Pichia pastoris culture medium and fermentation method

Abstract

The invention discloses a pichia pastoris fermentation medium and a pichia pastoris fermentation method, and belongs to the technical field of biological fermentation. Aiming at the technical problem of unstable nitrogen source supply caused by uncontrollable reaction rate of lactic acid and sodium nitrite in the existing pichia pastoris fermentation process, the invention provides the culture medium containing the dynamic nitrogen source slow-release particles. The culture medium comprises corncob hydrolysate, L yeast extract, a trace element solution, polyol, methanol, lactic acid, citric acid diamine, an amino acid mixture and dynamic nitrogen source slow-release particles according to the total volume, the dynamic nitrogen source slow-release particles are formed by loading 25-27% by mass of sodium nitrite on zeolite with the aperture of 0.48-0.52 nm, and the mass ratio of the lactic acid to the loaded sodium nitrite is 1: (1.8-2.2). According to the design, reaction kinetics of lactic acid and sodium nitrite is accurately regulated and controlled through a zeolite pore channel confinement effect, and continuous and stable supply of a nitrogen source in a fermentation process is realized. The method is mainly applied to the process of producing recombinant protein by high-density fermentation of pichia pastoris.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology. More specifically, this invention relates to a Pichia pastoris fermentation culture medium and fermentation method. Background Technology

[0002] Maintaining a stable nitrogen supply is a key challenge in ensuring efficient cell growth and recombinant protein expression during high-density Pichia pastoris fermentation. Current technologies utilizing the chemical reaction of lactic acid and sodium nitrite to generate nitrogen have significant drawbacks, primarily manifested in the highly uncontrollable reaction rate. This problem stems from three main factors: First, dynamic fluctuations in the fermentation environment (such as changes in temperature, pH, and ionic strength) significantly alter reaction kinetics. For instance, an increase in pH accelerates nitrite hydrolysis, and a temperature deviation of 0.5°C can cause orders-of-magnitude changes in the reaction rate. Second, uneven reactant concentrations can easily trigger localized violent reactions or stagnation. High-concentration areas may release excessive ammonia nitrogen instantaneously, leading to osmotic stress or metabolic inhibition in cells, while low-concentration areas may experience nitrogen shortages due to insufficient reaction. Third, the generation of byproducts such as toxic nitrosamines or excessive nitrogen gas can further contribute to this problem. The former directly damages cell activity, while the latter forms bubbles that disrupt the uniformity of dissolved oxygen transfer.

[0003] The aforementioned runaway reactions lead to intermittent fluctuations in nitrogen supply. Specifically, during cell growth, sudden nitrogen releases disrupt cellular metabolism and can even cause ammonia poisoning, while periods of nitrogen depletion force cells into a state of starvation, reducing specific growth rates. During protein expression, intermittent nitrogen supply causes fluctuations in translation efficiency, increasing the risk of target protein degradation and ultimately reducing yield. Particularly in large fermenters, decreased mixing efficiency further exacerbates spatial heterogeneity, resulting in differences in nitrogen concentrations across different regions of the fermenter, making stable process scale-up difficult.

[0004] To address this issue, existing technologies attempt to encapsulate sodium nitrite using slow-release carriers (such as alginate microcapsules), but these methods have fundamental limitations: the loading rate of sodium nitrite on the carrier is generally less than 15%, and leakage is prone to occur under high osmotic pressure environments; the degradation rate of the carrier and the lactic acid permeation rate are difficult to coordinate, resulting in initial burst release (release amount >50% in the first 2 hours) or insufficient release in the later stages; in addition, the carrier material may adsorb nutrients or clog pipelines, increasing the complexity of the process. Summary of the Invention

[0005] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0006] To achieve these and other advantages according to the present invention, a Pichia pastoris fermentation medium is provided, comprising a fermentation medium that includes the following components based on the total volume of the medium: The mixture consists of 16-20 g / L corn cob hydrolysate, 0.6-0.9 g / L yeast extract, 4.5-5.5 mL / L trace element solution, 1.5-2.5 g / L polyol, 7.5-8.5 g / L methanol, 0.04-0.07 g / L lactic acid, 0.45-0.55 g / L diamine citrate, 0.025-0.035 g / L amino acid mixture, and 0.95-1.05 g / L dynamic nitrogen source sustained-release particles. The dynamic nitrogen source sustained-release particles are composed of zeolite with a pore size of 0.48-0.52 nm and sodium nitrite loaded in the pores of the zeolite. The loading of sodium nitrite is 25-27% of the mass of zeolite, and the mass ratio of lactic acid to sodium nitrite loaded in zeolite is 1:1.8-2.2.

