CN112322664B

CN112322664B - Method for producing polyphosphate with high polymerization degree

Info

Publication number: CN112322664B
Application number: CN202011202446.4A
Authority: CN
Inventors: 魏炜; 潘鑫茹; 赵劲
Original assignee: Jiangsu Nanchuang Chemical And Life Health Research Institute Co ltd
Current assignee: Jiangsu Nanchuang Chemical And Life Health Research Institute Co ltd
Priority date: 2020-11-02
Filing date: 2020-11-02
Publication date: 2021-07-27
Anticipated expiration: 2040-11-02
Also published as: CN112322664A

Abstract

The invention discloses a method for producing high-polymerization-degree polyphosphate, which is characterized in that a 15L fermentation tank is used for preparing an active microbial inoculum through high-density fermentation, and a bioreactor is combined to realize intracellular synthesis of the high-polymerization-degree polyphosphate by using the active microbial inoculum, wherein the polymerization degree of the prepared polyphosphate is 15-130. In the method provided by the invention, polyphosphates with different polymerization degrees in microbial cells are separated and purified to obtain polyphosphates with different chain lengths, and the method is applied to the fields of medical treatment, daily chemicals, food and the like. Compared with the existing preparation method, the polyphosphate prepared by the biological method has high polymerization degree, can meet the scientific research and market requirements of polyphosphates with different chain lengths, and is green and environment-friendly in production method. The invention is the first domestic pioneering advanced technology for industrially preparing and separating and purifying the polyphosphate with different polymerization degrees in a pilot scale manner, and has great significance for basic scientific research and market development.

Description

Method for producing polyphosphate with high polymerization degree

Technical Field

The invention relates to a method for producing high-polymerization-degree polyphosphate, belonging to the technical field of production of high-polymerization-degree polyphosphate.

Background

Polyphosphates (polyps )_n) Is a linear inorganic polymer formed by connecting dozens to thousands of phosphate groups through high-energy phosphate bonds. The teaching of Arthur Kornberg, university of Stanford, Nobel, in the United states, 1957 proposes intracellular polyP_nThe synthesis of (A) is an enzymatic process, polyP_nPolyanions such as RNA and DNA are important molecular fossils in the evolution process. In recent years, it has been found that polyP_nNot only the indispensable bioenergy and phosphate group storage, the biomacromolecule with the highest negative charge density in the organism, the divalent cation chelating agent,also participate in regulating gene expression, regulating and controlling bacterial ion channels and DNA uptake, metabolism, etc. in the microorganism; and has close relation with physiological processes such as transient potential regulation, mitochondrial metabolism, cell calcification, blood coagulation and the like in mammals.

polyP_nThe polyP is widely present in prokaryotes and eukaryotes and in mammalian cells_nThe polymerization degree is about 60-100, and the poly P in prokaryotic cells_nThe polymerization degree can reach 1000. Polyps of different degrees of polymerization_nHas different physiological and pharmacological functions. Short-chain polyP_n(n<15) Has teeth whitening effect, and when hydroxyapatite is used as research material, polyP_nHas the functions of removing stains and preventing stains from being deposited on materials. When polyP_nWhen the degree of polymerization increases, medium-chain polyps_nAnd long chain polyP_nHas increased physiological functions. Medium chain polyP_n(n is 15 to 60): (1) FGF Activity-enhancing fibroblast factors FGF, FGF-1 and FGF-2 are cytokines that promote tissue regeneration, have been used to accelerate wound healing and treat burn scars, and polyP has been found_nExtending the half-life of FGF prevents its degradation and enhances the affinity of FGF for its receptor; (2) promote cell proliferation, polyP_nThe FGF activity is enhanced so as to promote the proliferation of fibroblasts, and the fibroblast proliferation promoter also has the effect of promoting the proliferation of human mesenchymal stem cells and human dental pulp cells; (3) promoting bone regeneration, and allowing osteoblast MC3T3-E1 to pass through polyP_nAfter induction, the differentiation capability of skeletal cells can be enhanced, and the Lijianlong team at Sichuan university finds that polyP is generated_nThe Ti coating treated by the solution is beneficial to the proliferation and differentiation of osteoblasts; (4) promoting periodontal regeneration and improving periodontitis; (5) promoting blood coagulation, and the platelet dense granule contains polyphosphate polymer and high-concentration calcium, and polyP with different lengths after platelet activation_nIs released and activates FXI and FV factors, initiating the coagulation pathway. Long chain polyP_n(n>60): (1) resisting fungi, and inhibiting growth of fungi such as Saccharomyces cerevisiae; (2) modulation of the immune System, polyP_nCan reduce NO inflammatory factor release induced by lipopolysaccharide, and can also be used as intracellular signal molecule to regulate innate immunity; (3) inhibiting bone resorption; (4) molecular chaperones, polyps under pressure_nWith eggsWhite matter forms a stable complex, keeping the protein in a foldable state; (5) enhancing epithelial cell barrier function; (6) maintaining intestinal homeostasis.

