Aureobasidium pullulans, its applications, and a method for producing medium molecular weight pullulan polysaccharide

Abstract

The present invention discloses Aureobasidium pullulans and its application and a method for producing medium-molecular-weight pullulan polysaccharide, belonging to the technical field of microorganisms. The Aureobasidium pullulans of the present invention is Aureobasidium pullulans ( Aureobasidium pullulans ) SYCY-018, which was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on December 26, 2022, with the deposit number of CGMCC NO. 40467. In the present invention, the Aureobasidium pullulans SYCY-018 is inoculated into a fermentation medium for fermentation to produce pullulan polysaccharide. The yield of medium-molecular-weight pullulan polysaccharide is as high as over 150 g / L, and it also has good genetic stability and high temperature resistance. Even at a high temperature of 40 °C, the yield of pullulan polysaccharide can still be higher than 130 g / L, which is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly relates to Aureobasidium pullulans, its application, and a method for producing medium-molecular-weight pullulan polysaccharide. Background Art

[0002] The information disclosed in the background art of the present invention is only intended to enhance the overall understanding of the present invention, and does not necessarily be regarded as an admission or imply in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Pullulan polysaccharide, also known as pullulan or aureobasidin, is an important metabolite of Aureobasidium pullulans; it is a colorless, odorless, tasteless, non-toxic, edible polymer substance with good safety, heat resistance, solubility, salt and acid-base resistance, adhesiveness, strong plasticity, natural degradability, and good film-forming property. Pullulan polysaccharide is composed of maltotriose as the basic unit and is repeatedly connected by α-(1→6) glycosidic bonds. The unique connection method endows it with unique physical and chemical properties, so it is widely used in various fields of medicine and food.

[0004] The molecular weight of pullulan polysaccharide is not fixed, generally between 5.0×10 4 ~5.0×10 6 Da, and the degree of polymerization is generally in the range of 100 - 5000. The molecular weight and degree of polymerization are closely related to the type of strain and fermentation conditions. Medium-molecular-weight (2.0×10 5 ~1.0×10 6 Da) pullulan polysaccharide has good market prospects in the fields of capsule application and food preservation.

[0005] Currently, the vitality of Aureobasidium pullulans strains used for fermentative production of pullulan polysaccharide is generally low. For example, in patents CN115141757A and CN114381381A, the yield of pullulan polysaccharide is less than 100 g / L. The patent with the authorized publication number CN115197857B discloses an Aureobasidium pullulans that can improve the yield of pullulan polysaccharide and produce medium-molecular-weight pullulan polysaccharide, which is a recombinant bacterium obtained by knocking out or weakening the poly(malic acid) synthase gene in the metabolic pathway of Aureobasidium pullulans with the preservation number CGMCC NO. 23807. After 120 h of fermentation, the yield of pullulan polysaccharide of this strain can reach 129.1 g / L. Although its yield is about 50% higher than that of the original strain, it is still relatively low. In addition, the fermentation temperature of this strain is 28±2°C, which is likely to have a significant impact on the yield of pullulan polysaccharide when the summer temperature is high; moreover, the genetic stability of this recombinant bacterium is not disclosed in this patent.

[0006] Therefore, it is an urgent problem to provide a Aureobasidium pullulans with high production of medium and high molecular weight pullulan polysaccharide, good temperature tolerance and genetic stability. Summary of the Invention

[0007] In view of this, the present invention provides an Aureobasidium pullulans, its application and a method for producing medium molecular weight pullulan polysaccharide. Using the Aureobasidium pullulans of the present invention to produce pullulan polysaccharide, the yield of pullulan polysaccharide in the fermenter can be as high as more than 150 g / L, and it has good temperature tolerance and genetic stability.

[0008] In the first aspect, the present invention provides an Aureobasidium pullulans, which is Aureobasidium pullulans ( Aureobasidium pullulans ) SYCY-018. This strain was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on December 26, 2022, and the deposit number is CGMCC NO. 40467.

[0009] The Aureobasidium pullulans SYCY-018 provided by the present invention is a highly viable strain screened from a corn farmland in Zhanhua, Binzhou, Shandong. It belongs to Aureobasidium pullulans through morphological and molecular biological identification.

[0010] In the second aspect, the present invention provides the application of the above-mentioned Aureobasidium pullulans, and the application is the application in the production of medium molecular weight pullulan polysaccharide.

