CN116445564A - Method for producing pullulan with low nitrogen content - Google Patents
Method for producing pullulan with low nitrogen content Download PDFInfo
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- CN116445564A CN116445564A CN202310597708.9A CN202310597708A CN116445564A CN 116445564 A CN116445564 A CN 116445564A CN 202310597708 A CN202310597708 A CN 202310597708A CN 116445564 A CN116445564 A CN 116445564A
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- 229920001218 Pullulan Polymers 0.000 title claims abstract description 79
- 239000004373 Pullulan Substances 0.000 title claims abstract description 79
- 235000019423 pullulan Nutrition 0.000 title claims abstract description 79
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 title claims abstract description 58
- 229910052757 nitrogen Inorganic materials 0.000 title claims abstract description 29
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 26
- 238000000855 fermentation Methods 0.000 claims abstract description 53
- 230000004151 fermentation Effects 0.000 claims abstract description 53
- 241000223678 Aureobasidium pullulans Species 0.000 claims abstract description 23
- 241001052560 Thallis Species 0.000 claims abstract description 22
- 238000006243 chemical reaction Methods 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 10
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 13
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 9
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 9
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 9
- 238000001556 precipitation Methods 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 9
- 230000001954 sterilising effect Effects 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 238000011218 seed culture Methods 0.000 claims description 7
- 238000004659 sterilization and disinfection Methods 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- 229930006000 Sucrose Natural products 0.000 claims description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 239000005720 sucrose Substances 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 4
- 230000003213 activating effect Effects 0.000 claims description 4
- 238000009423 ventilation Methods 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- 244000061456 Solanum tuberosum Species 0.000 claims description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 3
- 238000005273 aeration Methods 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims description 2
- 238000011084 recovery Methods 0.000 claims description 2
- 239000002699 waste material Substances 0.000 abstract description 6
- 239000012620 biological material Substances 0.000 abstract description 2
- 150000004676 glycans Chemical class 0.000 description 7
- 229920001282 polysaccharide Polymers 0.000 description 7
- 239000005017 polysaccharide Substances 0.000 description 7
- 239000004382 Amylase Substances 0.000 description 4
- 108010065511 Amylases Proteins 0.000 description 4
- 102000013142 Amylases Human genes 0.000 description 4
- 108090000637 alpha-Amylases Proteins 0.000 description 4
- 235000019418 amylase Nutrition 0.000 description 4
- 238000002329 infrared spectrum Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 3
- 238000004064 recycling Methods 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000007813 chromatographic assay Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- LUEWUZLMQUOBSB-UHFFFAOYSA-N UNPD55895 Natural products OC1C(O)C(O)C(CO)OC1OC1C(CO)OC(OC2C(OC(OC3C(OC(O)C(O)C3O)CO)C(O)C2O)CO)C(O)C1O LUEWUZLMQUOBSB-UHFFFAOYSA-N 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- UYQJCPNSAVWAFU-UHFFFAOYSA-N malto-tetraose Natural products OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(O)C(CO)O2)O)C(CO)O1 UYQJCPNSAVWAFU-UHFFFAOYSA-N 0.000 description 1
- LUEWUZLMQUOBSB-OUBHKODOSA-N maltotetraose Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@H](CO)O[C@@H](O[C@@H]2[C@@H](O[C@@H](O[C@@H]3[C@@H](O[C@@H](O)[C@H](O)[C@H]3O)CO)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-OUBHKODOSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
- C12P19/10—Pullulan
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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Abstract
The invention discloses a method for producing pullulan with low nitrogen content, and relates to the technical field of biological fermentation. And after the batch fermentation production of the pullulan is finished, the Aureobasidium pullulans fermentation thalli are recovered and used for the conversion production of the pullulan. The method of the invention utilizes the waste thalli generated in the fermentation process to produce pullulan, can improve the utilization rate of thalli and avoid resource waste caused by directly discarding thalli. The pullulan obtained by the invention has high molecular weight and low nitrogen content, and is suitable for development of high-added-value products such as biological materials.
Description
Technical Field
The invention relates to the technical field of biological fermentation, in particular to a method for producing pullulan with low nitrogen content.