Currently polyP_nThe mainstream preparation method of the method is a chemical synthesis method, and the finished product of the polyphosphate is obtained by melting and polymerizing phosphoric acid and ammonia gas at the temperature of 200-350 ℃. But the chemical method has high production cost, large energy consumption and more byproducts, and products with low polymerization degree, such as sodium tripolyphosphate, sodium pyrophosphate and sodium hexametaphosphate, can be obtained by separation and purification. The majority of the currently marketed patents are for the oligomerization of polyP_n(n<10) The patent CN 110787121A discloses whitening toothpaste and its preparation method, wherein polyphosphate (pentasodium/potassium tripolyphosphate) and pyrophosphate are added as effective components_nThe metal chelating agent can be used for complexing with calcium and magnesium ions, and the complex is separated from the tooth surface, so that tooth stains are removed, and dental calculus is avoided. Patent CN 111296823A discloses a food quality compound modifier and application thereof in fish bone paste additive food, polyP_nThe water retention agent is a water retention agent with strong hydrophilicity and can retain the water content in food, wherein the additive is a compound preparation of sodium tripolyphosphate, sodium hexametaphosphate, sodium pyrophosphate, dipotassium hydrogen phosphate, trisodium phosphate, potassium chloride, disodium dihydrogen pyrophosphate, potassium dihydrogen phosphate and sodium carbonate. Patent CN 105442352 a discloses a dye for plain thread of catgut suture, which is added with sodium tripolyphosphate to increase the luster of the suture. Patent CN105030815A discloses a surgical irrigating fluid and a method for preparing the same, wherein polyphosphate is added to the irrigating fluid, but the polymerization degree is not specified.

polyP_nThe biosynthesis method has mild reaction conditions and no byproduct generation, and can directly synthesize natural high-polymerization-degree polyP by using waste phosphorus sources_n. The phosphorus accumulating small moon bacterium engineering bacteria (CN 105368838A), Acinetobacter johnsonii (CN 109022328A), transgenic synechococcus (CN 106916775A) and Citrobacter freundii (CN 104531599B) all contain polyphosphate kinase coding gene Ppk1, can actively absorb extracellular phosphorus component under aerobic condition and use polyP_nForms are stored in vivo, but polyps are not described in the patent_nDegree of polymerization of (A) and poly-P of different chain lengths_nIs divided intoIsolation and purification method of polyP in bacteria other than Citrobacter freundii_nThe yield is low. Extraction of polyP from Yeast cells (CN 103687582B)_nCan obtain mixed product with polymerization degree of 20-300, and can be used as effective component for developing hair tonic, cosmetic, oral cavity cleaning agent, etc., but the preparation process takes more than 72 hr, and per gram of yeast polyP_nThere were only 0.22 mmol.

Although the diversity of the biological functions of polyphosphates has attracted considerable attention, economical, environmentally friendly, and efficient preparation of polyps_nThe production method of (1) has not yet established a production scale; in addition, polyps of different chain lengths_nHas different functions, and the specific separation and purification method thereof needs to be explored; the method is urgently needed to establish standards and quality standards of different chain lengths and develop and add the polyP with different chain lengths_nThe product of (1) and can be applied to the fields of medical treatment, daily chemicals and food.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a method for producing high-polymerization-degree polyphosphate, which is used for obtaining polyphosphate with different chain lengths, developing standards with different chain lengths, applying to the fields of medical treatment, daily chemicals, food and the like, realizing development of related functional products and filling up the blank of domestic markets.