[0011] In the third aspect, the present invention provides a method for producing medium molecular weight pullulan polysaccharide, and the method includes:

[0012] Inoculate the above-mentioned Aureobasidium pullulans SYCY-018 on YPD solid medium for activation culture, and then inoculate the activated cells into YPD liquid medium for subculture to obtain a seed solution; inoculate the seed solution into the fermentation medium for fermentation culture to obtain a fermentation broth, and obtain medium molecular weight pullulan polysaccharide through purification.

[0013] Preferably, the temperature of the fermentation culture is 28~35 °C, the time of the fermentation culture is 100~120 h; the inoculation amount of inoculating the seed solution into the fermentation medium is 4~10% (v / v).

[0014] Preferably, the rotation speed during the fermentation culture process is 400~600 rpm, the pH is 6.0~8.0, and the dissolved oxygen is controlled at 30~40%.

[0015] Preferably, the composition of the fermentation medium is as follows: sucrose 100 - 130 g / L, yeast extract powder 3 - 6 g / L, ammonium sulfate 0.5 - 2 g / L, dipotassium hydrogen phosphate 3 - 6 g / L, magnesium sulfate heptahydrate 0.3 - 0.6 g / L, sodium chloride 0.5 - 2 g / L, and the solvent is water; during the fermentation process, a sucrose solution is supplemented to make the total sucrose content in the medium 140 - 170 g / L.

[0016] Preferably, the temperature for the activation culture is 28 - 35°C, and the time is 65 - 72 h.

[0017] Preferably, the temperature for the subculture is 28 - 35°C, and the time is 32 - 48 h.

[0018] Further, the process of the subculture is specifically as follows: inoculate the activated bacterial cells into a test tube containing YPD liquid medium, culture at 28 - 35°C and 180 - 250 rpm for 16 - 24 h, then transfer to a flask containing YPD liquid medium, and culture at 28 - 35°C and 180 - 250 rpm for 16 - 24 h to obtain a seed solution.

[0019] Preferably, the steps of purification are as follows: centrifuge the fermentation broth, collect the supernatant, add ethanol and let it stand for precipitation, dry the obtained precipitate after centrifugation, and thus obtain medium - molecular - weight pullulan polysaccharide.

[0020] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0021] (1) The Aureobasidium pullulans strain SYCY - 018 screened in the present invention has high vitality. Using it to produce pullulan polysaccharide, the yield of pullulan polysaccharide can be as high as over 150 g / L, with high conversion rate, good genetic stability, and strong temperature stability. Even at a high temperature of 40°C, the yield of pullulan polysaccharide is still higher than 130 g / L, which is suitable for industrial production.

[0022] (2) The average molecular weight of the pullulan polysaccharide produced by the Aureobasidium pullulans strain screened in the present invention through fermentation is 5×10 5 ~8×10 5 Da, and it has a broad market prospect in the production of medium - molecular - weight pullulan polysaccharide. Detailed Embodiments

[0023] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0024] The technical solution of the present invention will be further described below in conjunction with specific embodiments. Unless otherwise specified, the present invention has no special restrictions on the sources of the reagents used in the following embodiments, and commercially available products well-known to those skilled in the art can be used.

[0025] In the following embodiments, the specific culture media used are as follows:

[0026] (1) The composition of the YPD solid medium is as follows: glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L, agar 20 g / L, the solvent is water, and the pH is 7.0.

[0027] (2) The composition of the YPD liquid medium is as follows: glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L, the solvent is water, and the pH is 7.0.

[0028] (3) The composition of the fermentation medium is as follows: sucrose 120 g / L, yeast extract powder 5 g / L, ammonium sulfate 1 g / L, dipotassium hydrogen phosphate 5 g / L, magnesium sulfate heptahydrate 0.5 g / L, sodium chloride 1 g / L, the solvent is water, and the pH is 7.0.

[0029] Example 1

[0030] This example provides the screening of strains.

[0031] (1) Dilute the samples collected from the natural environment with sterile water, take a part of the diluted solution and spread it on the YPD solid medium, culture at 30 °C for 70 h, observe the growth of colonies, screen out the strains with obvious drawing effect according to the colony morphology, color and colony drawing situation, pick them with a sterile toothpick and streak culture on a new YPD solid medium, culture at 30 °C for 70 h, after the colonies grow, pick them out with a sterile toothpick and inoculate them into a 24-well deep well plate containing the fermentation medium, the liquid loading volume in each well is 2 mL, and culture at 30 °C and a rotation speed of 250 rpm for 110 h to obtain the fermentation broth.

