CN105420127B - High-yield strain of high-molecular-weight pullulan and method for producing high-molecular-weight pullulan by using high-yield strain - Google Patents
High-yield strain of high-molecular-weight pullulan and method for producing high-molecular-weight pullulan by using high-yield strain Download PDFInfo
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Abstract
The invention relates to a high-yield strain of high-molecular-weight pullulan and a method for producing the high-molecular-weight pullulan by using the same, wherein the high-yield strain is classified and named as Aureobasidium pullulansAureobasidium pullulans) SWP35, which is preserved in China general microbiological culture Collection center (CGMCC) at 11 months and 11 days in 2015 with the preservation number of CGMCC NO. 11602; inoculating the strain on an inclined plane at 4 ℃ to a PDA culture medium for culture, inoculating a strain block to a seed culture solution for culture to prepare a seed solution, inoculating 1-5% (V/V) to a fermentation culture solution, fermenting in an airlift fermentation tank with the liquid loading amount of 40-70%, and performing aftertreatment on the fermentation liquid to obtain a white pullulan product. The culture medium used in the invention has no organic nitrogen source, so that the cost of the culture medium is reduced on one hand, the pigment in the culture medium is reduced on the other hand, the pressure of pullulan alcohol decoloration in fermentation post-treatment is relieved, and the obtained aureobasidium pullulan CGMCC No.11602 can be fermented to produce Mw>2560000, and the yield of the pullulan is more than 35 g/L.
Description
Technical Field
The invention belongs to the technical field of industrial biology, and particularly relates to a high-yield strain of high-molecular-weight pullulan and a method for producing the high-molecular-weight pullulan by using the strain.
Background
Pullulan (also called Pullulan) is prepared from Aureobasidium pullulans (Aureobasidium pullulans)Aureobacidium pullulans) The unique glycosidic linkage mode of pullulan polysaccharide makes it possess high structure flexibility and good water solubility, thus possessing high structure flexibility and good water solubilityUnique, excellent physical properties not found in other polysaccharides, including good cohesive properties, fiberizability, film formation and moldability. Meanwhile, pullulan is easily chemically modified to change its water solubility or provide reactive groups. Therefore, the pullulan and the derivatives thereof are widely applied to a plurality of fields such as food, medicine, chemical industry, electronics and the like, and are a novel multifunctional biological product with great development value and prospect.
At present, the main application of the pullulan is as food additives, capsule materials and cosmetic raw materials. In recent years, because pullulan has the characteristics of good biocompatibility, no toxicity of both a polymer and a degradation product, controllable degradation speed, proper mechanical property, excellent formability and the like, the application research of pullulan in drug embedding carriers, microcapsule preparation materials, tissue engineering applications and medical encapsulation polymer materials is greatly concerned. As a high molecular biological material, the molecular weight and the distribution thereof are important parameters of microbial polysaccharides, and influence the physical and mechanical properties and degradation rate of application products (such as tissue engineering scaffolds and the like) thereof. On the other hand, high molecular weight products can significantly increase solution viscosity. Therefore, the application field of the pullulan polysaccharide can be greatly expanded and the added value of the product can be improved when the high molecular weight pullulan polysaccharide is produced.
Microbial fermentation is the main production method of pullulan, and the molecular weight of pullulan is greatly changed due to the difference of strains, the components of a culture medium and the fermentation conditions. There have been many reports on the fermentation production of pullulan having different molecular weights. Patent 97121608.8 discloses a new method for producing pullulan by fermentation, wherein the average molecular weight of the produced pullulan is 10 ten thousand daltons; patent 200810052070.6 discloses a fermentation process for producing pullulan to obtain pullulan having a molecular weight of 20 to 60 ten thousand daltons; patent 200910148764.4 discloses a method for producing pullulan with different molecular weights, which can produce pullulan with average molecular weight of 8-48 ten thousand by adjusting the concentration of organic nitrogen source and inorganic nitrogen source in the fermentation medium. From these reports and other published methods for producing pullulan, the molecular weight of the produced pullulan is low, and is below 60 ten thousand daltons, which greatly limits the production and application of pullulan. Currently, only Shandong Furuida Biotech Co., Ltd (patent application No. 201210555219.9) in the literature obtains pullulan with molecular weight of 50 to 200 ten thousand by adjusting the concentration of phosphate and initial pH in the culture medium, but the yield of high molecular weight pullulan is not reported, and different culture schemes are not designed for different molecular weight products.
