A method for producing pullulan

Abstract

A new method for producing and separating pullulan is provided, which mainly uses Ausidia terreus EP1001 as the starting strain, and carries out transformation culture on sugar containing molecular markers with glucose as the basic unit. The transformation product is subjected to specific directional separation of pullulan with molecular markers in the transformation liquid under the action of magnetic force. The separated product is subjected to enzymatic hydrolysis to separate the magnetic bead and pullulan complex, and further decompose the molecular markers. After precipitation by an organic solvent, high-purity pullulan is finally obtained. The separation and extraction process does not require steps such as flocculant pretreatment, activated carbon adsorption decolorization, oxidation decolorization, protease decomposition and desalting by ion exchange. The experimental process is simple, the product yield and purity are high, and the product quality is excellent. The yield of the finally obtained pullulan is as high as 94.58 g / L, the purity is 99.80%, and the whiteness value is 99.62.

Description

Technical Field

[0001] This invention belongs to the field of microbial pharmaceuticals, specifically relating to a method for producing pullulan polysaccharide via biotransformation of *Brucea buddingis* and a method for separating and purifying pullulan polysaccharide. Background Technology

[0002] Pullulan is a novel high-molecular-weight biomaterial with excellent film-forming, fiber-forming, gas-barrier, adhesive, easy-processing, and non-toxic properties, and has been widely used in food, medicine, light industry, chemical, and petroleum fields. Existing research shows that pullulan is initially formed by glucose binding to maltotriose via α-1,4 glycosidic bonds, followed by further binding of the maltotriose at both ends via α-1,6 glycosidic bonds, with an α-(1-4) bond to α-(1-6) bond ratio of 2:1. Therefore, carbon source is a significant factor in the fermentation production of pullulan by *Brachystomata*. Available carbon sources for pullulan synthesis include glucose, fructose, sucrose, mannose, galactose, lactose, maltodextrin, and agricultural waste. These can be used as nutrients by cells or participate in the construction of pullulan molecules. Sucrose has been proven to be the most ideal carbon source for pullulan fermentation production.

[0003] Publicly available methods for extracting pullulan include precipitation and direct drying. The most mature precipitation methods are alcohol precipitation and salt precipitation. Commonly used alcohol precipitants include methanol, ethanol, and isopropanol; commonly used salt precipitants include quaternary ammonium salts, calcium chloride, calcium hydroxide, and aluminum chloride. The advantage of salt precipitation is that it uses 2-3 times less alcohol than alcohol precipitation, but the product quality is slightly lower. Direct drying involves evaporating water from the fermentation broth and directly drying it into a solid product. Freeze-drying is the best method to achieve this in the laboratory. Industrially, products obtained by rotary drying, forced-air drying, and spray drying are crude pullulan products, containing a large amount of bacterial cells, inorganic salts, organic residues, and other impurities. These products have poor water solubility, dark color, and poor rheological properties, limiting their application range.

[0004] The extraction steps of pullulan mainly include pretreatment, decolorization, deproteinization, and desalting. Decolorization methods mainly include activated carbon adsorption, ion exchange, oxidative decolorization, and metal complexation. Although activated carbon decolorization is thorough, it results in polysaccharide loss due to adsorption. Ion exchange utilizes weakly basic resins to adsorb pigments; this method is effective for free negatively charged pigments but less effective for pigments bound to sugars. Oxidative decolorization mainly uses oxidants to remove pigments under appropriate conditions. Deproteinization methods mainly include the Sevage method, trichloroacetic acid method, tannic acid method, trifluorotrichloroethane method, and protease method. After decolorization and deproteinization, the polysaccharide solution can be separated using ethanol fractionation precipitation, and membrane technology can be used to remove salts and small molecules.