[0032] (2) Centrifuge the fermentation broth in step (1), transfer the supernatant to a new 15 mL centrifuge tube (weigh the centrifuge tube in advance), slowly add 2 times of absolute ethanol, let it stand for 8 h for precipitation, centrifuge again, place the obtained precipitate in the oven and dry it for 24 h, weigh it, and subtract the weight of the empty centrifuge tube to obtain the mass of pullulan polysaccharide, and further obtain the yield of pullulan polysaccharide.

[0033] A total of more than 2,600 strains were screened from more than 120 samples, and finally a high-vitality Aureobasidium pullulans strain was obtained from the corn farmland in Zhanhua, Binzhou, Shandong. This strain was named Aureobasidium pullulans ( Aureobasidium pullulans)SYCY-018 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on December 26, 2022, with the deposit number CGMCC NO. 40467.

[0034] Example 2

[0035] This example provides a method for producing pullulan using Aureobasidium pullulans SYCY-018 screened in Example 1.

[0036] (1)Activation culture: Streak inoculate the Aureobasidium pullulans SYCY-018 strain on YPD solid medium and culture at 30 °C for 72 h to obtain the activated Aureobasidium pullulans strain.

[0037] (2)Subculture: Pick the activated Aureobasidium pullulans in step (1) and inoculate it into a test tube containing YPD liquid medium. Culture at 30 °C and 200 rpm for 20 h, then transfer it to a shake flask containing YPD medium and culture at 30 °C and 200 rpm for 20 h to obtain the seed liquid.

[0038] (3)Fermentation culture: Inoculate the seed liquid obtained in step (2) into the fermentation medium (250 L fermenter) with an inoculation amount of 5% (v / v). Set the fermentation temperature at 30 °C and the rotation speed at 500 rpm. In the first 48 h of fermentation, control the fermentation pH = 7.0, then control the fermentation pH = 6.0. The dissolved oxygen throughout the fermentation is controlled at about 35%. Ferment for 110 h, and add sucrose solution during the fermentation process with a total addition amount of 50 g / L. End the fermentation when there is no sugar residue in the fermentation broth. After detection, the yield of pullulan is 154.5 g / L.

[0039] (4)Extraction of pullulan: Centrifuge the fermentation broth at the end of fermentation in step (3) to remove the cells. Take the supernatant of the fermentation broth and slowly add 2 times the amount of absolute ethanol. Let it stand for 8 h for precipitation, then centrifuge again. Place the obtained white precipitate in an oven and dry it for 36 h to obtain pullulan.

[0040] (5)Determination of the molecular weight of pullulan: It is determined by high performance gel filtration chromatography (HPGFC). Instrument: Waters 2695 high performance liquid chromatograph (equipped with a 2410 refractive index detector and an Empower workstation). The chromatographic column used is: Ultrahydrogel Linear 300mm×7.8mm, the mobile phase is 0.1 M sodium nitrate, the flow rate is 0.5 mL / min, and the column temperature is controlled at 40 °C. Sample treatment: Weigh 20 mg of the dried sample, dissolve it with the mobile phase and make up the volume to 10 mL. The injection volume is 10 μL. The weight average molecular weight (M w) is 7.2×10 5 Da.

[0041] Example 3

[0042] This example provides a genetic stability test for Aureobasidium pullulans SYCY-018 screened in Example 1.

[0043] The Aureobasidium pullulans SYCY-018 strain was streaked on YPD solid medium to obtain the first-generation strain. According to this method, the first-generation strain was continuously streaked to obtain the second-generation strain, and so on to obtain the third, fourth, fifth, sixth, seventh, and eighth-generation strains. Each generation of strains was activated, and the activation process was the same as in Example 2. Then, they were respectively inoculated into the fermentation medium (5 L fermenter), the fermentation temperature was set at 30 °C, the rotation speed was 500 rpm. In the first 48 h of fermentation, the fermentation pH was controlled at 7.0, and after 48 h, the fermentation pH was then controlled at 6.0. The dissolved oxygen was controlled at about 35% during the whole fermentation process. Fermentation was carried out for 110 h, and a sucrose solution was supplemented during the fermentation process, with a total supplementation amount of 50 g / L. Fermentation could be ended when there was no sugar residue in the fermentation broth. After fermentation ended, the content of pullulan polysaccharide was detected. The detection results are shown in Table 1.