The specificity of the whole fermentation process is determined by the fact that the high molecular weight pullulan fermentation liquor has high viscosity and shows typical non-Newtonian fluid characteristics particularly in the later fermentation period, so that the oxygen transfer in the later fermentation period is influenced. At present, a mechanical stirring type fermentation tank is mostly adopted for producing the pullulan; however, the high molecular weight pullulan is severely limited in dissolved oxygen in the late fermentation stage, the fermentation liquid is almost in a static state outside the stirring paddle, and a large amount of electricity is consumed for improving the stirring effect.
Disclosure of Invention
The invention aims to provide a high-yield strain of high-molecular-weight pullulan and a method for producing the high-molecular-weight pullulan by using the strain, so that the high-molecular-weight pullulan can be obtained in a large scale.
The technical scheme adopted by the invention is as follows:
the high-yield strain of high molecular weight pullulan is characterized in that:
the classification of the high-producing strain is named as aureobasidium pullulans (A)Aureobasidium pullulans) SWP35, which is preserved in China general microbiological culture Collection center (CGMCC) at 11 months and 11 days in 2015 with the preservation number of CGMCC NO. 11602.
The method for producing the pullulan polysaccharide by using the high-yield strain of the high-molecular-weight pullulan polysaccharide is characterized by comprising the following steps of:
the method comprises the following steps:
(1) activating strains:
culturing at 4 deg.C under slant to budMold (A), (B), (C), (Aureobasidium pullulans) Transferring the SWP35 to a PDA culture medium, and culturing for 4-6 days at 28 +/-2 ℃;
(2) seed culture:
selecting a pure culture flat plate, inoculating the bacterium blocks into a seed culture solution by using an aseptic puncher and an aseptic inoculating needle, and culturing for 24-48 h at the temperature of 28 +/-2 ℃ and the rotating speed of 200 +/-30 rpm to prepare a seed solution;
(3) fermentation culture:
inoculating the seed solution prepared in the step (2) into a fermentation culture solution in a ratio of 1-5% (V/V), fermenting in an airlift fermentation tank with a liquid loading amount of 40-70%, wherein the fermentation temperature is 28 +/-2 ℃, the ventilation amount is 0.5-1.5 VVM, and fermenting for 60-96 h;
(4) post-treatment of fermentation liquor:
and diluting the fermentation liquor, filtering and sterilizing the fermentation liquor by using a plate-and-frame filter, clarifying and filtering the fermentation liquor, precipitating the fermentation liquor by using alcohol, washing the fermentation liquor by using alcohol, removing the organic solvent by using filter pressing or centrifugation to obtain a solid matter, and drying and crushing the solid matter to obtain a white pullulan polysaccharide product.
In the step (1), the formula of the PDA culture medium is as follows:
200g/L of potato, 20g/L of glucose, 20g/L of agar and the balance of water;
sterilizing at 121 deg.C for 20min with natural initial pH.
In the step (2), the formula of the seed culture solution is as follows:
50g/L glucose, 0.6g/L ammonium sulfate, 1.7g/L yeast powder, 0.2g/L magnesium sulfate, 1.0g/L sodium chloride and 5.0g/L dipotassium hydrogen phosphate;
the initial pH was 6.5, 121 ℃ and sterilized for 20 min.