[0005] Currently, most pullulan production methods still use *Brachystomata buddingus* as the starting strain and organic carbon sources such as glucose, fructose, sucrose, mannose, galactose, lactose, and maltodextrin as the backbone. Pullulan is produced through microbial fermentation. The fermentation broth undergoes a series of steps including flocculant pretreatment, activated carbon adsorption decolorization, oxidative decolorization, protease deproteinization, ion exchange desalting, and acetone or ethanol precipitation. The entire production process is very cumbersome. To improve product purity, the precipitated pullulan often undergoes multiple dissolution and precipitation processes, consuming large amounts of organic solvents, resulting in a long processing cycle and high production costs. Using hot air or drum drying, if temperature control is not properly managed, can easily cause the product to turn yellow, affecting product quality. Summary of the Invention

[0006] To overcome the above problems, one of the objectives of this invention is to provide a method for producing pullulan polysaccharide using *Aureobasidium pullullans* via conversion. This method utilizes molecularly labeled sugars with glucose as the basic unit as the carbon source for conversion. The *Aureobasidium pullullans* strain was deposited on April 19, 2022, at the China General Microbiological Culture Collection Center (CGMCC); accession number CGMCC No. 40158, classified as *Aureobasidium pullullans*, and located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] Another objective of this invention is to provide a composition of the conversion solution for producing pullulan polysaccharide using the *Brachystomum buddingum* method. The conversion solution contains nuclease inhibitors and / or protease inhibitors, primarily to inhibit the activity of ribonucleases and / or deoxyribonucleases and / or proteases produced during the *Brachystomum buddingum* conversion process. This prevents the ribonucleases and / or deoxyribonucleases and / or proteases from degrading the substrate (molecularly labeled sugars with glucose as the basic unit) and / or the molecular markers linked to the product (pullulan polysaccharide), thus affecting the separation and extraction of the pullulan polysaccharide product.

[0008] Another objective of this invention is to provide a method for separating and extracting pullulan. A molecular marker coated on the surface of magnetic beads binds to the molecularly marked pullulan in the product to form a complex. Under the action of an external magnetic force, the pullulan in the conversion solution is specifically and directionally separated. The separated product is then subjected to enzymatic hydrolysis to dissociate the molecular marker, ultimately yielding high-purity pullulan.

[0009] Another objective of this invention is to provide a composition of an enzymatic hydrolysate for decomposing molecular markers. The hydrolysate contains nucleases and / or proteases, primarily for the purpose of dissociating the magnetic beads and pullulan complex and further decomposing the molecular markers linked to the pullulan molecules, thus preparing for the subsequent precipitation of high-quality pullulan using organic solvents.

[0010] The pullulan extraction method provided by this invention utilizes the interaction of nucleic acids and / or proteins and / or antibodies and / or other identifiable markers coated on the surface of magnetic beads with pullulan polysaccharides carrying the same nucleic acids and / or proteins and / or antibodies and / or other identifiable markers in the product to form a complex. Under magnetic force, the molecularly labeled pullulan polysaccharides in the conversion solution are specifically and directionally separated. The separated product is then subjected to enzymatic hydrolysis to dissociate the magnetic beads and pullulan polysaccharide complex, further decompose the molecular markers, and finally precipitate with an organic solvent to obtain high-purity pullulan polysaccharides. The entire separation and extraction process does not require flocculant pretreatment, activated carbon adsorption decolorization, oxidative decolorization, protease deproteinization, ion exchange desalting, etc. The experimental procedure is simple, and the product yield and purity are high, with excellent quality. After adsorption, desorption, enzymatic hydrolysis, and ethanol precipitation, the final pullulan polysaccharide yield is as high as 94.58 g / L, with a purity of 99.80% and a whiteness value of 99.62 (Table 1).

[0011] This invention provides a method for producing pullulan, which mainly includes the following steps.

[0012] Steps for producing pullulan via the budding short-stem mold conversion method.