[0044] Table 1 Yield and molecular weight of pullulan polysaccharide of strains of different generations

[0045]

[0046] It can be seen from Table 1 that with the increase in the number of passages, the yield of pullulan polysaccharide is relatively stable, indicating that the genetic stability of Aureobasidium pullulans SYCY-018 is good.

[0047] Example 4

[0048] This example provides a verification of the high-temperature tolerance of Aureobasidium pullulans SYCY-018 screened in Example 1.

[0049] In the process of industrial production, it is difficult to accurately control the temperature, and there are also problems such as difficulty in cooling in summer. To further verify the high-temperature tolerance of Aureobasidium pullulans SYCY-018, the fermentation temperature was controlled at 30-40 °C, and the yield of pullulan polysaccharide was measured.

[0050] For the activation and scale-up culture process, refer to Example 2. Inoculate the seed solution into the fermentation medium (5 L fermenter) at an inoculation amount of 5% (v / v). Set the fermentation temperatures at 30 °C / 35 °C / 40 °C, the rotation speed at 500 rpm. In the first 48 h of fermentation, control the fermentation pH at 7.0, and then control the fermentation pH at 6.0. Control the dissolved oxygen at about 35%. Ferment for 110 h, and add sucrose solution during the fermentation process with a total addition amount of 50 g / L. End the fermentation when there is no sugar residue in the fermentation broth. Measure the production of pullulan polysaccharide after the fermentation ends. The test results are shown in Table 2.

[0051] Table 2 Experimental results of the heat resistance of the strain

[0052]

[0053] Judging from the test results, when the fermentation temperature is increased to 40 °C, the production of pullulan polysaccharide can still be maintained at 137.6 g / L, indicating that the Aureobasidium pullulans SYCY-018 strain has good temperature stability.

[0054] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A Aureobasidium pullulans ( Aureobasidium pullulans ), characterized in that The Aureobasidium pullulans is Aureobasidium pullulans SYCY-018, and this strain was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on December 26, 2022, with the deposit number CGMCC NO. 40467.

2. The application of Aureobasidium pullulans according to claim 1, wherein The application is the application in the production of pullulan.

3. A method for producing pullulan, characterized in that, The method includes: Inoculating the Aureobasidium pullulans SYCY-018 described in Claim 1 onto a YPD solid medium for activation culture, and then inoculating the activated cells into a YPD liquid medium for scale-up culture to obtain a seed solution; inoculating the seed solution into a fermentation medium for fermentation culture to obtain a fermentation broth, and purifying it to obtain pullulan.

4. The method according to claim 3, wherein The temperature of the fermentation culture is 28 - 35 °C, and the time of the fermentation culture is 100 - 120 h; the inoculation amount of inoculating the seed solution into the fermentation medium is 4 - 10% (v / v).

5. The method according to claim 3, wherein The rotation speed during the fermentation culture process is 400 - 600 rpm, the pH is 6.0 - 8.0, and the dissolved oxygen is controlled at 30 - 40%.

6. The method according to claim 3, wherein The composition of the fermentation medium is: sucrose 100 - 130 g / L, yeast extract powder 3 - 6 g / L, ammonium sulfate 0.5 - 2 g / L, dipotassium hydrogen phosphate 3 - 6 g / L, magnesium sulfate heptahydrate 0.3 - 0.6 g / L, sodium chloride 0.5 - 2 g / L, and the solvent is water; a sucrose solution is supplemented during the fermentation process so that the total sucrose content in the medium is 140 - 170 g / L.

7. The method according to claim 3, characterized in that, The temperature of the activation culture is 28 - 35 °C, and the time is 65 - 72 h.

8. The method according to claim 3, wherein The temperature of the scale-up culture is 28 - 35 °C, and the time is 32 - 48 h.

9. The method according to claim 8, wherein The specific process of the scale-up culture is: inoculating the activated cells into a test tube containing YPD liquid medium, culturing at 28 - 35 °C and 180 - 250 rpm for 16 - 24 h, and then transferring it to a shake flask containing YPD liquid medium, culturing at 28 - 35 °C and 180 - 250 rpm for 16 - 24 h to obtain a seed solution.

10. The method according to claim 3, wherein The steps of purification are: centrifuging the fermentation broth, collecting the supernatant, adding ethanol and standing for precipitation, and drying the obtained precipitate after centrifugation to obtain pullulan.

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