In the step (3), the formula of the fermentation culture solution is as follows:
50-100 g/L of sucrose, 1.2-2.1 g/L of ammonium sulfate, 0.2g/L of magnesium sulfate, 1.0g/L of sodium chloride, 5.0g/L of dipotassium hydrogen phosphate and the balance of water;
the initial pH was 6.5, 121. + -. 5 ℃ and sterilized for 20 min.
The invention has the following advantages:
the high molecular weight pullulan produced by the technical scheme of the invention has high yield (more than 35 g/L) and weight average molecular weight of more than 200 ten thousand, and can be used in the tissue engineering fields of hydrogel, artificial bone and the like; when the pullulan composite is used for food thickening, the addition amount of pullulan can be obviously reduced, and the use cost is reduced.
Compared with a stirring type fermentation tank, the air-lift type fermentation tank is lower in energy consumption, and the obtained pullulan polysaccharide is higher in molecular weight; the fermentation medium used in the invention does not contain an organic nitrogen source, has definite medium components, and simple post-extraction process, can obtain a large amount of high molecular weight pullulan, and is suitable for industrial production.
Drawings
FIG. 1 is an infrared chromatogram of pullulan prepared in the first example.
FIG. 2 is an infrared spectrum of pullulan prepared in example II.
Fig. 3 is an infrared spectrum of pullulan standard (Sigma).
Fig. 4 shows the pullulan molecular weight standard (Sigma) GPC results.
Fig. 5 is a pullulan molecular weight standard curve obtained by third-order fitting.
FIG. 6 is a chart of molecular weight measurement in accordance with the example.
In fig. 6, the peak time of the example sample is 14.278, and the molecular weight Mw =2359232 of the prepared pullulan is estimated by a third-order fitting equation.
FIG. 7 is a chart of the measurement of molecular weight in the example.
In FIG. 7, the peak time of the sample of example two is 14.067, and the molecular weight of the obtained pullulan is 3330502 kDa by estimation of a third-order fitting equation.
Detailed Description
The present invention will be described in detail with reference to specific embodiments.
The experimental methods in the following are all conventional methods unless otherwise specified; the experimental materials used in the following examples are all conventional biochemical reagents unless otherwise specified.
The invention utilizes a composite mutagenesis technology to screen and obtain a high-yield strain of high molecular weight pullulan polysaccharide, which is classified and named as buddingAureobasidium pullulans (A), (B), (C)Aureobasidium pullulans) SWP35 Aureobasidium pullulans (A. pullulans) separated from apple surface in Luochuan region of Shaanxi province in ChinaAureobsidium pullulans) SW2009AP5 is obtained by performing ultraviolet and nitrite compound mutagenesis on an original strain, is preserved in the common microorganism center of China general microbiological culture Collection management Committee 11.11.2015, has the address of No. 3 Xilu No.1 Beichen in the Korean area of Beijing and has the preservation number of CGMCC NO. 11602.
The method for producing the pullulan by using the high-yield strain of the high-molecular-weight pullulan comprises the following steps of:
(1) activating strains:
aureobasidium pullulans (Aureobasidium pullulans) strain preserved on inclined plane at 4 ℃Aureobasidium pullulans) Transferring the SWP35 to a PDA culture medium, and culturing for 4-6 days at 28 +/-2 ℃;
(2) seed culture:
selecting a pure culture flat plate, inoculating the bacterium blocks into a seed culture solution by using an aseptic puncher and an aseptic inoculating needle, and culturing for 24-48 h at the temperature of 28 +/-2 ℃ and the rotating speed of 200 +/-30 rpm to prepare a seed solution;
(3) fermentation culture:
inoculating the seed solution prepared in the step (2) into a fermentation culture solution in a ratio of 1-5% (V/V), fermenting in an airlift fermentation tank with a liquid loading amount of 40-70%, wherein the fermentation temperature is 28 +/-2 ℃, the ventilation amount is 0.5-1.5 VVM, and fermenting for 60-96 h;
(4) post-treatment of fermentation liquor:
and diluting the fermentation liquor, filtering and sterilizing the fermentation liquor by using a plate-and-frame filter, clarifying and filtering the fermentation liquor, precipitating the fermentation liquor by using alcohol, washing the fermentation liquor by using alcohol, removing the organic solvent by using filter pressing or centrifugation to obtain a solid matter, and drying and crushing the solid matter to obtain a white pullulan polysaccharide product.