[0013] Seed plate culture: The EP1001 strain, preserved at the China General Microbiological Culture Collection Center with accession number CGMCCNo.40158, was inoculated into PDA agar plates using an inoculation needle for activation culture. The specific steps were as follows: the budding short-stem fungus strain EP1001 was taken out of the -80°C freezer, allowed to thaw naturally on ice, and then transferred to PDA agar plates using an inoculation needle on a sterile laminar flow hood. The plates were then incubated at 30°C in the dark for 72 hours.

[0014] Seed liquid culture: The activated *Brucea buddingis* strain EP1001 from the above steps was transferred to liquid seed culture medium and cultured for 24 hours. Specifically, a single colony of the culture activated on PDA plates for 72 hours was inoculated into a 500ml Erlenmeyer flask containing 100ml of liquid seed culture medium. The culture was incubated at 30°C with a shaker speed of 220rpm for 24 hours to obtain the liquid culture of *Brucea buddingis* strain.

[0015] Transformation culture: 5.0L bioreactor, liquid volume 0.5L-2.0L, inoculum 5%-25%, stirring speed 200rpm-600rpm, aeration rate 1.0:1.0-2.0 (V / V), tank pressure 0.01Mpa-0.02Mpa, temperature 27℃-28℃, pH 2.8-3.0, fed conversion broth at the start of fermentation at a rate of 1.0g / L / h-3.0g / L / h (based on glucose as the basic unit of sugar), transformation culture 60hr-72hr.

[0016] Furthermore, the conversion solution consists of 0.0 g / L-100 g / L of molecularly labeled sugars based on glucose, 100 U / L-2500 U / L of nuclease inhibitors, and 1 μmol / L-1.0 mmol / L of protease inhibitors.

[0017] Furthermore, the concentration of molecularly labeled sugars with glucose as the basic unit in the conversion solution is 0.0 g / L-100 g / L.

[0018] Furthermore, when a glucose-based sugar containing molecular markers is added to the bioreactor in one go, its concentration in the conversion solution is 100 g / L.

[0019] Furthermore, when molecularly labeled sugars based on glucose are added to the bioreactor via a continuous feed method, their initial concentration in the conversion solution is 0.0 g / L, and their final concentration is 100 g / L, with a feed rate of 1.0 g / L / h to 3.0 g / L / h (based on molecularly labeled glucose).

[0020] Furthermore, when the rate of addition of molecularly labeled sugars with glucose as the basic unit during the conversion process is 1.0 g / L•h-3.0 g / L•h, the final volume of the conversion solution is 2.5 L-3.5 L, and the conversion cycle is 60 hr-72 hr.

[0021] Furthermore, the 100 U / L-2500 U / L nuclease inhibitor and / or 1 μmol / L-1.0 mmol / L protease inhibitor in the conversion solution mainly inhibit the activity of ribonuclease and / or deoxyribonuclease and / or protease produced during the transformation of *Brachystomia buddingensis*, thus preventing the ribonuclease and / or deoxyribonuclease and / or protease from degrading the molecular markers linked to the substrate (molecularly labeled sugars with glucose as the basic unit) and / or product (pullulan), which would affect the final separation and extraction of pullulan.

[0022] Furthermore, molecules used to label carbohydrates include nucleic acids, proteins, antibodies, peptides, etc., which can be labeled individually or in combination with two or more.

[0023] Furthermore, nucleic acids used to label carbohydrates include DNA, RNA, cDNA, RLFP, VNTR, SSR, RAPD, AFLP, oligonucleotides, etc., which can be labeled individually or in combination with two or more.

[0024] Furthermore, proteins used to label carbohydrates include 6xHIS, Flag, GST, Myc, eGFP, eCFP, eYFP, mCherryeGFP, HA, SUMO, vimentin, nestin, PAX6, HES1, HES5, GFAP, GLAST, BLBP, TN-C, N-cadherin, SOX2, etc., which can be labeled individually or in combination with two or more.

[0025] Furthermore, the antibody molecules used to label carbohydrates are prepared from the protein molecules in the above steps as an immunogenic source.

