onto PDA slant, and incubate at 28°C for 36 hours. Scrape the budding short-stem fungus NCPS-D9187 cells from the slant culture medium into the seed culture medium and incubate at 28°C and 220 rpm for 24 hours. Transfer the seed culture to a 500 mL shake flask containing 80 mL of fermentation medium, with an inoculum size of 4%, and incubate at 28°C and 220 rpm on a rotary shaker for 7 days.

[0024] Pullulan yield determination: After fermentation, 10 mL of fermentation broth was pipetted into a 15 mL centrifuge tube and centrifuged at 8000×g for 10 min. The supernatant was placed in 30 mL of pre-cooled anhydrous ethanol and precipitated in a 4℃ refrigerator for 6 h. After precipitation, the precipitate was centrifuged at 8000×g for 10 min. The obtained pullulan was dried in an 80℃ oven until constant weight, weighed, and the pullulan yield was calculated.

[0025] Cell dry weight determination: The cells (precipitate) obtained after centrifugation of the fermentation broth were also placed in an 80℃ oven to dry to constant weight, weighed, and the cell biomass was calculated.

[0026] pH measurement of fermentation broth: The pH value of the fermentation broth was measured using a pH meter.

[0027] Reducing sugar determination: Take 2 mL of sample (which may be diluted appropriately), add 1.5 mL of 3,5-dinitrosalicylic acid (DNS), heat in a boiling water bath for 5 min, remove and cool to room temperature, and dilute to 25 mL with distilled water. Measure the absorbance at 540 nm wavelength.

[0028] Table 1. Yield of pullulan under different carbon source concentrations in Examples 1-7 Carbon source concentration (g / L) Pullulan yield (g / L) Biomass (g / L) Reducing sugar (g / L) pH value Example 1 130 112.00 24.00 0.14 5.48 Example 2 150 125.67 27.33 0.19 5.40 Example 3 180 151.33 32.67 0.17 5.25 Example 4 200 162.00 34.00 0.17 3.80 Example 5 230 171.67 31.00 5.97 3.59 Example 6 250 189.67 33.33 7.84 3.58 Example 7 270 167.33 29.67 11.26 3.55

[0029] The data in Table 1 are combined Figure 1 It is evident that increasing the concentration of glucose, the carbon source in the fermentation medium, from 130 g / L to 270 g / L resulted in a gradual decrease in pullulan yield after 7 days of fermentation. The highest pullulan yield (189.67 g / L) was achieved at a glucose concentration of 250 g / L. When the carbon source concentration was further increased to 270 g / L, the pullulan yield decreased to only 167.33 g / L. Furthermore, the pH of the fermentation broth decreased with increasing carbon source concentration, while the reducing sugar content increased, indicating that the *Brachystomata buddingii* NCPS-D9187 strain could not fully utilize glucose, leading to a decrease in pullulan yield.

[0030] Example 8: Determination of the molecular weight of pullulan synthesized by *Bacillus buddingus* strain NCPS-D9187 The molecular weight of pullulan produced by *Bacillus buddingus* strain NCPS-D9187 under the conditions of Example 6 was determined by gel permeation chromatography (HPGPC-MALLS). The specific procedure is as follows: Pullulan obtained by drying was pulverized in a pulverizer. 10 mg of the sample was weighed and dissolved in 1 mL of ultrapure water, resulting in a sample concentration of 10 mg / mL. Simultaneously, 10 mg of dextran standard (35000~45000 Da) was weighed and dissolved in 1 mL of ultrapure water, resulting in a standard sample concentration of 10 mg / mL. The prepared standard and sample solutions were filtered through a 0.2 μm filter membrane, and the resulting liquid was placed in a liquid chromatography vial for analysis. The molecular weight of the produced pullulan was determined using an Agilent 1260 high-performance liquid chromatography (HPLC) system equipped with a differential detector and a laser detector. Two gel chromatography columns (Shodex OHpak SB-805 HQ and SB-803 HQ) were used, with physiological saline as the mobile phase, a flow rate of 0.5 mL / min, a sample loading volume of 20 μL, a standard detection time of 1 h, and a sample detection time of 2 h. The detection results are shown in Table 2 and below. Figure 2 As shown.

[0031] Table 2 combined with Figure 2 Experimental data showed that the Mw, Mn, and Mp of pullulan produced by fermentation of *Brucea buddingis* NCPS-D9187 were 2.490 × 10⁻⁶. 6 (±2.271%), 9.954×10 5 (±3.113%) and 4.881×10 5 (±3.440%), Mw / Mn ratio is 2.502 (±3.853%).

[0032] Table 2. Determination of pullulan molecular weight by gel permeation chromatography Molecular weight Mw (g / mol) 2.490 x 10 6 (±2.271%)]]> Mn (g / mol) 9.954 x 10 5 (±3.113%)]] Mp (g / mol) 4.881 x 10 5 (±3.440%)]]> Mw / Mn 2.502 (±3.853%)

[0033] In summary, the *Brucea buddingis* NCPS-D9187 strain provided by this invention, with an optimized carbon source culture medium and a carbon source concentration of 250 g / L, yields pullulan with a molecular weight as high as 2.49 MDa and a yield as high as 189.67 g / L. This achieves a simultaneous increase in pullulan yield and molecular weight, providing a superior fermentation candidate strain for the industrial production of high molecular weight pullulan.

[0034] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. Budding short-stalked fungus NCPS-D9187, characterized in that, The aforementioned *Brood-producing Short-stalked Pterygospermum* NCPS-D9187 is preserved at the China Center for Type Culture Collection and classified as: *Brood-producing Short-stalked Pterygospermum* (NCPS-D9187). Aureobasidiu pullulans NCPS-D9187, accession number: CCTCC NO: M 20252577.

2. The application of the budding short-stem mold NCPS-D9187 as described in claim 1 in the fermentation preparation of high molecular weight pullulan polysaccharide.

3. The application as described in claim 2, characterized in that, The high molecular weight pullulan polysaccharide has a weight-average molecular weight of 2.4~2.5 MDa.

4. The application as described in any one of claims 2 to 3, characterized in that, The high molecular weight pullulan was obtained by activating, seed culture, and fermentation culture of the budding short-stalked mold NCPS-D9187 strain. The fermentation medium used in the fermentation culture contained glucose, and the glucose concentration was 230~260 g / L.

5. The application as described in claim 4, characterized in that, The concentration of glucose in the fermentation medium used during fermentation culture was 250 g / L.

6. The application as described in claim 4, characterized in that, The fermentation medium comprises: 250 g / L glucose, 8-12 g / L acid-hydrolyzed soybean meal, 7-8 g / L potassium chloride, 0.5-1 g / L ammonium sulfate, 0.5-1.5 g / L sodium chloride, 0.1-0.3 g / L magnesium sulfate, and 1-3 g / L light calcium carbonate.

7. The application as described in claim 4, characterized in that, The fermentation conditions are: a culture temperature of 25~30℃ and a culture time of 6~8 days.

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