[0007] Preferably, the preparation method of dynamic nitrogen source sustained-release particles includes the following steps: Zeolite with a pore size of 0.48~0.52nm was immersed in a sodium nitrite aqueous solution with a mass concentration of 6.5~7.5% for 25~28 minutes under a vacuum of -0.09 to -0.095MPa. After impregnation, filter the residue and dry it in two stages: first at 45~48℃ for 30~40 minutes, and then at 52~55℃ for 20~25 minutes. The dried granules were placed in a muffle furnace and heated to 280-300°C at a rate of 3-5°C / min. After holding at this temperature for 8-10 min, the granules were cooled to room temperature. After cooling, the surface of the particles is sprayed with alumina sol with a coating thickness of 0.5~0.8μm and cured at 110~115℃ for 15~20min. Then, the particles are vacuum sealed. The vacuum-sealed particles are sterilized by steam at 110~115℃ for 8~12min before use.

[0008] Preferably, the surface of the dynamic nitrogen source slow-release particles is coated with a sodium carboxymethyl cellulose layer, the coating thickness is 0.5~0.8μm and the degree of substitution is 0.65~0.70; The sodium carboxymethyl cellulose layer is formed by the following method: the dynamic nitrogen source slow-release particles obtained by steam sterilization are immersed in an aqueous solution of sodium carboxymethyl cellulose with a mass concentration of 2.5~3.5% for 5~8 minutes, and then cured at 110~115℃ for 15~20 minutes.

[0009] Preferably, the amino acid mixture comprises glutamic acid, L-methionine, glycine, asparagine and tyrosine, with the molar ratio of each amino acid being glutamic acid:L-methionine:glycine:asparagine:tyrosine = 1:0.5:0.6:1:0.3, and L-methionine accounts for 30-35% of the total mass of the amino acid mixture.

[0010] Preferably, the trace element solution comprises 0.1~0.3 g / L biotin, 0.05~0.2 g / L cobalt chloride hexahydrate, 35~40 g / L ferric chloride, and 1.5~2.5 g / L cuprous oxide; The solvents used for the fermentation medium and trace element solution are weakly acidic ionized water with a redox potential of -150 to -200 mV and a pH of 5.0 to 5.5. The polyol is one or more of xylitol, sorbitol, ethylene glycol or pentaerythritol.

[0011] A method for fermenting Pichia pastoris using the aforementioned fermentation medium is provided, comprising the following steps: S1. Prepare Pichia pastoris seed culture. Inoculate the Pichia pastoris seed culture into a fermenter containing Pichia pastoris fermentation medium at an inoculation rate of 8-10% for initial culture. During the initial culture, maintain the dissolved oxygen in the fermenter above 40%. S2. When the dissolved oxygen in the fermenter is greater than 75%, add 50v / v% glycerol solution to the fermenter at a rate of 10mL / (L·h). When the cell OD... 600 When the concentration exceeds 180, stop adding glycerol solution. During the process of adding glycerol solution, maintain the dissolved oxygen in the fermenter at 23-27%. S3. One hour after the addition of glycerol solution, add the inducing agent to the fermenter for induction culture. Take samples every 2-4 hours for testing. When the increase in expression level is less than 20 mg / (L·h), stop fermentation. During the induction culture, control the dissolved oxygen in the fermenter to 10-50%. The preparation method of the inducer is as follows: A1. Mix sodium alginate solution, sodium carboxymethyl cellulose, culture medium base solution and deionized water in a volume ratio of 8:2:1:12 to obtain the first mixture. The culture medium base solution consists of the components of the fermentation medium except for methanol and polyol. A2. Mix lactic acid and sodium alginate solution at a mass ratio of 1:3~5 to obtain a gel precursor solution containing lactic acid; A3. Using a pipette, the first mixture in A1 is added dropwise to anhydrous calcium chloride solution to form primary microcapsules containing dynamic nitrogen source sustained-release particles. After filtration and washing, the primary microcapsules are immersed in the gel precursor solution of A2, and calcium chloride solution is added dropwise a second time to form double-layer microcapsules. A4. Disperse the bilayer microcapsules obtained in A3 in methanol to obtain a suspension. Mix the suspension with glycerol at a volume ratio of 1:0.1~2 to obtain the inducer. The suspension contains 0.5 g of bilayer microcapsules per liter of methanol.

[0012] Preferably, during the induction culture, a fortifying solution is continuously fed into the fermenter at a flow rate of 0.8 to 1.2 mL / (L·h), and the dissolved oxygen is maintained at 10 to 15% during the feeding. The fortifying solution consists of 0.5 to 1.5 g / L lactic acid and 2.0 to 3.0 g / L sodium carboxymethyl cellulose.

[0013] Preferably, a segmented temperature control strategy is adopted during the induction culture in step S3: For 0-6 hours after induction begins, maintain the fermenter temperature at 28-30℃. After 6 hours of induction until the end of fermentation, raise the temperature to 33-35℃.

[0014] Preferably, the bilayer microcapsules formed in step A3 further comprise a pH buffer layer: Before the primary microcapsules are immersed in the gel precursor solution, they are first sprayed with a 0.1-0.3 mol / L disodium hydrogen phosphate-citric acid buffer solution to form a buffer coating with a thickness of 1-2 μm. The ionic strength of the buffer coating is 0.15-0.25 M, and its pH value is 0.3-0.5 units lower than that of the fermentation medium.