[0026] Furthermore, the aforementioned molecular markers are located at the OH groups of C2 and / or C3 and / or C6 of the basic structural unit of glucose. - and / or H + Location.

[0027] Furthermore, sugars with glucose as the basic unit, including glucose, fructose, sucrose, mannose, galactose, lactose, maltodextrin, honey, and other sugars that can be converted by budding short-stem mold, can be used alone or in combination of two or more.

[0028] Furthermore, the Aureobasidium pullulans strain used for transformation was deposited on April 19, 2022, at the China General Microbiological Culture Collection Center (CGMCC); the accession number is CGMCC No. 40158, the classification name is Aureobasidium pullulans, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0029] Steps for separating and extracting pullulan.

[0030] S1 bacterial culture separation: Take 10 ml of conversion solution, centrifuge at 12000 rpm for 15 minutes, and transfer the supernatant to a new sample tube for later use.

[0031] S2 magnetic bead activation: A solution of magnetic beads coated with nucleic acids, proteins, or antibodies at a concentration of 1.0 mg / mL to 10.0 mg / mL is prepared. The solution is thoroughly mixed to ensure complete and uniform suspension of the beads within the tube. Adsorption occurs when the molecular markers coated on the magnetic beads interact with the molecular markers in the pullulan product to form a complex. This complex is then separated from the conversion solution by the attraction between the magnetic bead and the magnetic bead using a magnetic device. The diameter of the magnetic beads ranges from 50 nm to 1000 nm.

[0032] S3 Preparation of Complex: Add 1.0-5.0 times the volume of the magnetic bead solution to the supernatant of the separated conversion liquid, and agitate or vortex for 5-15 seconds, then allow to stand at room temperature for 5-30 minutes to bind.

[0033] S4 Adsorption Complex: Place the sample tube containing the complex on the magnetic device for about 1 min-10 min or until the magnetic beads are completely adsorbed by the magnetic device. Keep the sample tube stationary on the magnetic frame, use a pipette to aspirate and discard the supernatant, and remove all the liquid in the tube. Avoid touching the magnetic bead cluster during the operation. The central magnetic induction intensity of the magnetic device should be ≥50 mT.

[0034] S5 Separation of the complex: Remove the sample tube from the magnetic device and let it stand at room temperature for 5 min-30 min. Add 100 μL-1000 μL of distilled water to the tube for elution, and gently pipette 5-10 times to resuspend and mix the magnetic beads. Let it stand at room temperature for 5 min-30 min. During the operation, care should be taken to avoid generating air bubbles.

[0035] S6 complex removal of molecular markers: The complex solution collected in the above steps is separated and collected, and the nucleic acids and / or proteins and / or antibodies coated on the surface of the magnetic beads are decomposed with an enzymatic digestion solution. The concentration of nuclease used is 50 U / L-2000 U / L, and the concentration of protease used is 10 μmol / L-2.5 mmol / L. The digestion is carried out at 37℃ for 1.0hr-5.0hr.

[0036] Obtaining S7 pullulan: The delabeled complex solution obtained in the above steps was centrifuged at 12,000 rpm for 15 minutes at 4°C. The precipitate is the dissociated magnetic bead. The supernatant was transferred to a new sample tube, and 1.5-5.0 times the volume of anhydrous ethanol was added. The mixture was inverted 30 times and then centrifuged at 12,000 rpm for 20 minutes in a high-speed centrifuge. The supernatant was discarded, and the precipitate was dried at 120°C for 24 hours and then weighed to obtain crude pullulan P1.

[0037] Determination of pullulan purity (S7): Weigh 50.0 mg of the dried crude pullulan (P1) and prepare a 50.0 mg / L pullulan solution. The total sugar content was determined using the sulfuric acid-anthrone method, as described in the reference (Zhang Shuihua. Food Analysis. Beijing: China Light Industry Press, 2004). The amount of pullulan (P2, based on total sugar) was calculated using the standard curve. Pullulan purity % = P2 / P1 * 100.