The technical scheme of the invention is as follows:

the invention provides a method for producing polyphosphate, which comprises the following steps:

(1) activated polyphosphate-transgenic Citrobacter freundii (Citrobacter freundii);

(2) transferring the activated phosphorus-transgenic Citrobacter freundii into a fermentation tank for high-density fermentation, centrifuging the obtained fermentation liquor, and taking bacterial sludge for precipitation to obtain an active microbial inoculum;

(3) injecting a phosphorus-containing culture medium into a sequencing batch bioreactor, and adding an active microbial inoculum to enable the initial OD600 to be 0.2-0.3;

(4) starting the sequencing batch bioreactor at the room temperature of 15-30 ℃, and culturing for 16-19h to obtain a bacterial liquid containing polyphosphate;

the conditions of the high-density fermentation in the step (2) are as follows: the liquid filling amount of the tank body is 70-80%, aerobic culture is carried out for 10-16h at the temperature of 30-37 ℃, and the formula of the used culture medium is as follows: yeast extract 0.5-3%, peptone 0.5-3%, sodium chloride 0.5-2%, inorganic salt ion, and adjusting pH to neutral.

Further, the sequencing batch bioreactor in the step (4) is operated in three steps: a. feeding water, and pre-culturing for 4-6 h; b. continuously feeding water and continuously performing suction filtration for 12-13 h; c. concentrating thallus, discharging thallus for 1-2 hr, and aerating at 0.5-1L/min.

Furthermore, the Citrobacter freundii with the transphosphorylation gene in the step (1) takes Citrobacter freundii ATCC 8090 as a host, and a polyphosphate kinase coding gene Ppk1 of the host is introduced; the genome DNA of the Citrobacter freundii only contains one polyphosphate kinase coding gene Ppk1, and the regulation mode of the polyphosphate kinase coding gene Ppk1 and the exo-polyphosphate coding gene Ppx on the genome is bicistronic cotranscription.

Further, the step (1) is specifically: activating an LB flat plate, namely, activating the phosphate-transgenic Citrobacter freundii preserved at the temperature of-80 ℃, selecting a single clone, inoculating the single clone to 50mL of LB liquid culture medium, and culturing at the temperature of 30-37 ℃ and at the speed of 180-220rpm for 10-12 h; inoculating into 500mL LB liquid culture medium according to 1% inoculum size, culturing at 30-37 deg.C and 180-220rpm for 10-12 h.

Further, the phosphorus-containing medium in the step (3) contains per liter: 0.1-0.5g of glucose, 0.05-0.3g of peptone, 0.01-0.1g of yeast powder, 0.05-0.3g of anhydrous sodium acetate, 0.01-0.3g of sodium chloride, 0.1-0.5g of magnesium sulfate heptahydrate, 0.45-0.75g of dipotassium hydrogen phosphate trihydrate, 0.05-0.5g of ammonium chloride and 6-10mg/L of phosphorus content.

Further, the method also comprises the step of separating polyphosphates with different chain lengths, and comprises the following steps:

(5) centrifuging bacteria liquid containing polyphosphate, freezing bacteria mud at-20 deg.C, thawing, heating in 70-100 deg.C water bath for 10-30min, cooling to room temperature, centrifuging at 10000g for 5min, collecting supernatant, and measuring volume to obtain V1;

(6) adding 0.05V1 volume of hydrochloric acid solution, mixing uniformly, centrifuging for 20min at 10000g, collecting supernatant, adjusting to neutrality with sodium hydroxide solution, and adding sodium chloride to make the final concentration of sodium chloride in the mixed solution be 100 mM;

(7) isolation of medium chain polyphosphate: adding 96% ethanol into the solution, mixing, standing, incubating for 1 hr, centrifuging at 10000g for 5min, and precipitating to obtain medium chain polyphosphate;