[0015] The present invention has at least the following beneficial effects: First, this invention solves the problem of uncontrollable lactic acid-sodium nitrite reaction rate by loading a specific amount of sodium nitrite onto zeolite with a specific pore size in the fermentation medium, forming a precise mass ratio with free lactic acid. The zeolite pore size matches the diameter of sodium nitrite molecules, and the nano-confining effect restricts its diffusion, avoiding excessively high local concentrations and explosive reactions. This ensures that the rate of lactic acid permeation matches the rate of sodium nitrite release, transforming the violent reaction into a slow and controllable "permeation-reaction" process.

[0016] Second, the present invention defines the preparation process of dynamic nitrogen source slow-release particles as "vacuum impregnation - two-stage drying - high-temperature heat treatment - alumina coating encapsulation": vacuum impregnation allows sodium nitrite to fully fill the core pores of zeolite; two-stage drying and high-temperature heat treatment remove moisture from the pores, stabilize the crystal structure, and prevent sodium nitrite from deliquescing and being lost in the high humidity environment of fermentation; the alumina coating forms a dense physical barrier, enhances the mechanical stability under high osmotic pressure environment, prevents particle breakage leading to sudden release of active substances, and ensures the reliability and repeatability of the nitrogen source slow-release kinetic curve.

[0017] Third, this invention adds a sodium carboxymethyl cellulose (CMC) coating layer with a substitution degree of 0.65~0.70. Its hydrophilicity and film-forming properties form a hydrated gel layer on the outer layer of the particles: on the one hand, it blocks the adsorption and covering of macromolecules such as proteins and cell fragments in the fermentation broth, avoiding pore blockage and resulting in a decrease in the release rate; on the other hand, it provides a mild microenvironment for lactic acid permeation, buffers and smooths the transfer rate, enhances the system's ability to resist the interference of slight fluctuations in pH and ionic strength, and makes the nitrogen release curve more stable.

[0018] Fourth, the core of the fermentation method provided by this invention lies in the special preparation process of the inducer: the inducer is not a simple methanol mixture, but rather a double-layered microcapsule with a core-shell structure formed by encapsulating dynamic nitrogen source slow-release particles, lactic acid and other culture medium components in a sodium alginate-calcium chloride system.

[0019] Fifth, this invention continuously adds a fortifying solution containing lactic acid and sodium carboxymethyl cellulose (CMC) during the induction period to achieve "fine-tuning" of reactant concentration: low concentration of lactic acid replenishes the reaction consumption, maintains a stable lactic acid concentration in the system, and ensures the continuous progress of the nitrate reduction reaction; CMC maintains the rheological properties of the fermentation broth, prevents cell aggregation, and works synergistically with the CMC layer on the surface of the inducer microcapsules to further stabilize the drug release microenvironment.

[0020] Sixth, this invention sprays a buffer coating with a pH 0.3 to 0.5 lower than the fermentation medium onto the surface of the primary microcapsules to establish a locally stable acidic microenvironment. On the one hand, this maintains the morphology of lactic acid molecules and promotes their penetration into the capsule core; on the other hand, it optimizes the pH conditions for the reaction between sodium nitrite and lactic acid. Even if the pH of the external fermenter fluctuates slightly, the internal reaction rate remains constant, further enhancing the robustness and anti-interference ability of the sustained-release system and making the nitrogen release kinetic curve more predictable and controllable.

[0021] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0023] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0024] The embodiments, comparative examples, and blank groups of this invention all used the same Pichia pastoris strain (GS115 / pPIC9K-α-amylase) and were carried out in a 5L fermenter with an initial liquid volume of 3L. The dissolved oxygen in the fermenter of this invention was controlled by adjusting the stirring speed and the amount of air introduced.

[0025] <Example 1> The preparation method of dynamic nitrogen source sustained-release particles includes the following steps: Take 100g of zeolite particles with a pore size of 0.50±0.02nm, immerse them in a 7.0% sodium nitrite aqueous solution for 26 minutes under a vacuum of -0.092MPa, filter, and then dry them in two stages: first at 47℃ for 35 minutes, and then at 54℃ for 22 minutes. Place the dried particles in a muffle furnace, heat them to 290℃ at a rate of 4℃ / min and hold for 9 minutes. After cooling to room temperature, spray an alumina sol layer (coating thickness 0.7μm), cure it at 115℃ for 18 minutes, and finally vacuum seal it. Before use, sterilize it with steam at 113℃ for 10 minutes.

[0026] <Example 2> A method for preparing dynamic nitrogen source sustained-release particles with a surface coated with a sodium carboxymethyl cellulose layer includes the following steps: The dynamic nitrogen source slow-release particles prepared in Example 1 and obtained by steam sterilization were immersed in a 3.0% sodium carboxymethyl cellulose aqueous solution (degree of substitution 0.68) for 6 minutes, and then removed and cured at 115°C for 18 minutes to form a coating layer with a thickness of 0.65 μm.