[0038] Determination of whiteness of S8 pullulan polysaccharide: The whiteness was measured using a blue light whiteness meter in accordance with GB / T 22427.6-2008.

[0039] In this invention, the term "pullulan" refers to a polysaccharide composed of maltotriose units linked together. Maltotriose is produced by linking glucose units with α-1,4 bonds, and pullulan is produced by linking maltotriose units with α-1,6 bonds. Pullulan is also known as pullulan, short-stem polysaccharide, or styrax polysaccharide.

[0040] In this invention, the term "Aureobasidium pullulans" refers to a yeast-like fungus that is widely distributed in nature.

[0041] In this invention, the term "transformation" refers to a biochemical reaction that utilizes certain enzymes or enzyme systems in the microbial metabolic process to convert a compound into a product containing a specific functional group. Specifically, microbial transformation involves microbial cells modifying the structure of a complex substrate, transforming it into another organic compound with a similar structure.

[0042] In this invention, the term "sugar with glucose as the basic unit" refers to various disaccharides, oligosaccharides, polysaccharides, etc., composed of a single glucose unit. Any sugar containing a single glucose structure is a sugar with glucose as the basic unit.

[0043] In this invention, the term "RFLP" refers to Restriction Fragment Length Polymorphism; the term "VNTR" refers to Variable Number of Tandem Repeats, also known as Minisatellite DNA; the term "RAPD" refers to Random Amplified Polymorphism DNA; and the term "AFLP" refers to Amplified Fragment Length Polymorphism. Detailed Implementation

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0046] Unless otherwise stated, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0047] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0048] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0049] Unless otherwise specified, all raw materials used in this invention are commercially available products. Unless otherwise specified, all methods used in this invention are conventional methods in the field. All substances used in this invention are of conventional usage quality. Unless otherwise specified, all materials were purchased from commercial channels or are publicly available. Example 1

[0050] See section

[0013] for seed plate culture and section

[0014] for seed liquid culture.

[0051] 500 ml of the activated liquid seed culture was inoculated into a 5.0 L bioreactor containing 2.0 L of conversion solution for pullulan production. The glucose concentration in the conversion solution was 100 g / L, and the total reaction volume was 2.5 L.

[0052] Transformation culture control process: In a 5.0L bioreactor, the stirring speed was controlled at 600rpm, the aeration rate 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 using 1.0M hydrochloric acid solution and 1.0M sodium hydroxide solution, respectively. The transformation culture was completed after 72 hours.

[0053] Pullulan was isolated and extracted by centrifuging 10 ml of transformation culture medium at 12,000 rpm for 15 minutes. The supernatant was transferred to a new sample tube, and anhydrous ethanol (3.0 times the volume of the supernatant) was added. The mixture was inverted 30 times and then centrifuged at 12,000 rpm for 20 minutes. The supernatant was discarded, and the precipitate was dried at 120°C for 24 hours and weighed. The yield of pullulan was 83.6 g / L, with a purity of only 85.12% (Table 1).

[0054] The whiteness of pullulan was determined using a blue light whiteness meter according to GB / T 22427.6-2008. The whiteness value of the product was 65.5 (Table 1). Example 2

[0055] See Example 1 for the seed culture procedure.

[0056] 500 ml of activated liquid seed culture was inoculated into a 5.0 L bioreactor containing 2.0 L of conversion medium for pullulan production. The concentration of glucose (C2 hydroxyl group replaced by 9 thymines (5'-TTTTTTTTT-3')) in the conversion medium was 100 g / L, the concentration of nuclease inhibitor was 100 U / L, the concentration of protease inhibitor was 1 μmol / L, and the total reaction volume was 2.5 L.

[0057] Transformation culture control process: Same as in Example 1.