(8) isolation of short-chain polyphosphates: collecting the supernatant obtained after the centrifugation in the step (7), adding absolute ethyl alcohol, and fully suspending; standing the solution for 1h, centrifuging the solution for 10min at 10000g, and precipitating the solution to obtain short-chain polyphosphate;

(9) isolation of long-chain polyphosphates: collecting the supernatant obtained after centrifugation in the step (8), adding ultrapure water to enable the volume fraction of ethanol to be 70%, standing the solution for 1h, centrifuging 10000g for 10min, and precipitating to obtain long-chain polyphosphate; washing with 50% ethanol to remove salt residue in the precipitate; placing the product on a dryer, and drying for one week;

the short chain represents the degree of polymerization of less than 15, the medium chain represents the degree of polymerization of 15-60, and the long chain represents the degree of polymerization of 60-130.

The invention also provides the application of the method in the fields of medical treatment, daily chemicals and food, wherein the application is not aimed at the diagnosis or treatment of diseases.

Compared with the prior art, the invention has the advantages that:

(1) the invention provides a pilot plant production method of high polymerization degree poly P_nThe biological preparation method of (1) realizes the biological mass production of polyP in China for the first time_nEach gram of the dry thallus contains 140mg of polyP_nI.e. 4.5mmol of polyP_n(ii) a The phosphorus source required by the production can be taken from wastewater, the production efficiency is high, the reaction condition is mild, and the environment-friendly and resource-renewable preparation of the product is achieved. In contrast to chemical methods, biological methods for preparing polyP_nThe components are natural, the safety is higher, and the polymerization degree can reach 15-130; the production cost is lower, a high-temperature and high-pressure device is not needed, and the preparation process is simple.

(2) The invention provides a poly-P with different chain lengths_nThe separation and purification method of (1) realizes short-chain, medium-chain and long-chain polyP for the first time in China_nThe separation of the standard substance can realize the construction of the standard substance and meet different scientific research and market requirements. According to the different chain lengths of polyP_nHas different functions, and can be developed by aiming at the polyP_nIs a functional product with effective components, fills the domestic market blank, and has significant outstanding implementation effect and commercial value.

Drawings

FIG. 1: preparation of polyps of different chain lengths_nThe process flow diagram of (1).

FIG. 2: schematic diagram of a sequencing batch membrane bioreactor.

FIG. 3: intracellular polyP of Citrobacter freundii_nDAPI stained microscopic pictures.

FIG. 4: thallus OD in 15L fermentation tank₆₀₀And (4) changing.

FIG. 5: intracellular polyP of Citrobacter freundii_nThe content was varied.

FIG. 6: detection of separated and purified polyP by 15% TBE-Urea gum_nChain length.

Detailed Description

The "room temperature" referred to in the present invention means 15 to 30 ℃.

Citrobacter freundii ATCC 8090 mentioned in the present invention is a commercial strain and can be obtained by purchase.

The following examples are only for specifically illustrating the present invention, but the scope of the present invention is not limited to the following examples.

Example 1

The selection process of the organic carbon nitrogen source culture medium comprises the following steps:

(1) a50 mg/L Kan-containing LB plate was activated and the P-transgenic Citrobacter freundii (disclosed in patent CN 104531599A) stored at-80 ℃ was monoclonally inoculated into 50mL of LB liquid medium and cultured at 30 ℃ and 200rpm for 12 hours. Inoculating to four organic carbon nitrogen source culture media (shown in Table 1) according to 1% inoculum size, culturing at 30 deg.C and 200rpm for 12 hr, centrifuging, and collecting bacteria;

TABLE 1 organic carbon nitrogen source medium formulation

(2) Respectively transferring the active microbial inoculum into 100mL of phosphorus-containing culture medium, wherein the initial phosphorus content is 20mg/L, antibiotics are not required, and the initial OD600 is about 0.2;

(3) culturing at 30 deg.C for 14 hr, wherein phosphorus content in consumed culture medium is shown in Table 1, phosphorus consumption of bacterial sludge of yeast extract culture medium is the most, and intracellular polyP is generated_nAnd correspondingly most.