[0027] <Example 3> Pichia pastoris culture medium, which includes a fermentation medium, comprising the following components by total volume of the culture medium: The following ingredients were used: 16.0 g / L corn cob hydrolysate, 0.60 g / L yeast extract, 4.5 mL / L trace element solution (containing 0.2 g / L biotin, 0.12 g / L cobalt chloride hexahydrate, 38 g / L ferric chloride, and 2.0 g / L cuprous oxide), 1.5 g / L polyol (xylitol to sorbitol mass ratio 1:1), 7.5 g / L methanol, 0.040 g / L lactic acid, 0.45 g / L diamine citrate, 0.025 g / L amino acid mixture (glutamic acid:L-methionine:glycine:asparagine:tyrosine molar ratio = 1:0.5:0.6:1:0.3, L-methionine accounting for 33%), and 0.95 g / L dynamic nitrogen source slow-release particles (prepared in Example 2). All solvents used were weakly acidic deionized water with a redox potential of -180 mV and a pH of 5.3.

[0028] The fermentation method of Pichia pastoris using the above-mentioned fermentation medium includes the following steps: S1, OD600 The Pichia pastoris seed culture with a concentration of 8.5 was inoculated at a rate of 9% into the fermenter containing the Pichia pastoris fermentation medium for initial culture. During the initial culture, the dissolved oxygen in the fermenter was kept above 40% and cultured at 30°C for 18 hours. S2. When the dissolved oxygen in the fermenter is greater than 75%, add 50v / v% glycerol solution to the fermenter at a rate of 10mL / (L·h). When the cell OD... 600 Greater than 180 (measured OD) 600 When the concentration reaches 185, stop adding glycerol solution. During the process of adding glycerol solution, maintain the dissolved oxygen in the fermenter at 25% at all times. S3. One hour after the addition of glycerol solution is completed, add an inducing agent to the fermenter for induction culture. The preparation method of the inducing agent includes the following steps: A1: Mix sodium alginate solution, sodium carboxymethyl cellulose, culture medium base solution (same as fermentation medium but without methanol and polyols) and deionized water in a volume ratio of 8:2:1:12 to obtain the first mixture; A2: Lactic acid and sodium alginate solution are mixed at a mass ratio of 1:4 to obtain a gel precursor solution; A3: The first mixture of A1 is dropped into anhydrous CaCl2 solution to form primary microcapsules (embedding dynamic particles). The surface of the primary microcapsules is sprayed with 0.2 mol / L disodium hydrogen phosphate-citric acid buffer (pH 4.8, ionic strength 0.20M) to form a 1.5 μm buffer coating. Then, it is immersed in the gel precursor solution of A2 and CaCl2 solution is dropped in a second time to form a bilayer microcapsule. A4: Disperse the bilayer microcapsules in methanol (0.5 g microcapsules / L methanol), and then mix them with glycerol at a volume ratio of 1:1; S4. A solution containing 1.0 g / L lactic acid and 2.5 g / L sodium carboxymethyl cellulose was continuously added at a rate of 1.0 mL / (L·h), with dissolved oxygen controlled at 10% during the addition. S5, Induction 0-6h: Maintain 29℃ for 6h until fermentation ends, then raise the temperature to 34℃, take samples every 3h for testing, and end fermentation when the increase in protein expression level is <20mg / (L·h) (total 72h).

[0029] <Example 4> Pichia pastoris culture medium, which includes a fermentation medium, comprising the following components by total volume of the culture medium: The following ingredients were used: 18.0 g / L corn cob hydrolysate, 0.75 g / L yeast extract, 5.0 mL / L trace element solution (containing 0.2 g / L biotin, 0.12 g / L cobalt chloride hexahydrate, 38 g / L ferric chloride, and 2.0 g / L cuprous oxide), 2.0 g / L ethylene glycol, 8.0 g / L methanol, 0.055 g / L lactic acid, 0.50 g / L diamine citrate, 0.030 g / L amino acid mixture (glutamic acid:L-methionine:glycine:asparagine:tyrosine molar ratio = 1:0.5:0.6:1:0.3, with L-methionine accounting for 33%), and 0.95 g / L dynamic nitrogen source slow-release particles (prepared in Example 2). All solvents used were weakly acidic deionized water with a redox potential of -180 mV and a pH of 5.3.

