Pichia pastoris fermentation medium for expressing bovine lactoferrin and fermentation method

Abstract

The invention provides a pichia pastoris fermentation medium for expressing bovine lactoferrin and a fermentation method, and mainly relates to the technical field of biochemistry, each liter of the medium comprises the following components: 2-6 mmol / L of farnesol, 8-12 g / L of a yeast extract, 2-6 ml / L of a PTM1 solution, 0.8-1.2 g / L of calcium sulfate, 17-19 g / L of potassium sulfate, 6-8 g / L of magnesium sulfate, 3.5-4.5 g / L of potassium hydroxide, 25-30 g / L of a phosphoric acid aqueous solution, 30-35 g / L of glycerin and 0.5-1.5 ml / L of a defoaming agent, and the balance of water. The culture medium is obtained under the synergistic effect of the components and concentration of the culture medium, so that the subsequent pichia pastoris can be better fermented, the bovine lactoferrin can be expressed in a better growth environment, the activity and the stability of the bovine lactoferrin are improved, the quality of a final bovine lactoferrin product is further improved, and the content of the bovine lactoferrin can reach 6.3 g / L.

Description

Technical Field

[0001] The invention mainly relates to the technical field of biochemistry, and in particular to a Pichia yeast fermentation medium for expressing bovine lactoferrin and a fermentation method. Background Art

[0002] At present, bovine lactoferrin is mainly obtained by extracting from bovine milk, and the commonly used methods include acid precipitation, centrifugation and membrane separation, etc. Although these methods can effectively extract bovine lactoferrin, they have problems such as high cost, complex process and low yield, which limits their application in industry.

[0003] With the development of gene editing technology, bovine lactoferrin can now be expressed from a variety of genetically engineered bacteria. As a eukaryotic expression system, Pichia pastoris has been widely used in the expression of exogenous proteins in recent years. Studies have shown that Pichia pastoris can effectively express a variety of recombinant proteins under high-density fermentation conditions. However, existing fermentation processes are usually not fully optimized for bovine lactoferrin, resulting in yield and activity that fail to meet expectations. Summary of the invention

[0004] In order to solve the technical problems in the prior art, the first aspect of the present invention provides a Pichia yeast fermentation medium for expressing bovine lactoferrin, wherein the medium is composed of the following per liter: 2-6 mmol / L of farnesol, 8-12 g / L of yeast extract, 2-6 ml / L of PTM1 solution, 0.8-1.2 g / L of calcium sulfate, 17-19 g / L of potassium sulfate, 6-8 g / L of magnesium sulfate, 3.5-4.5 g / L of potassium hydroxide, 25-30 g / L of phosphoric acid aqueous solution, 30-35 g / L of glycerol and 0.5-1.5 ml / L of defoaming agent, and the balance is water.

[0005] Compared with the prior art, firstly, the Pichia yeast fermentation medium of the present invention contains yeast extract, which contains various nutrients required for the growth of Pichia yeast and is easily directly utilized by Pichia yeast, and has an obvious effect in the bacterial cell amplification stage; secondly, the medium also contains farnesol, and the synergistic effect of farnesol and yeast extract can have a significant effect on the hyphae morphology and biological characteristics of Pichia yeast; finally, the medium obtained under the synergistic effect of various components and concentrations of the above-mentioned medium enables the subsequent Pichia yeast to ferment better, express bovine lactoferrin in a more optimal growth environment, improve its activity and stability, and thus improve the quality of the final bovine lactoferrin product, and the bovine lactoferrin content can be as high as 6.3g / L.

[0006] Furthermore, it also includes a feed medium, and the composition of the feed medium per liter is as follows: 400-600 g / L of glycerol, 10-14 ml / L of PTM1 solution, and the balance is water.

[0007] Furthermore, it also includes an induction-feed medium, and the composition of the induction-feed medium per liter is as follows: 900-1100 g / L methanol, 10-14 ml / L PTM1 solution, 2-6 mmol / L farnesol, 2-4 g / L glutamic acid, and the balance is water.