[0058] Pullulan was isolated and extracted using two methods. The control group was extracted using the method described in Example 1, and the yield of pullulan was 85.56 g / L, the purity was 80.29%, and the whiteness value was 61.56 (Table 1). The experimental group was extracted using the following method.

[0059] S1 bacterial culture separation: Take 10 ml of transformation culture medium, centrifuge at 12000 rpm for 15 minutes, and transfer the supernatant to a new sample tube for later use.

[0060] S2 magnetic beads are activated by coating the surface of the magnetic beads with 9 adenine molecules (5'-AAAAAAAAA3') in a solution at a concentration of 5.0 mg / mL. The solution is thoroughly mixed and shaken to ensure that the magnetic beads in the tube are completely and uniformly suspended.

[0061] To prepare the complex in S3, add a magnetic bead solution twice the volume of the supernatant separated in S1 to the supernatant, and agitate or vortex for 15 seconds. Allow it to bind at room temperature for 30 minutes.

[0062] To adsorb the S4 adsorption complex, place the sample tube on the magnetic rack for 10 minutes or until the magnetic beads are completely adsorbed by the magnetic rack device. Keep the sample tube on the magnetic rack and let it stand. Use a pipette to aspirate and discard the supernatant, and remove all the liquid from the tube.

[0063] To separate the S5 complex, remove the sample tube from the magnetic rack and let it stand at room temperature for 30 minutes. Add 1000 μL of distilled water to the tube for elution, and gently pipette 5-10 times to resuspend and mix the magnetic beads. Let it stand at room temperature for 30 minutes. During the operation, care should be taken to avoid generating air bubbles.

[0064] After removing the label from the S6 complex, the complex collected in the above steps was decomposed with 2000 U / L nuclease to break down the adenine (5'-AAAAAAAAA3') coated on the surface of the magnetic beads and the thymine (5'-TTTTTTTTT-3') labeled on the pullulan polysaccharide product. The reaction was carried out at 37°C for 1.0 hr.

[0065] To obtain pullulan S7, the unlabeled complex solution obtained in the above steps was centrifuged at 12,000 rpm for 15 minutes at 4°C. The precipitate was the molecularly unlabeled magnetic beads. The supernatant was transferred to a new centrifuge tube, and 5.0 times the volume of anhydrous ethanol was added. The mixture was inverted 30 times and then centrifuged at 12,000 rpm for 20 minutes in a high-speed centrifuge. The supernatant was discarded, and the precipitate was dried at 120°C for 24 hours and then weighed. The yield of pullulan was 84.93 g / L, with a purity of 97.95% (Table 1).

[0066] The whiteness of S8 pullulan was determined using a blue light whiteness meter according to GB / T 22427.6-2008, and the whiteness value was 99.26 (Table 1). Example 3

[0067] See Example 1 for the seed culture procedure.

[0068] 500 ml of activated liquid seed culture was inoculated into a 5.0 L bioreactor containing 2.0 L of conversion medium for pullulan production. The concentration of glucose (with the C3 hydroxyl group replaced by Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu polypeptide) in the conversion medium was 100 g / L, the concentration of nuclease inhibitor was 2500 U / L, the concentration of protease inhibitor was 1 mmol / L, and the total reaction volume was 2.5 L.

[0069] Transformation culture control process: see Example 1.

[0070] Isolation and extraction of Lulan polysaccharide.

[0071] S1 bacterial culture isolation, see S1 in Example 2.

[0072] S2 magnetic bead activation involves an antibody magnetic bead solution containing Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu polypeptide coated on the surface of the magnetic beads at a concentration of 1.0 mg / mL. The magnetic bead solution is thoroughly mixed and shaken to ensure that the magnetic beads in the tube are completely and uniformly suspended.

[0073] S3 was used to prepare the complex, see Example 2.

[0074] S4 adsorption complex, see Example 2.