[0030] The fermentation method of Pichia pastoris using the above-mentioned fermentation medium includes the following steps: S1, OD 600 The Pichia pastoris seed culture with a concentration of 8.5 was inoculated at a rate of 9% into the fermenter containing the Pichia pastoris fermentation medium for initial culture. During the initial culture, the dissolved oxygen in the fermenter was kept above 40% and cultured at 30°C for 18 hours. S2. When the dissolved oxygen in the fermenter is greater than 75%, add 50v / v% glycerol solution to the fermenter at a rate of 10mL / (L·h). When the cell OD... 600 Greater than 180 (measured OD) 600 When the concentration reaches 185, stop adding glycerol solution. During the process of adding glycerol solution, maintain the dissolved oxygen in the fermenter at 25% at all times. S3. One hour after the addition of glycerol solution is completed, add an inducing agent to the fermenter for induction culture. The preparation method of the inducing agent includes the following steps: A1: Mix sodium alginate solution, sodium carboxymethyl cellulose, culture medium base solution (same as fermentation medium but without methanol and polyols) and deionized water in a volume ratio of 8:2:1:12 to obtain the first mixture; A2: Lactic acid and sodium alginate solution are mixed at a mass ratio of 1:4 to obtain a gel precursor solution; A3: The first mixture of A1 is dropped into anhydrous CaCl2 solution to form primary microcapsules (embedding dynamic particles). The surface of the primary microcapsules is sprayed with 0.2 mol / L disodium hydrogen phosphate-citric acid buffer (pH 4.8, ionic strength 0.20M) to form a 1.5 μm buffer coating. Then, it is immersed in the gel precursor solution of A2 and CaCl2 solution is dropped in a second time to form a bilayer microcapsule. A4: Disperse the bilayer microcapsules in methanol (0.5 g microcapsules / L methanol), and then mix them with glycerol at a volume ratio of 1:1; S4. A solution containing 1.0 g / L lactic acid and 2.5 g / L sodium carboxymethyl cellulose was continuously added at a rate of 1.0 mL / (L·h), with dissolved oxygen controlled at 10% during the addition. S5, Induction 0-6h: Maintain 29℃ for 6h until fermentation ends, then raise the temperature to 34℃, take samples every 3h for testing, and end fermentation when the increase in protein expression level is <20mg / (L·h) (total 72h).

[0031] <Example 5> A culture medium for Pichia pastoris, comprising a fermentation medium, wherein the fermentation medium comprises the following components based on the total volume of the culture medium: The following ingredients were used: 20.0 g / L corn cob hydrolysate, 0.90 g / L yeast extract, 5.5 mL / L trace element solution (containing 0.2 g / L biotin, 0.12 g / L cobalt chloride hexahydrate, 38 g / L ferric chloride, and 2.0 g / L cuprous oxide), 2.5 g / L pentaerythritol, 8.5 g / L methanol, 0.070 g / L lactic acid, 0.55 g / L diamine citrate, 0.035 g / L amino acid mixture (glutamic acid:L-methionine:glycine:asparagine:tyrosine molar ratio = 1:0.5:0.6:1:0.3, with L-methionine accounting for 33%), and 1.05 g / L dynamic nitrogen source slow-release granules (prepared in Example 2). All solvents used were weakly acidic ionized water with a redox potential of -180 mV and a pH of 5.3.

[0032] The fermentation method of Pichia pastoris using the above-mentioned fermentation medium includes the following steps: S1, OD 600 The Pichia pastoris seed culture with a concentration of 8.5 was inoculated at a rate of 9% into the fermenter containing the Pichia pastoris fermentation medium for initial culture. During the initial culture, the dissolved oxygen in the fermenter was kept above 40% and cultured at 30°C for 18 hours. S2. When the dissolved oxygen in the fermenter is greater than 75%, add 50v / v% glycerol solution to the fermenter at a rate of 10mL / (L·h). When the cell OD... 600 Greater than 180 (measured OD) 600 When the concentration reaches 185, stop adding glycerol solution. During the process of adding glycerol solution, maintain the dissolved oxygen in the fermenter at 25% at all times. S3. One hour after the addition of glycerol solution is completed, add an inducing agent to the fermenter for induction culture. The preparation method of the inducing agent includes the following steps: A1: Mix sodium alginate solution, sodium carboxymethyl cellulose, culture medium base solution (same as fermentation medium but without methanol and polyols) and deionized water in a volume ratio of 8:2:1:12 to obtain the first mixture; A2: Lactic acid and sodium alginate solution are mixed at a mass ratio of 1:4 to obtain a gel precursor solution; A3: The first mixture of A1 is dropped into anhydrous CaCl2 solution to form primary microcapsules (embedding dynamic particles). The surface of the primary microcapsules is sprayed with 0.2 mol / L disodium hydrogen phosphate-citric acid buffer (pH 4.8, ionic strength 0.20M) to form a 1.5 μm buffer coating. Then, it is immersed in the gel precursor solution of A2 and CaCl2 solution is dropped in a second time to form a bilayer microcapsule. A4: Disperse the bilayer microcapsules in methanol (0.5 g microcapsules / L methanol), and then mix them with glycerol at a volume ratio of 1:1; S4. A solution containing 1.0 g / L lactic acid and 2.5 g / L sodium carboxymethyl cellulose was continuously added at a rate of 1.0 mL / (L·h), with dissolved oxygen controlled at 10% during the addition. S5, Induction 0-6h: Maintain 29℃ for 6h until fermentation ends, then raise the temperature to 34℃, take samples every 3h for testing, and end fermentation when the increase in protein expression level is <20mg / (L·h) (total 72h).