Example 2

Preparation of polyps of different chain lengths_nThe process flow is shown in figure 1, and the specific implementation steps are as follows:

(1) activating LB plate containing 50mg/L Kan-Citrobacter freundii preserved at-80 deg.C, selecting single clone, inoculating into 50mL LB liquid culture medium, culturing at 30 deg.C and 200rpm for 12 h. Inoculating to 500mL LB liquid culture medium according to 1% inoculum size, culturing at 30 deg.C and 200rpm for 12 h;

(2) transferring the strain into a 15L seeding tank, wherein the liquid filling amount of the tank body is 70-80%, selecting yeast extract 0.5-3% (v/v), peptone 0.5-3% (v/v), sodium chloride 0.5-2% (v/v), and organic carbon nitrogen source culture medium of inorganic salt ions to perform high-density fermentation on the polyphosphate gene citrobacter freundii in a fermentation tank, fermenting for 12h at 30 ℃, wherein the OD600 of the strain in the tank can reach 120 as shown in figure 4, discharging the strain from the tank to collect fermentation liquor, and centrifuging for 15min at 6000g to obtain 4Kg of active microbial inoculum;

(3) injecting phosphorus-containing culture medium into 85L sequencing batch bioreactor (shown in figure 2), and adding appropriate amount of above zymogenic active microbial inoculum to make initial OD600 be 0.2-0.3;

(4) starting the sequencing batch bioreactor at room temperature, and a, feeding water and pre-culturing (the time is 4-6 h); b. continuously feeding water and continuously filtering (the time length is 12-13 h); c. thallus concentration and polyP enrichment_nThe thallus is discharged (the time is 1-2h), and the aeration rate is 0.5-1L/min. FIG. 3 shows the cultured cells subjected to DAPI staphyloscopy, in which the intracellular black granules are polyP_n(ii) a Bacteria detected by potassium persulfate digestion ammonium molybdate spectrophotometry (GB11893-89)Intracellular polyP_nThe content was varied (as shown in FIG. 5), and the maximum yield was 140mg of polyP per gram of dry cell weight_nI.e. 4.5mmol of polyP_n；

(5) The thus obtained polyP-containing polymer_nThe recombinant strain is centrifuged and frozen at-20 ℃, thawed, heated at 100 ℃ for 15min, cooled to room temperature, centrifuged at 10000g for 5min, and the supernatant is taken and measured for volume V1;

(6) adding 0.05V1 volume of hydrochloric acid solution (2.5M), mixing well and centrifuging at 10000g for 20min, collecting supernatant and adjusting to neutrality with sodium hydroxide solution (2.5M), adding sodium chloride to make the final concentration of sodium chloride in the mixture 100 mM;

(7) isolation of medium chain polyphosphate: adding a proper amount of 96% ethanol (v/v) into the solution obtained in the step (6), uniformly mixing, standing and incubating for 1h, centrifuging for 5min at 10000g, and precipitating to obtain medium-chain polyphosphate;

(8) isolation of short-chain polyphosphates: collecting the supernatant obtained in the step (7), adding a proper amount of absolute ethyl alcohol, and fully suspending; standing the solution for 1h, centrifuging the solution for 10min at 10000g, and precipitating the solution to obtain short-chain polyphosphate;

(9) isolation of long-chain polyphosphates: collecting the supernatant obtained in the step (8), adding a proper amount of ultrapure water to ensure that the volume fraction of ethanol is 70% (v/v), standing the solution for 1h, centrifuging 10000g for 10min, and precipitating to obtain long-chain polyphosphate; a gentle 50% ethanol (v/v) wash removed the salt residue from the precipitate. The product was placed on a desiccator (a device filled with dry silica) and dried for one week.

The polyP isolated and purified in example 2 was detected with 15% TBE-Urea gel_nAs a result of the chain length, as shown in FIG. 6, the positions of the long, medium and short chains separated by this company on the urea gel were the same as those of the polyps having polymerization degrees of 130, 60 and 14 provided by the Japanese RegeneTiss company_nThe polyP in the same position but not separated_nA long band is shown on the urea gel to indicate the isolated and purified polyP_nCan realize long chain (n is 60-130), medium chain (n is 15-60) and short chain (n is 60-130)<15) Separation of the chain lengths of_nAre mixed together.