Background
Pullulan (Pullulan), also known as aureobasidium pullulans, pullulan, is a class of water-soluble extracellular neutral polysaccharides secreted by aureobasidium pullulans. The polysaccharide is polymaltritose (containing a small amount of maltotetraose) connected by alpha-1, 6-glycosidic bond, generally has no branch structure and is linear polysaccharide. The unique glycosidic bond connection mode of the pullulan enables the pullulan to have high structural flexibility and good water solubility, so that the pullulan has unique and excellent physical properties which are not available for other polysaccharides, including good adhesive property, fiber forming property, film forming property and plasticity, and a film made of the pullulan has excellent oxygen blocking property. In addition, pullulan is susceptible to chemical modification to alter its water solubility or to provide reactive groups. Therefore, the pullulan and the derivatives thereof are widely used in various fields of food, medicine, chemical industry, electronics and the like, and are multifunctional novel biological products with great development value and prospect.
The pullulan production process comprises the following steps: (1) Pullulan is produced by using Aureobasidium pullulans in batch or fed-batch fermentation; (2) removing thalli in the fermentation liquor; (3) extracting pullulan by post-treatment of supernatant; (4) drying, crushing and packaging. However, after the fermentation production of the pullulan is finished, the fermentation thalli still have good pullulan synthesis capability, and the direct removal of the waste causes not only the resource waste, but also the environmental pollution treatment pressure in the production process of the pullulan.
In addition, nitrogen sources must be added to the fermentation substrate during the pullulan production process, otherwise Aureobasidium pullulans cannot grow. In the process of extracting pullulan by alcohol precipitation, a nitrogen source in fermentation liquor is brought into polysaccharide precipitation. And the pullulan is affected by amylase, pullulanase and the like in the fermentation process, and the molecular weight of the pullulan cannot be further increased due to the existence of the amylase, the pullulanase and the like.
Therefore, the method for producing the pullulan with low nitrogen content and high molecular weight by recycling the Aureobasidium pullulans fermentation thalli is a problem which needs to be solved by the technicians in the field.
Disclosure of Invention
In view of this, the present invention provides a process for producing pullulan having a low nitrogen content.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for producing pullulan with low nitrogen content comprises the steps of recycling aureobasidium pullulans fermentation thalli after batch fermentation production of pullulan is finished, and using the aureobasidium pullulans fermentation thalli for conversion production of pullulan.
Further, the method comprises the following steps:
(1) Activating strains: transferring the strain of Aureobasidium pullulans preserved at the inclined plane of 4 ℃ to a PDA culture medium, and culturing for 4-6 days at the temperature of 28+/-2 ℃;
(2) Seed culture: selecting a pure culture flat plate, performing aseptic operation, inoculating the bacterial blocks activated in the step (1) into a seed culture solution, and culturing for 24-48h at 28+/-2 ℃ and the rotating speed of 200+/-30 rpm to obtain the seed solution;
(3) Fermentation production: inoculating the seed liquid in the step (2) into a fermentation culture liquid according to the inoculum size of 1-5% (V/V), fermenting in a fermentation tank at the stirring rotation speed of 300-1000rpm, wherein the fermentation temperature is 28+/-2 ℃, the ventilation rate is 1-1.5vvm, and fermenting for 72-96 hours to obtain a fermentation liquid;
(4) Recovery of Aureobasidium pullulans fermentation thalli: centrifugally separating the fermentation liquor prepared in the step (3), respectively collecting thalli and supernatant, extracting pullulan from the supernatant, and recovering aureobasidium pullulans fermentation thalli for converting and producing pullulan;
(5) Re-suspending the Aureobasidium pullulans fermentation bacteria recovered in the step (4) in pullulan conversion liquid, wherein the temperature is 28 ℃, the ventilation rate is 0.2-1.0 vvm, the stirring speed is 100-500 rpm, the conversion is carried out for 72-144 h, the bacteria are removed by centrifugation, and the supernatant is subjected to alcohol precipitation, drying and crushing to prepare the pullulan with low nitrogen content.
Further, the formula of the PDA culture medium in the step (1) is as follows: 200g/L of potato, 20g/L of glucose and 20g/L of agar;
the initial pH is natural, and the sterilization is carried out for 20min at 121 ℃.
Further, the formula of the seed culture solution in the step (2) is as follows: glucose 50g/L, ammonium sulfate 0.6g/L, yeast powder 1.7g/L, magnesium sulfate 0.2g/L, sodium chloride 1.0g/L and dipotassium hydrogen phosphate 5.0g/L;
the initial pH was 6.5 and sterilized at 121℃for 20min.
Further, the formula of the fermentation culture solution in the step (3) is as follows: 50-100g/L of sucrose, 0.9g/L of ammonium sulfate, 1.7g/L of yeast powder, 0.2g/L of magnesium sulfate, 1.0g/L of sodium chloride and 5.0g/L of dipotassium hydrogen phosphate;
the initial pH is 6.5, and the sterilization is carried out for 20min at 121+/-5 ℃.