In the step (1), the formula of the PDA culture medium is as follows:
200g/L of potato, 20g/L of glucose, 20g/L of agar and the balance of water;
sterilizing at 121 deg.C for 20min with natural initial pH.
In the step (2), the formula of the seed culture solution is as follows:
50g/L glucose, 0.6g/L ammonium sulfate, 1.7g/L yeast powder, 0.2g/L magnesium sulfate, 1.0g/L sodium chloride and 5.0g/L dipotassium hydrogen phosphate;
the initial pH was 6.5, 121 ℃ and sterilized for 20 min.
In the step (3), the formula of the fermentation culture solution is as follows:
50-100 g/L of sucrose, 1.2-2.1 g/L of ammonium sulfate, 0.2g/L of magnesium sulfate, 1.0g/L of sodium chloride, 5.0g/L of dipotassium hydrogen phosphate and the balance of water;
the initial pH was 6.5, 121. + -. 5 ℃ and sterilized for 20 min.
Obtaining high-molecular-weight pullulan high-yield strains:
(1) original strain SW2009AP5 and its properties: the original strain SW2009AP5 is aureobasidium pullulans (A) separated from the surface of apples in Luochuan area of Shaanxi province in ChinaAureobsidium pullulans). When the strain grows on a flat plate, the bacterial colony is gray white at the initial stage, gradually turns dark to brown, is leather-shaped, has wrinkles, is wet, and is in a mycelium shape by microscopic observation. After the liquid culture is carried out for 96 hours by adopting a culture medium containing an organic nitrogen source, the color of a fermentation liquid is yellow brown, most thalli are yeast-shaped, the yield of extracellular polysaccharide reaches 23.5g/L, the conversion rate of sucrose is 47 percent, and the weight average molecular weight of the obtained pullulan polysaccharide is about 20 ten thousand.
(2) Obtaining of a mutagenic strain: the original strain SW2009AP5 was subjected to ultraviolet and nitrous acid complex mutagenesis to obtain the mutagenized strain SWP 35.
1) Preparation of spore suspension: inoculating slant strains in a liquid culture medium, and culturing at 28 ℃ and 230r/min for 48h to reach a logarithmic phase. Placing into a triangular flask with glass ball, oscillating for 10-15 min, breaking spore block mass, filtering with absorbent cotton, centrifuging at 10000r/min for 10min, collecting sterile physiological saline solution twice, counting with blood counting plate and plate coating method, and adjusting spore concentration to 107one/mL, a spore suspension for mutagenesis treatment was prepared.
2) Ultraviolet mutagenesis treatment: 5mL of the prepared spore suspension was placed in a 9cm sterile plate. And (3) opening a 15W ultraviolet lamp for preheating for 20min, placing the dish containing the spore suspension on a magnetic stirrer, keeping the distance of the dish cover 20cm away from the lamp tube, opening the dish cover, and irradiating for 1-4 min while stirring. Diluting the treated spore suspension in different times in an ultraclean workbench, coating a flat plate, culturing for 3 days to observe results, calculating the lethality rate, selecting strains which grow fast and have large colonies, thick and colloidal colony periphery and faded colors, and evaluating the pullulan production capacity by adopting an organic nitrogen source-containing culture medium.