Application example 1

Short chains (n) obtained via examples 1 and 2 above<15) And medium chain polyP_n(n-15-60) and is added into oral care products as an effective ingredient, including toothpaste, mouthwash, tooth whitening liquid, and breath fresheners. Short-chain polyP_n(n<15) Has teeth whitening effect, and has effects of removing stains and preventing stain deposition; further, professor Toshikazu Shiba, Hokkaido university, Japan, found medium-chain polyP_nHas effect in improving periodontitis. Addition of polyP_n0.05-70%, preferably 0.1-50%, in the form of liquid, solid, gel, or concentrated solution, and short-chain polyP_nOr a medium chain polyP_nOr their mixture as effective component for developing tooth whitening toothpaste, repair toothpaste for preventing periodontitis, tooth whitening liquid, tooth whitening mouthwash, etc. The oral care products include, but are not limited to, the above-mentioned products.

Application example 2

Medium-chain (n-15-60) and long-chain polyP obtained by the above-described examples 1 and 2_n(n-60-130) and can be added into medical products as effective components, including operation washing liquid, suture, wound dressing, and hemostatic plaster. Medium chain polyP_nCan promote cell proliferation, promote wound healing, and promote blood coagulation; long chain polyP_nHas anti-inflammatory and antifungal effects. Adding polyP_nThe surgical washing liquid has the efficacy of stopping bleeding and reducing the amount of bleeding in the surgical process; added medium chain polyP_nThe suture line can reduce the inflammation symptoms at the suture wound after the suture, and promote the wound healing; added medium chain polyP_nThe wound dressing and the hemostatic plaster can promote the rapid blood coagulation of bleeding points, and are favorable for wound healing and repair. Addition of polyP_nThe total mass of the product is 0.05-70%, preferably 0.1-50%, and the product is added in liquid, solid, gel, paste, or concentrated solution state, and the product is added with medium chain polyP_nOr a long-chain polyP_nOr the mixture of the two is used as an effective component to develop surgical medical supplies. The medical product includes, but is not limited to, the above-mentioned products.

Application example 3

Medium-chain (n-15-60) and long-chain polyP obtained by the above-described examples 1 and 2_n(n-60-130) and is added into medical appliance products as an effective component, including orthopedic implant materials, dental materials and the like. Medium and Long-chain PolyPs_nCan promote bone regeneration, induce proliferation and differentiation of osteoblast, and is found by Lijianlong team of Sichuan university via polyP_nThe solution treated Ti coating facilitates osteoblast proliferation and differentiation. Warp polyP_nThe treated orthopedic implant material has better biocompatibility and is beneficial to bone restoration; warp polyP_nThe treated tooth material can realize better tooth restoration. Addition of polyP_nThe total mass of (A) is 0.05-70%, preferably 0.1-50%, and the implant material is soaked in polyP_nIn solution with medium chain poly-P_nOr a long-chain polyP_nOr the mixture of the two is used as an effective component to develop medical appliance products. The medical device product includes, but is not limited to, the above-mentioned products.

Application example 4

Short-chain polyP obtained via examples 1 and 2 above_n(n<15) And can be added into food as effective component, including chewing gum, breath-refreshing candy, food antistaling agent, and quality improver. Short-chain polyP_nHas the effects of whitening teeth and removing stains, and is also a metal chelating agent and a water-retaining agent. Adding polyP_nThe breath-freshening candy has the breath-freshening effect; a composition containing polyP_nThe food additive is beneficial to the preservation and the preservation of food and improves the mouthfeel. Addition of polyP_nThe total mass of the product is 0.05-70%, preferably 0.1-50%, and the product is added in the form of liquid, solid, gel, paste, or concentrated solution, and short-chain polyP_nAs an active ingredient, food additives and candies have been developed. The product includes the above-mentioned products but is not limited thereto.