[0075] To separate the S5 complex, remove the sample tube from the magnetic rack and let it stand at room temperature for 15 min. Add 1000 μL of PBS buffer (pH 7.0) to the tube for elution, and gently pipette 5-10 times to resuspend and mix the magnetic beads. Let it stand at room temperature for 30 min. During the operation, care should be taken to avoid generating air bubbles.

[0076] The S6 complex was delabeled, and 1000 μL of the complex collected in the above steps was used to break down the Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu polypeptide labeled on the pullulan polysaccharide product with 1.0 mmol / L protease, so as to separate the Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu polypeptide and the Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu antibody. The reaction was carried out at 37°C for 4.0 hours.

[0077] The pullulan S7 was obtained as described in Example 2, with a yield of 87.98 g / L and a purity of 98.89% (Table 1).

[0078] The whiteness of pullulan S8 was determined in Example 2, with a whiteness value of 99.35 (Table 1). Example 4

[0079] See Example 1 for the seed culture procedure.

[0080] 500 ml of activated liquid seed culture was inoculated into a 5.0 L bioreactor containing 2.0 L of conversion medium for pullulan production. The concentration of glucose (C3 hydroxyl group replaced by Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu polypeptide, C6 hydroxyl group replaced by 9 thymines (5'-TTTTTTTTT-3')) in the conversion medium was 100 g / L, the concentration of nuclease inhibitor was 100 U / L, the concentration of protease inhibitor was 1 μmol / L, and the total reaction volume was 2.5 L.

[0081] Transformation culture control process: see Example 2.

[0082] Isolation and extraction of Lulan polysaccharide.

[0083] S1 bacterial culture isolation, see S1 in Example 2.

[0084] S2 magnetic bead activation involves a magnetic bead solution coated with an antibody containing Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu polypeptide and 9 adenine molecules (5'-AAAAAAAAA3') at a concentration of 2.0 mg / mL. The magnetic bead solution is thoroughly mixed and shaken to ensure that the magnetic beads in the tube are completely and uniformly suspended.

[0085] S3 was used to prepare the complex, see Example 2.

[0086] S4 adsorption complex, see Example 2.

[0087] S5 separates the complex; see S5 in Example 3.

[0088] After removing the label from the S6 complex, 1000 μL of the complex collected in the above steps was used to decompose the nine thymine peptides (5'-TTTTTTTT-3' and Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu) labeled on the pullulan polysaccharide product using 50 U / L nuclease and 10 μmol / L protease, respectively, at 37°C for 3.0 hr.

[0089] The S7 pullulan was obtained as described in Example 2, with a yield of 88.36 g / L and a purity of 99.56% (Table 1).

[0090] The whiteness of pullulan S8 was determined in Example 2, and the whiteness value was 99.59 (Table 1). Example 5

[0091] See Example 1 for the seed culture procedure.

[0092] Centrifuge 2000 ml of the activated liquid seed culture at 12000 rpm for 15 minutes to separate the supernatant and bacterial cells. Transfer the bacterial cells to a 5.0 L bioreactor containing 500 ml of transformation medium. The concentration of glucose (C3 hydroxyl replaced by Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu, C6 hydroxyl replaced by 9 thymines (5'-TTTTTTTTT-3')) in the transformation medium is 100 g / L, the concentration of nuclease inhibitor is 500 U / L, the concentration of protease inhibitor is 0.5 mmol / L, the initial reaction volume is 500 ml, and 2.0 L of transformation medium with a concentration of 100 g / L is fed at the beginning of transformation at a flow rate of 2.0 g / L / h. Transformation culture is completed after 72 hours.

[0093] The transformation culture control process is the same as in Example 2.

[0094] The separation, extraction, and whiteness determination of Lulan polysaccharide were the same as in Example 4.

[0095] The final yield of pullulan was 94.58 g / L, with a purity of 99.80% and a whiteness value of 99.62 (Table 1).

[0096] Table 1. Results of pullulan yield, purity, and whiteness in Examples 1-5.