[0033] <Blank Group> Pichia pastoris culture medium was prepared using BSM basal medium (composition: 85% H3PO4 26.7 mL / L, CaSO4·2H2O 0.93 g / L, K2SO4 18.2 g / L, MgSO4·7H2O 14.9 g / L, KOH 4.13 g / L, glycerol 40.0 g / L). The fermentation method of Pichia pastoris involves feeding pure methanol during the induction phase, and the remaining steps are the same as in Example 1.

[0034] <Performance Test 1> The ammonia nitrogen fluctuation coefficient (%), nitrosamine accumulation (μg / L), dissolved oxygen uniformity (%), and protein expression level (g / L) of Pichia pastoris in Examples 3-5 and the blank group of the present invention were detected by the following methods: Method for detecting ammonia nitrogen fluctuation coefficient: Sampling is performed every 2 hours, and the concentration of ammonia nitrogen is determined by the indophenol blue method. The percentage of the standard deviation to the mean is calculated. Method for detecting nitrosamine accumulation: Sampling was taken at the fermentation endpoint, and the content of N-nitrosodimethylamine was detected by HPLC (chromatographic column: C18, mobile phase methanol:water = 30:70). Method for detecting dissolved oxygen uniformity: Record the dissolved oxygen probe readings of the upper, middle and lower layers of the fermenter, and calculate the percentage of the range to the mean; Methods for detecting protein expression levels: At the end of fermentation, cell lysate was collected, and the concentration of recombinant α-amylase was determined using the Bradford method; The test results are shown in Table 1: Table 1 Performance test of Pichia pastoris in Examples 3-5 and the blank group Among them, the fluctuation phenomenon in 8.9* is mainly caused by the inherent characteristics of the phosphate buffer system of the culture medium and the ammonia water addition operation carried out during the fermentation process to maintain pH stability. This is the basic fluctuation caused by the consumption and replenishment of conventional nitrogen sources in high-density fermentation.

[0035] As shown in Table 1, the ammonia nitrogen fluctuation coefficients of Examples 3-5 were significantly lower than those of the blank group, indicating that the dynamic nitrogen source sustained-release particles precisely regulated the reaction kinetics of lactic acid and sodium nitrite through the confinement effect of zeolite channels, thereby achieving a continuous and stable release of nitrogen source. The blank group, lacking a sustained-release design, experienced greater fluctuations in nitrogen source supply, and its protein expression level was only 60%-70% of that of the examples. The nitrosamine accumulation in Examples 3-5 was all below the detection limit (<5 μg / L), while although no nitrosamines were detected in the blank group, the ammonia nitrogen fluctuation coefficient was higher, indicating that the dynamic sustained-release particles effectively inhibited... While generating nitrosamines, the problem of localized severe reactions that may be caused by the traditional direct addition of sodium nitrite is effectively avoided; the dissolved oxygen uniformity of Examples 3-5 is better than that of the blank group, proving that the slow-release particles reduce the sudden release of nitrogen and avoid the interference of bubbles on the dissolved oxygen transfer process, among which Example 5 has the best effect; the protein expression levels of Examples 3-5 are 44%-67% higher than those of the blank group, confirming the key role of stable nitrogen source supply in the efficient expression of recombinant proteins; the protein expression level of Example 5 reaches its peak due to the optimization of culture medium concentration and synergistic effect with slow-release particles.

[0036] <Comparative Example 1> The Pichia pastoris culture medium differs from that in Example 3 in that dynamic slow-release particles are not added to the fermentation medium; instead, 0.95 g / L sodium nitrite is added directly. The remaining components and parameters are the same as in Example 3.

[0037] The fermentation method of Pichia pastoris differs from that of Example 3 in that the inducer is replaced by a mixture of methanol and glycerol in a volume ratio of 1:1, while the remaining steps are the same as in Example 3.

[0038] <Comparative Example 2> The Pichia pastoris culture medium differs from that in Example 3 in that the dynamically released particles are replaced with inducer microcapsules prepared in the prior art (CN115747087A). The specific preparation method is as follows: Add sodium alginate solution (8:2:1:12 by volume), sodium carboxymethyl cellulose, and fermentation medium (based on the total volume of the medium, the fermentation medium includes the following components: 15 g / L corn cob hydrolysate, 0.5 g / L yeast extract, 4 mL / L trace element solution (biotin 0.1 g / L, cobalt chloride hexahydrate 0.05 g / L, ferrous sulfate heptahydrate 45 g / L and copper sulfate pentahydrate 3 g / L), 10 g / L agar, 1 g / L polyol (a mixture of sorbitol and pentaerythritol in a 2:1 weight ratio), 7 g / L methanol, 0.03 g / L lactic acid, 0.01 g / L sodium nitrite, and 0 g / L diamine citrate). 0.4 g / L of an amino acid mixture (glutamic acid, L-cysteine, glycine, asparagine, and tyrosine in a weight ratio of 1:0.5:0.6:1:0.3) and 0.02 g / L of deionized water were mixed evenly to obtain a mixture. The solvent used for the fermentation medium and the trace element solution was weakly acidic deionized water with a pH of 6-7 and a redox potential of 200 mA. The mixture was pipetted into anhydrous calcium chloride solution drop by drop. After standing for 1 hour, the residue was filtered and collected. The residue was washed three times with deionized water to obtain microcapsules. The microcapsules were dispersed in methanol (0.5 g microcapsules / L methanol) to obtain a suspension, which was then mixed with glycerol at a volume ratio of 1:1. The remaining components and parameters are the same as in Example 3.