Further, the aeration rate in the step (5) is 0.5vvm, and the stirring rotating speed is 200rpm;
the pullulan conversion solution comprises the following components: 50-100g/L of sucrose, 0.2g/L of magnesium sulfate, 1.0g/L of sodium chloride and 5.0g/L of dipotassium hydrogen phosphate;
the initial pH is 6.5, and the sterilization is carried out for 20min at 121+/-5 ℃.
Compared with the prior art, the invention has the beneficial effects that:
the method of the invention utilizes the waste thalli generated in the fermentation process to produce pullulan, can improve the utilization rate of thalli and avoid resource waste caused by directly discarding thalli. Meanwhile, the conversion liquid contains no nitrogen source, and the post-extraction process is simple and suitable for industrial production. In addition, the pullulan obtained by the method has high molecular weight and low nitrogen content, and is suitable for developing high-added-value products such as biological materials.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a molecular weight distribution of a pullulan produced by fermentation in accordance with the present invention;
FIG. 2 is an infrared spectrum of pullulan produced by fermentation in accordance with the present invention;
FIG. 3 shows the molecular weight distribution of pullulan produced by the conversion of the present invention;
FIG. 4 is an infrared spectrum of pullulan produced by the conversion of the present invention;
FIG. 5 is a nitrogen content comparison of pullulan produced by fermentation and conversion in accordance with the present invention;
FIG. 6 is a technical scheme for producing pullulan by recycling Aureobasidium pullulans fermentation thalli.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1:
pullulan fermentation production process
1. Activating Aureobasidium pullulans (Aureobasidium pullulans) CGMCC No.11602 with PDA culture medium (potato 200g/L, glucose 20g/L, agar 20g/L; initial pH is natural, sterilization is performed at 121deg.C for 20 min), and activating temperature is 28deg.C for 4 days;
2. inoculating the pure cultured fungus blocks into seed culture solution (glucose 50g/L, ammonium sulfate 0.6g/L, yeast powder 1.7g/L, magnesium sulfate 0.2g/L, sodium chloride 1.0g/L, dipotassium hydrogen phosphate 5.0g/L; initial pH6.5, sterilizing at 121 ℃ for 20 min), culturing at 28deg.C and rotating speed 200rpm for 48h to obtain seed solution;
3. inoculating the prepared seed solution into a 250mL shaking flask containing 50mL of fermentation culture solution (sucrose 50g/L, ammonium sulfate 0.9g/L, yeast powder 1.7g/L, magnesium sulfate 0.2g/L, sodium chloride 1.0g/L, dipotassium hydrogen phosphate 5.0g/L, initial pH6.5, 121 ℃ for 20 min) according to the amount of 5% (V/V), and fermenting and culturing for 72h at 28 ℃ with the aeration rate of 1vvm and the rotating speed of 300 rpm;
4. after fermentation, centrifugal solid-liquid separation (3000 rpm,15 min) is carried out, supernatant is desalted and decolorized, alcohol precipitation, drying and crushing are carried out to obtain pullulan, and thallus precipitation is used for converting pullulan (fermentation stage).
The pullulan is produced by fermenting Aureobasidium pullulans for 48 hours, the yield is 32.68g/L, the conversion rate is 65.36 percent, and the nitrogen content is 4.7521g/kg. GPC measurement revealed that the weight average molecular weight of the prepared pullulan was 317kDa (FIG. 1); the infrared chromatographic assay shows that the prepared pullulan is consistent with the infrared spectrum of the pullulan of a Sigma standard (figure 2).
Pullulan conversion production process
Collecting Aureobasidium pullulans in a fermentation stage, suspending in 3.5L of pullulan conversion liquid (sucrose 50g/L, magnesium sulfate 0.2g/L, sodium chloride 1.0g/L, dipotassium hydrogen phosphate 5.0g/L, initial pH6.5, sterilizing at 121+/-5 ℃ for 20 min), 28 ℃, ventilating at 0.5vvm, stirring at 200rpm, converting for 96 hours, centrifuging to remove thalli, desalting and decolorizing supernatant, precipitating with alcohol, drying and pulverizing to obtain pullulan.
The pullulan is produced by transformation of Aureobasidium pullulans for 96 hours, the yield is 27.71g/L, the transformation rate is 55.42 percent, and the nitrogen content is 0.5137g/kg. GPC measurement revealed that the obtained pullulan had a weight average molecular weight of 2312kDa (FIG. 3); the infrared chromatographic assay showed that the prepared pullulan was consistent with the infrared spectrum of the Sigma standard pullulan (fig. 4). The nitrogen content of pullulan produced in the comparative fermentation stage is obviously reduced (figure 5), the molecular weight is obviously increased, the yield is not obviously reduced, and the results are shown in table 1.