[0008] Compared with the prior art, in the above technical solution, farnesol and glutamic acid are added to the induction-feed medium, and the content of each component is limited. The present invention introduces farnesol and glutamic acid as growth regulators into the fermentation process of bovine lactoferrin, and by adding glutamic acid and farnesol and limiting the concentration of the above components, the induction-medium thus obtained further optimizes the fermentation conditions of bovine lactoferrin, promotes the growth rate and protein yield of cells, and significantly improves the yield and quality of the obtained bovine lactoferrin.

[0009] Furthermore, the concentration of the phosphoric acid aqueous solution is 80-90wt%; and the defoaming agent is an organosilicon or polyether defoaming agent.

[0010] The second aspect of the present invention provides a Pichia yeast fermentation method for expressing bovine lactoferrin, using the above-mentioned culture medium, comprising the following steps:

[0011] S1: inoculating the working seeds of Pichia pastoris expressing bovine lactoferrin into the preculture medium and culturing to obtain seed liquid;

[0012] S2: aseptically introducing the seed liquid into a fermentation tank containing a Pichia yeast fermentation medium, and after the initial ventilation ratio, initial stirring speed, and initial relative tank pressure are stable, correcting the dissolved oxygen DO to 100%, and starting fermentation, controlling the dissolved oxygen DO to 30-50%, controlling the pH to 5.5-6.5, and the temperature to 26-35° C. during the process;

[0013] S3: During the fermentation process, feed medium was added to the culture medium regularly to maintain bacterial growth and the expression of bovine lactoferrin. 600 When the value reaches 200-250, stop feeding and starve for 0.5-1h;

[0014] S4: After the starvation culture, the induction-feed medium is added, and the induction time is 24-36 hours;

[0015] After the fermentation, the cells were collected by centrifugation to measure the bovine lactoferrin content.

[0016] Compared with the prior art, the preparation method of the present invention uses the above-mentioned Pichia yeast fermentation medium, feed medium, and induction-feed medium during the fermentation process, controls the dissolved oxygen content DO, pH, temperature and other parameter conditions during the fermentation process, and realizes high-density fermentation through genetic engineering technology, which not only enables Pichia yeast to express bovine lactoferrin in a more optimal growth environment, improves its activity and stability, and improves the quality and yield of the final product, but also reduces the cumbersome steps of extracting bovine lactoferrin from bovine milk to meet the needs of industrial production, reduces the cost of raw materials and operating expenses, thereby making the production of bovine lactoferrin more economically feasible, and also reduces pollution to the environment, and promotes green production.

[0017] Furthermore, in the step S1, the bovine lactoferrin is expressed intracellularly in the Pichia pastoris, and the copy number of the bovine lactoferrin gene in the genome of the Pichia pastoris is 3-8.

[0018] Furthermore, in step S1, the pre-culture medium is BMGY culture medium or BMMY culture medium or YPG culture medium; the culture temperature when culturing the seed solution is 26-35°C, the stirring speed is 200-300rpm, and the culture period is 24-36h; the OD of the seed solution is 600 Is 15-20.

[0019] Furthermore, in step S2, the volume of the Pichia yeast fermentation medium in the fermenter accounts for 60-80% of the volume of the fermenter, and the volume of the seed liquid is 8-10% of the volume of the Pichia yeast fermentation medium in the fermenter; the initial ventilation volume is 2-4 L / min, the initial stirring speed is 200-400 rpm, and the initial relative tank pressure is 0.03-0.06 MPa.

[0020] Furthermore, in step S3, the rate of adding the feed medium is 5-20 g / L per hour based on the volume of the Pichia yeast fermentation medium in the fermenter; the condition for adding the feed medium to the culture medium at regular intervals is: when the pH reaches 7.5-9.0 or the dissolved oxygen DO reaches 55%-70%, start feeding; adjust the feed amount according to the dissolved oxygen DO value in the fermentation liquid, so that the dissolved oxygen DO is controlled at 20-40%, and the pH of the fermentation liquid is controlled between 5.5-6.5.