3) Nitrite mutagenesis treatment: screening out bacterial strain by ultraviolet mutagenesis treatment to prepare 2mL spore suspension, adding 1mL0.1mol/L NaNO2The solution was added with 1mL of 1mol/L acetic acid buffer (NaNO) of pH 4.52The concentration is 0.025 mol/L), and the temperature is kept at 28 ℃ for 30-120 min. After a certain period of treatment, 2mL of the treated solution was added with Na having a pH of 8.62HPO4The solution was adjusted to pH 7.0 and the mutagenesis was terminated. Diluting according to different times, coating a flat plate, culturing for 3 days to observe results, calculating lethality, selecting strains which grow fast, have large bacterial colonies, are thick and colloidal at the periphery of the bacterial colonies and are faded in color, and evaluating the pullulan production capacity by adopting a culture medium without an organic nitrogen source.
4) And (3) evaluating the pullulan production capacity of the mutant strain: respectively inoculating the strains screened in the step 2) or the step 3) into a seed culture medium, carrying out shaking culture for 48h at the temperature of 28 ℃ and the speed of 220r/min, inoculating 5 percent of the strains into a fermentation culture medium, fermenting for 4d at the temperature of 28 ℃ and the speed of 220r/min, measuring the yield, the purity and the molecular weight of the pullulan, and re-screening a high-yield high-molecular-weight pullulan strain swp 35.
5) The fermentation medium is divided into a medium containing an organic nitrogen source and a medium without the organic nitrogen source. The organic nitrogen source-containing culture medium is used for evaluating the pullulan production capacity of the strain subjected to ultraviolet mutagenesis treatment; and the culture medium without organic nitrogen source is used for evaluating the pullulan production capacity of the strain subjected to nitrite mutagenesis treatment.
Organic nitrogen source-containing medium: 50g/L of sucrose, 0.6g/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 phosphate;
organic nitrogen source-free culture medium: 100g/L of sucrose, 0.6g/L of ammonium sulfate, 0.2g/L of magnesium sulfate, 1.0g/L of sodium chloride and 5.0g/L of dipotassium phosphate.
(3) Mutagenic strain swp35 characteristics: when the bacterial strain grows on a flat plate, the color of a bacterial colony is white, the bacterial colony is circular, the center is raised, the surface is smooth, the bacterial colony is wet, and the microbial thalli are microscopically observed to be in a hypha shape. After the culture medium without organic nitrogen source is adopted for liquid culture for 96 hours, the color of the fermentation liquid is light pink, the thalli are mostly yeast-shaped, the yield of extracellular polysaccharide reaches 32.8g/L, the conversion rate of sucrose reaches 32.8%, and the weight average molecular weight of the obtained pullulan polysaccharide reaches more than 200 ten thousand.
The first embodiment is as follows:
(1) aureobasidium pullulans (Aureobasidium pullulans) strain preserved on inclined plane at 4 ℃Aureobsidium pullulans) The CGMCC No.11602 is activated by using a PDA culture medium at 28 ℃ for 4 days;
(2) inoculating the pure cultured fungus blocks into a seed culture solution (the formula is the same as above) through an aseptic puncher and an inoculating needle, and culturing for 48h at 28 ℃ and 200rpm to prepare a seed solution;
(3) inoculating the prepared seed liquid into a 1L triangular flask filled with 200mL of fermentation culture liquid according to the amount of 5% (V/V), wherein the culture medium comprises the following components: 75g/L of sucrose, 1.5g/L of ammonium sulfate, 0.2g/L of magnesium sulfate, 1.0g/L of sodium chloride, 5.0g/L of dipotassium phosphate, initial pH of 6.5, sterilization at 121 ℃ for 20min, fermentation culture at 28 ℃ for 96h in a shaking table at the rotating speed of 180 rpm;
(4) diluting the fermentation liquor by 1 time with water with the same volume, stirring uniformly, rotating at 6000rpm, centrifuging at normal temperature for 20min, discarding the precipitate to leave supernatant, clarifying and filtering the obtained supernatant with diatomite to remove fine solid matters not removed in the centrifuging process;
(5) adding 1.5 times volume of 95% industrial alcohol into the clear filtrate for precipitation, wherein the precipitation operation temperature is 25 ℃, and the precipitation time is 5 hours; centrifuging, washing the solid substance with 95% industrial alcohol for 1 time, filtering to remove organic solvent to obtain solid substance, vacuum drying and pulverizing the solid substance to obtain pullulan polysaccharide product;
(6) aureobasidium pullulans (Aureobasidium pullulans) CGMCC No.11602 is fermented to produce pullulan with the yield of 30g/L, and the content of maltotriose is analyzed by a DNS method after pullulanase enzymolysis, which shows that the purity of the prepared pullulan reaches 96.8%. Infrared chromatographic determination shows that the prepared pullulan polysaccharide has the same infrared spectrum with a pullulan polysaccharide standard Sigma; GPC measurement showed that the weight average molecular weight was 235 ten thousand.