[0097] Example Crude yield of pullulan (g / L) Purity of pullulan (%) Net yield of pullulan (g / L) Pullulose whiteness Carbon source consumed (g / L) Example 1 83.6 85.12 71.16 65.5 100.0 Example 2 Control Group 85.56 80.29 68.69 61.56 100.0 Example 2 Experimental Group 84.93 97.95 83.19 99.26 100.0 Example 3 87.98 98.89 86.99 99.35 100.0 Example 4 88.36 99.56 87.97 99.59 100.0 Example 5 94.58 99.80 94.39 99.62 100.0 .

[0098] The advantages of this invention lie in providing a novel conversion method for producing pullulan and separating pullulan from the conversion solution. The method primarily uses EP1001, a strain deposited at the China General Microbiological Culture Collection Center (CGMCC No. 40158), as the starting strain. It transforms glucose-based sugars containing molecular markers. Under magnetic force, the conversion product specifically and directionally separates the molecularly marked pullulan molecules from the conversion solution. The separated product is then subjected to enzymatic hydrolysis to dissociate the magnetic beads and pullulan complex, further decomposing the molecular markers. After precipitation with an organic solvent, high-purity pullulan is finally obtained. The entire separation and extraction process eliminates the need for flocculant pretreatment, activated carbon adsorption decolorization, oxidative decolorization, protease decomposition, ion exchange desalting, and other steps. The experimental procedure is simple, resulting in high product yield and purity, and excellent quality. After adsorption, desorption, enzymatic hydrolysis, and ethanol precipitation, the final yield of pullulan polysaccharide reached 94.58 g / L, with a purity of 99.80% and a whiteness value of 99.62.

Claims

1. A method for producing pullulan, characterized in that, Includes the following steps: S1, Aureobasidium pullulans strain, was deposited at the China General Microbiological Culture Collection Center on April 19, 2022; the accession number is CGMCC No.40158, the classification name is Aureobasidium pullulans, and the deposit address is No.3, No.1 Courtyard, Beichen West Road, Chaoyang District, Beijing. S2, transformation culture medium, including 100 g / L of glucose with C2-position hydroxyl and / or C3-position hydroxyl and / or C6-position hydroxyl replaced by 9 thymine and / or Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu polypeptide, 100 U / L to 2500 U / L of nuclease inhibitor, and 1 μmol / L to 1.0 mmol / L of protease inhibitor; S3. The transformation culture conditions are as follows: 5.0L bioreactor, liquid volume 0.5L-2.0L, inoculum size 5.0%-25.0%, stirring speed 200rpm-600rpm, aeration rate 1.0:1.0-2.0 (V / V), tank pressure 0.01Mpa-0.02Mpa, temperature 27℃-28℃, pH 2.8-3.0, transformation solution is fed at the beginning of transformation at a flow rate of 2.0g / L / h, where 2.0g is the mass of glucose in the transformation solution, and transformation culture is carried out for 72 hours. S4. The product obtained from the above transformation is subjected to adsorption, desorption, enzymatic hydrolysis, and organic solvent precipitation steps to separate and purify pullulan polysaccharide.

2. The method for producing pullulan polysaccharide according to claim 1, characterized in that, The adsorption is achieved by the interaction of adenine and / or Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu antibodies coated on the surface of magnetic beads with thymine and / or Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu polypeptides carried in pullulan polysaccharide products to form a complex. The complex in the conversion solution is separated by a magnetic device with a diameter of 50nm-1000nm and a central magnetic induction intensity of ≥50 mT.

3. The method for producing pullulan polysaccharide according to claim 1, characterized in that, The desorption is achieved by eliminating the interaction force between the magnetic device and the magnetic beads.

4. The method for producing pullulan polysaccharide according to claim 1, characterized in that, The enzymatic hydrolysate includes 50 U / L-2000 U / L nuclease and 10 μmol / L-1.0 mmol / L protease, and is hydrolyzed at 37°C for 1.0 hr-5.0 hr.