[0039] The fermentation method of Pichia pastoris is the same as in Example 3.

[0040] <Comparative Example 3> The Pichia pastoris culture medium differs from that in Example 3 in that the addition of lactic acid is omitted, and the dynamic slow-release particles are replaced with urea loaded with an equal amount of nitrogen. The remaining components and parameters are the same as in Example 3.

[0041] The fermentation method of Pichia pastoris is the same as in Example 3.

[0042] <Comparative Example 4> The Pichia pastoris culture medium differs from that in Example 3 in that the dynamic sustained-release particles were prepared in Example 1, while the other components and parameters are the same as in Example 3.

[0043] The fermentation method of Pichia pastoris uses the Pichia pastoris culture medium of Comparative Example 4 as the basal solution of the inducer, but removes methanol and polyols. The remaining steps are the same as in Example 3.

[0044] <Performance Test 2> The ammonia nitrogen fluctuation coefficient (%), nitrosamine accumulation (μg / L), dissolved oxygen uniformity (%), and protein expression level (g / L) of Pichia pastoris in Comparative Examples 1-4 of this invention were detected and compared with those in Example 3 and the blank group. The results are shown in Table 2 below: Table 2 Performance tests of Pichia pastoris in Comparative Examples 1-4, Example 3, and the blank group According to the data in Table 2, compared with Example 3, Comparative Example 1 (direct addition of sodium nitrite) showed significantly increased ammonia nitrogen fluctuation coefficient and nitrosamine concentration, significantly worsened dissolved oxygen uniformity, and significantly decreased protein expression, directly confirming the defect of "uncontrollable reaction rate leading to localized severe reaction" mentioned in the background art. Compared with Example 3, Comparative Example 2 (using existing microencapsulation technology) still showed significantly increased ammonia nitrogen fluctuation and nitrosamine concentration, confirming that traditional carriers cannot achieve synergistic regulation of lactic acid permeation and degradation rate, resulting in initial burst release and insufficient supply in the later stage. Although no nitrosamine was detected in Comparative Example 3 (urea replacement), ammonia nitrogen fluctuation and protein expression were still significantly lower than in Example 3, indicating that the urea system could not reproduce the controllable reaction kinetics of lactic acid-sodium nitrite in the zeolite confined space. Comparative Example 4 (without sodium carboxymethyl cellulose coating) showed higher ammonia nitrogen fluctuation than Example 3, and protein expression decreased by 6%, indicating that the surface coating layer can effectively enhance the stability of particle structure, prevent leakage of active substances under high osmotic pressure environment, and thus optimize the stability of nitrogen release curve.

[0045] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A Pichia pastoris fermentation medium, characterized in that, The fermentation medium, by total volume, comprises the following components: The mixture consists of 16-20 g / L corn cob hydrolysate, 0.6-0.9 g / L yeast extract, 4.5-5.5 mL / L trace element solution, 1.5-2.5 g / L polyol, 7.5-8.5 g / L methanol, 0.04-0.07 g / L lactic acid, 0.45-0.55 g / L diamine citrate, 0.025-0.035 g / L amino acid mixture, and 0.95-1.05 g / L dynamic nitrogen source sustained-release particles. The dynamic nitrogen source sustained-release particles are composed of zeolite with a pore size of 0.48-0.52 nm and sodium nitrite loaded in the pores of the zeolite. The loading of sodium nitrite is 25-27% of the mass of zeolite, and the mass ratio of lactic acid to sodium nitrite loaded in zeolite is 1:1.8-2.

2.

2. The Pichia pastoris fermentation medium as described in claim 1, characterized in that, The preparation method of dynamic nitrogen source sustained-release particles includes the following steps: Zeolite with a pore size of 0.48~0.52nm was immersed in a sodium nitrite aqueous solution with a mass concentration of 6.5~7.5% for 25~28 minutes under a vacuum of -0.09 to -0.095MPa. After impregnation, filter the residue and dry it in two stages: first at 45~48℃ for 30~40 minutes, and then at 52~55℃ for 20~25 minutes. The dried granules were placed in a muffle furnace and heated to 280-300°C at a rate of 3-5°C / min. After holding at this temperature for 8-10 min, the granules were cooled to room temperature. After cooling, the surface of the particles is sprayed with alumina sol with a coating thickness of 0.5~0.8μm and cured at 110~115℃ for 15~20min. Then, the particles are vacuum sealed. The vacuum-sealed particles are sterilized by steam at 110~115℃ for 8~12min before use.