TABLE 1 pullulan yield and product index
Pullulan is affected by amylase, pullulanase, etc. during fermentation, the presence of which negatively affects molecular weight. The nitrogen source is lacking in the transformation culture medium prepared by the application, so that the synthesis of biological enzymes in thalli is limited to a certain extent, and therefore, the influence of amylase and pullulanase on the molecular weight of pullulan is weakened, and the molecular weight of pullulan is obviously increased. In addition, in the fermentation medium, the nitrogen source in the fermentation liquid can be brought into polysaccharide precipitation in the alcohol precipitation process, and because the nitrogen source is lack in the conversion medium, when the pullulan alcohol is precipitated, the nitrogen source is not contained in the fermentation liquid wrapped in the polysaccharide precipitation process, so that the nitrogen source content of the pullulan obtained by conversion is lower.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (6)
1. A method for producing pullulan with low nitrogen content is characterized in that after the batch fermentation production of pullulan is finished, aureobasidium pullulans fermentation thalli are recovered and are used for the conversion production of pullulan.
2. The method for producing pullulan with low nitrogen content according to claim 1, which comprises the following steps:
(1) Activating strains: transferring the strain of Aureobasidium pullulans preserved at the inclined plane of 4 ℃ to a PDA culture medium, and culturing for 4-6 days at the temperature of 28+/-2 ℃;
(2) Seed culture: selecting a pure culture flat plate, performing aseptic operation, inoculating the bacterial blocks activated in the step (1) into a seed culture solution, and culturing for 24-48h at 28+/-2 ℃ and the rotating speed of 200+/-30 rpm to obtain the seed solution;
(3) Fermentation production: inoculating the seed liquid in the step (2) into a fermentation culture liquid according to the inoculum size of 1-5% (V/V), fermenting in a fermentation tank at the stirring rotation speed of 300-1000rpm, wherein the fermentation temperature is 28+/-2 ℃, the ventilation rate is 1-1.5vvm, and fermenting for 72-96 hours to obtain a fermentation liquid;
(4) Recovery of Aureobasidium pullulans fermentation thalli: centrifugally separating the fermentation liquor prepared in the step (3), respectively collecting thalli and supernatant, extracting pullulan from the supernatant, and recovering aureobasidium pullulans fermentation thalli for converting and producing pullulan;
(5) Re-suspending the Aureobasidium pullulans fermentation bacteria recovered in the step (4) in pullulan conversion liquid, wherein the temperature is 28 ℃, the ventilation rate is 0.2-1.0 vvm, the stirring speed is 100-500 rpm, the conversion is carried out for 72-144 h, the bacteria are removed by centrifugation, and the supernatant is subjected to alcohol precipitation, drying and crushing to prepare the pullulan with low nitrogen content.
3. A process for the production of pullulan having a low nitrogen content according to claim 2, wherein the PDA medium of step (1) is formulated as follows: 200g/L of potato, 20g/L of glucose and 20g/L of agar;
the initial pH is natural, and the sterilization is carried out for 20min at 121 ℃.
4. The method for producing pullulan having a low nitrogen content according to claim 2, wherein the seed culture in step (2) is formulated as follows: glucose 50g/L, ammonium sulfate 0.6g/L, yeast powder 1.7g/L, magnesium sulfate 0.2g/L, sodium chloride 1.0g/L and dipotassium hydrogen phosphate 5.0g/L;
the initial pH was 6.5 and sterilized at 121℃for 20min.
5. The method for producing pullulan having a low nitrogen content according to claim 2, wherein the fermentation broth of step (3) has the formula: 50-100g/L of sucrose, 0.9g/L of ammonium sulfate, 1.7g/L of yeast powder, 0.2g/L of magnesium sulfate, 1.0g/L of sodium chloride and 5.0g/L of dipotassium hydrogen phosphate;
the initial pH is 6.5, and the sterilization is carried out for 20min at 121+/-5 ℃.
6. A process for the production of pullulan having a low nitrogen content according to claim 2, wherein the aeration rate of step (5) is 0.5vvm, and the stirring speed is 200rpm;
the pullulan conversion solution comprises the following components: 50-100g/L of sucrose, 0.2g/L of magnesium sulfate, 1.0g/L of sodium chloride and 5.0g/L of dipotassium hydrogen phosphate;
the initial pH is 6.5, and the sterilization is carried out for 20min at 121+/-5 ℃.
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