Example two:
(1) aureobasidium pullulans (Aureobasidium pullulans) strain preserved on inclined plane at 4 ℃Aureobsidium pullulans) The CGMCC No.11602 is activated by using a PDA culture medium at 28 ℃ for 4 days;
(2) inoculating the pure cultured fungus blocks into a seed culture solution (the formula is the same as above) through an aseptic puncher and an inoculating needle, and culturing for 48h at 28 ℃ and 200rpm to prepare a seed solution;
(3) inoculating the prepared seed liquid into a 10L airlift fermentation tank filled with 7L fermentation culture solution according to the amount of 5% (V/V), wherein the culture medium comprises the following components: 100g/L of sucrose, 1.8g/L of ammonium sulfate, 0.2g/L of magnesium sulfate, 1.0g/L of sodium chloride, 5.0g/L of dipotassium phosphate, initial pH6.5, sterilization at 121 ℃ for 20min, 28 ℃, ventilation capacity of 1VVM, tank pressure of 0.5Mpa, and fermentation culture for 72 h;
(4) diluting the fermentation liquor by 1 time with water of the same volume in a storage tank, stirring uniformly, removing thalli by adopting plate and frame filtration treatment, and then clarifying and filtering the obtained fermentation liquor supernatant by adopting a diatomite filter;
(5) adding 1.5 times volume of 95% industrial alcohol into the clear filtrate for precipitation, wherein the precipitation operation temperature is 20 ℃, and the precipitation time is 5 hours; separating solid and liquid phases by adopting filter pressing, washing the solid substance for 1 time by using a small amount of the corresponding ethanol, removing the organic solvent by filter pressing to obtain a solid substance, and drying and crushing the solid substance in vacuum to obtain a pullulan product;
(6) the yield of pullulan produced by fermenting Aureobasidium pullulans (Aureobasidium pullulans) with CGMCC No.11602 is 35g/L, and the purity of the pullulan obtained by preparation reaches 97.5 percent by analyzing the maltotriose content by a DNS method after pullulanase enzymolysis. Infrared chromatographic determination shows that the prepared pullulan polysaccharide has the same infrared spectrum with a pullulan polysaccharide standard Sigma; GPC measurement showed that the weight average molecular weight of the pullulan obtained by the preparation was 333 ten thousand.
The culture medium used in the invention has no organic nitrogen source, so that the cost of the culture medium is reduced, the pigment in the culture medium is reduced, and the pressure of pullulan alcohol decoloration in fermentation post-treatment is relieved. By adopting the culture medium, the aureobasidium pullulans CGMCC No.11602 can ferment and produce pullulan with Mw more than 2560000, and the yield can reach more than 35 g/L. In previous researches, the yield is generally sacrificed to obtain high molecular weight, so that the production cost is high, the authorized strain is pigment-free, an airlift fermentation tank is adopted, the energy consumption generated by stirring is reduced, the produced pullulan meets the requirement of high molecular weight, the high yield is ensured, and the pullulan is suitable for industrial development as a novel ultrahigh molecular polymer in the fields of biological materials, drug carriers, beauty treatment and the like.