3. The Pichia pastoris fermentation medium as described in claim 2, characterized in that, The surface of the dynamic nitrogen source slow-release particles is coated with a sodium carboxymethyl cellulose layer with a thickness of 0.5~0.8μm and a degree of substitution of 0.65~0.

70. The sodium carboxymethyl cellulose layer is formed by the following method: the dynamic nitrogen source slow-release particles obtained by steam sterilization are immersed in an aqueous solution of sodium carboxymethyl cellulose with a mass concentration of 2.5~3.5% for 5~8 minutes, and then cured at 110~115℃ for 15~20 minutes.

4. The Pichia pastoris fermentation medium as described in claim 1, characterized in that, The amino acid mixture contains glutamic acid, L-methionine, glycine, asparagine and tyrosine. The molar ratio of each amino acid is glutamic acid:L-methionine:glycine:asparagine:tyrosine = 1:0.5:0.6:1:0.3, and L-methionine accounts for 30 to 35% of the total mass of the amino acid mixture.

5. The Pichia pastoris fermentation medium as described in claim 1, characterized in that, The trace element solution includes 0.1~0.3 g / L biotin, 0.05~0.2 g / L cobalt chloride hexahydrate, 35~40 g / L ferric chloride, and 1.5~2.5 g / L cuprous oxide; The solvents used for the fermentation medium and trace element solution are weakly acidic ionized water with a redox potential of -150 to -200 mV and a pH of 5.0 to 5.

5. The polyol is one or more of xylitol, sorbitol, ethylene glycol or pentaerythritol.

6. A method for fermenting Pichia pastoris using the fermentation medium according to any one of claims 1 to 5, characterized in that, Includes the following steps: S1. Prepare Pichia pastoris seed culture. Inoculate the Pichia pastoris seed culture into a fermenter containing Pichia pastoris fermentation medium at an inoculation rate of 8-10% for initial culture. During the initial culture, maintain the dissolved oxygen in the fermenter above 40%. S2. When the dissolved oxygen in the fermenter is greater than 75%, add 50v / v% glycerol solution to the fermenter at a rate of 10mL / (L·h). When the bacterial OD... 600 When the concentration exceeds 180, stop adding glycerol solution. During the process of adding glycerol solution, maintain the dissolved oxygen in the fermenter at 23-27%. S3. One hour after the addition of glycerol solution, add the inducing agent to the fermenter for induction culture. Take samples every 2-4 hours for testing. When the increase in expression level is less than 20 mg / (L·h), stop fermentation. During the induction culture, control the dissolved oxygen in the fermenter to 10-50%. The preparation method of the inducer is as follows: A1. Mix sodium alginate solution, sodium carboxymethyl cellulose, culture medium base solution and deionized water in a volume ratio of 8:2:1:12 to obtain the first mixture. The culture medium base solution consists of the components of the fermentation medium except for methanol and polyol. A2. Mix lactic acid and sodium alginate solution at a mass ratio of 1:3~5 to obtain a gel precursor solution containing lactic acid; A3. Using a pipette, the first mixture in A1 is added dropwise to anhydrous calcium chloride solution to form primary microcapsules containing dynamic nitrogen source sustained-release particles. After filtration and washing, the primary microcapsules are immersed in the gel precursor solution of A2, and calcium chloride solution is added dropwise a second time to form double-layer microcapsules. A4. Disperse the bilayer microcapsules obtained in A3 in methanol to obtain a suspension. Mix the suspension with glycerol at a volume ratio of 1:0.1~2 to obtain the inducer. The suspension contains 0.5 g of bilayer microcapsules per liter of methanol.

7. The fermentation method as described in claim 6, characterized in that, During the induction culture, a fortifying solution is continuously fed into the fermenter at a rate of 0.8–1.2 mL / (L·h), and the dissolved oxygen is maintained at 10–15% during the feeding. The fortifying solution consists of 0.5–1.5 g / L lactic acid and 2.0–3.0 g / L sodium carboxymethyl cellulose.

8. The fermentation method as described in claim 6, characterized in that, During the induction culture in step S3, a segmented temperature control strategy was adopted: For 0-6 hours after induction begins, maintain the fermenter temperature at 28-30℃. After 6 hours of induction until the end of fermentation, raise the temperature to 33-35℃.

9. The fermentation method as described in claim 6, characterized in that, The bilayer microcapsules formed in step A3 further include a pH buffer layer: Before the primary microcapsules are immersed in the gel precursor solution, they are first sprayed with a 0.1-0.3 mol / L disodium hydrogen phosphate-citric acid buffer solution to form a buffer coating with a thickness of 1-2 μm. The ionic strength of the buffer coating is 0.15-0.25 M, and its pH value is 0.3-0.5 units lower than that of the fermentation medium.

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