Application example 5

Medium-chain (n-15-60) and long-chain polyP obtained by the above-described examples 1 and 2_n(n-60-130) and can be added into cosmetics and cleaning and caring products as effective component, such as emulsion, cosmetic water, color cosmetic, shampoo, and bath shampooBath lotion. Medium chain polyP_nCan promote cell proliferation, provide moisture to stratum corneum and keep moisture. Adding polyP_nThe lotion or lotion has better moisturizing effect and is easy to be absorbed by skin; adding polyP_nThe shampoo and the shower gel have good water retention effect on skin. Addition of polyP_nThe total mass of the product is 0.05-70%, preferably 0.1-50%, and the product is added in liquid, solid, gel, paste, or concentrated solution state, and the product is added with medium chain polyP_nOr a long-chain polyP_nOr their mixture as effective component for developing cosmetics and daily chemical products. The cosmetic and daily chemical products include the above products but are not limited thereto.

The invention firstly treats the main inorganic polyanion-polyphosphate (polyP) in organisms_n) The high-yield preparation is realized in prokaryotes, and the phosphorus source required by the production can be sourced from wastewater, so that the environment-friendly and resource-renewable preparation of products is achieved. At present, no biological pilot-scale production of polyP is found in the domestic market_nIn addition, with a high degree of polymerization of the polyP_nThe development of functional products as active ingredients is still in the blank stage.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. Poly P with different chain lengths_nThe method for separating and purifying, characterized in that the method comprises the following steps:

(1) activated transphosphorylation gene Citrobacter freundii (B.), (Citrobacter freundii）；

The Citrobacter freundii with the transphosphorylation gene takes Citrobacter freundii ATCC 8090 as a host, and a polyphosphate kinase coding gene Ppk1 of the host is introduced; only one polyphosphate kinase coding gene Ppk1 exists in the genomic DNA of the Citrobacter freundii, and the regulation mode of the polyphosphate kinase coding gene Ppk1 and the exo-polyphosphate coding gene Ppx on the genome is bicistronic cotranscription;

(5) centrifuging the bacterial liquid containing polyphosphate prepared in the step (4), freezing bacterial mud at the temperature of minus 20 ℃, thawing, heating in a water bath at the temperature of 70-100 ℃ for 10-30min, cooling to room temperature, centrifuging at 10000g for 5min, taking supernatant, and measuring the volume to obtain V1;

2. The method according to claim 1, wherein the conditions of the high-density fermentation in step (2) are as follows: the liquid filling amount of the tank body is 70-80%, aerobic culture is carried out for 10-16h at the temperature of 30-37 ℃, and the formula of the used culture medium is as follows: yeast extract 0.5-3%, peptone 0.5-3%, sodium chloride 0.5-2%, inorganic salt ion, and adjusting pH to neutral.

3. The method of claim 1, wherein the sequencing batch bioreactor of step (4) is operated in three steps: a. feeding water, and pre-culturing for 4-6 h; b. continuously feeding water and continuously performing suction filtration for 12-13 h; c. concentrating thallus, discharging thallus for 1-2 hr, and aerating at 0.5-1L/min.

4. The method according to claim 1, characterized in that step (1) is in particular: activating an LB flat plate, namely, activating the phosphate-transgenic Citrobacter freundii preserved at the temperature of-80 ℃, selecting a single clone, inoculating the single clone to 50mL of LB liquid culture medium, and culturing at the temperature of 30-37 ℃ and at the speed of 180-220rpm for 10-12 h; inoculating into 500mL LB liquid culture medium according to 1% inoculum size, culturing at 30-37 deg.C and 180-220rpm for 10-12 h.

5. The method of claim 1, wherein the phosphorus-containing medium in step (3) contains per liter: 0.1-0.5g of glucose, 0.05-0.3g of peptone, 0.01-0.1g of yeast powder, 0.05-0.3g of anhydrous sodium acetate, 0.01-0.3g of sodium chloride, 0.1-0.5g of magnesium sulfate heptahydrate, 0.45-0.75g of dipotassium hydrogen phosphate trihydrate, 0.05-0.5g of ammonium chloride and 6-10mg/L of phosphorus content.

6. Use of the method according to any of claims 1 to 5 in the medical, daily chemical, food fields, wherein said use is not aimed at the diagnosis or treatment of a disease.