The invention is not limited to the examples, and any equivalent changes to the technical solution of the invention by a person skilled in the art after reading the description of the invention are covered by the claims of the invention.
Claims (3)
1. The high-yield strain of high molecular weight pullulan is characterized in that: the classification of the high-producing strain is named as aureobasidium pullulans (A)Aureobasidium pullulans) SWP35, which is preserved in China general microbiological culture Collection center (CGMCC) at 11 months and 11 days in 2015 with the preservation number of CGMCC NO. 11602;
the weight-average molecular weight of the obtained pullulan polysaccharide is 333 ten thousand, and the yield reaches more than 35 g/L.
2. A method for producing pullulan using the high-yielding strain of high molecular weight pullulan according to claim 1, characterized in that:
the method comprises the following steps:
(1) activating strains:
aureobasidium pullulans (Aureobasidium pullulans) strain preserved on inclined plane at 4 ℃Aureobasidium pullulans) SWP35 was transferred to PDA medium and cultured at 28 ℃ for 4 days;
(2) seed culture:
selecting a pure culture flat plate, inoculating the bacterium blocks into a seed culture solution by using an aseptic puncher and an aseptic inoculating needle, and culturing for 48 hours at the temperature of 28 ℃ and the rotating speed of 200rpm to prepare a seed solution;
the formula of the seed culture solution is as follows:
50g/L glucose, 0.6g/L ammonium sulfate, 1.7g/L yeast powder, 0.2g/L magnesium sulfate, 1.0g/L sodium chloride and 5.0g/L dipotassium hydrogen phosphate; sterilizing at initial pH of 6.5 and 121 deg.C for 20 min;
(3) fermentation culture:
inoculating the seed solution prepared in the step (2) into a fermentation culture solution in a ratio of 5% (V/V), fermenting in an airlift fermentation tank with a liquid filling amount of 70%, wherein the fermentation temperature is 28 ℃, the ventilation amount is 1VVM, and fermenting for 72 h;
the formula of the fermentation culture solution is as follows:
100g/L of sucrose, 1.8g/L of ammonium sulfate, 0.2g/L of magnesium sulfate, 1.0g/L of sodium chloride, 5.0g/L of dipotassium hydrogen phosphate and the balance of water; sterilizing at initial pH of 6.5, 121 deg.C for 20min
(4) Post-treatment of fermentation liquor:
and diluting the fermentation liquor, filtering and sterilizing the fermentation liquor by using a plate-and-frame filter, clarifying and filtering the fermentation liquor, precipitating the fermentation liquor by using alcohol, washing the fermentation liquor by using alcohol, removing the organic solvent by using filter pressing or centrifugation to obtain a solid matter, and drying and crushing the solid matter to obtain a white pullulan polysaccharide product.
3. The method for producing pullulan using a high-yielding strain of high molecular weight pullulan according to claim 2, wherein:
in the step (1), the formula of the PDA culture medium is as follows:
200g/L of potato, 20g/L of glucose, 20g/L of agar and the balance of water;
sterilizing at 121 deg.C for 20min with natural initial pH.
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| CN112430549A (en) * | 2020-12-11 | 2021-03-02 | 中国海洋大学 | Natural bacterial strain for producing pullulan and application thereof |
| CN113881734A (en) * | 2021-11-16 | 2022-01-04 | 江苏力凡胶囊有限公司 | Culture medium for producing pullulan through microbial fermentation and application of culture medium |
| CN116426391B (en) * | 2023-03-28 | 2024-02-23 | 陕西省微生物研究所 | Aureobasidium pullulans Aureobasidium pullulans P1 and application thereof |
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| CN1609188A (en) * | 2004-11-16 | 2005-04-27 | 江南大学 | Prussian blue producing strain and prussian blue producing process with the strain |
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