[0021] Furthermore, in step S4, the rate of addition of the induction-feed medium is 1-20 g / L per hour, based on the volume of the Pichia yeast fermentation medium in the fermenter; during the induction period, the pH is controlled at 6.0±0.2, the temperature is controlled at 22±0.5°C, the dissolved oxygen content is controlled at 10-30%, and the tank pressure is controlled at 0.03-0.06 MPa.

[0022] Furthermore, in the steps S2 to S4, the stirring speed is 200-1000 rpm, and the relative tank pressure is 0.02-1 MPa; the dissolved oxygen content in the steps S2 to S4 is controlled by the stirring speed and the ventilation volume, when the speed is 350-450 rpm, the ventilation volume is adjusted to 2.8-3.2 L / min; when the speed is 450-550 rpm, the ventilation volume is adjusted to 3.3-3.7 L / min; when the speed is 550-650 rpm, the ventilation volume is adjusted to 3.8-4.2 L / min; when the speed is 650-750 rpm, the ventilation volume is adjusted to 4.3-4.7 L / min; when the speed is 750-850 rpm; the ventilation volume is adjusted to 4.8-5.2 L / min. DETAILED DESCRIPTION

[0023] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0024] In a first aspect, an embodiment of the present invention provides a Pichia yeast fermentation medium for expressing bovine lactoferrin, wherein each liter of the medium is composed of: 2-6 mmol / L of farnesol, 8-12 g / L of yeast extract, 2-6 ml / L of PTM1 solution, 0.8-1.2 g / L of calcium sulfate, 17-19 g / L of potassium sulfate, 6-8 g / L of magnesium sulfate, 3.5-4.5 g / L of potassium hydroxide, 25-30 g / L of aqueous phosphoric acid solution, 30-35 g / L of glycerol and 0.5-1.5 ml / L of defoaming agent, and the balance is water.

[0025] For example, in the fermentation medium of Pichia yeast expressing bovine lactoferrin, the concentration of farnesol can be 2mmol / L, 3.0mmol / L, 3.6mmol / L, 3.7mmol / L, 3.8mmol / L, 3.9mmol / L, 4.0mmol / L, 4.1mmol / L, 4.2mmol / L, 4.3mmol / L, 4.4mmol / L, 4.5mmol / L, 5.0mmol / L, and 6.0mmol / L. The preferred concentration of farnesol is 3.8-4.2 mmol / L; the concentration of yeast extract is 8 g / L, 8.5 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, and the preferred concentration of yeast extract is 9-11 g / L; the concentration of PTM1 solution is 2 ml / L, 2.5 ml / L, 3.0 ml / L, 4 ml / L, 4.5 ml / L, 5 ml / L, 6 ml / L; the concentration of calcium sulfate is 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.05 g / L, 1.1 g / L, 1.2 g / L; the concentration of potassium sulfate is 1 The concentration of magnesium sulfate can be specifically 6g / L, 7g / L, 7.5g / L, 8g / L; potassium hydroxide 3.5g / L, 3.8g / L, 4.0g / L, 4.2g / L, 4.5g / L; the concentration of phosphoric acid aqueous solution can be specifically 25g / L, 26g / L, 27g / L, 29g / L, 30g / L; the concentration of glycerol can be specifically 30g / L, 31g / L, 32g / L, 34g / L, 35g / L; the concentration of defoaming agent can be specifically 0.5ml / L, 0.8ml / L, 1.0ml / L, 1.2ml / L, 1.5ml / L.

[0026] Experimental verification shows that when the optimal concentration of farnesol is 3.8-4.2 mmol / L and the optimal concentration of yeast extract is 9-11 g / L, the two can work synergistically and together with other components form a culture medium to achieve the best culture results for Pichia yeast fermentation.

[0027] It should be noted that the main components of the conventional BSM basic inorganic salt medium aqueous solution are: phosphoric acid aqueous solution, calcium sulfate, potassium sulfate, magnesium sulfate, potassium hydroxide, glycerol, and PTM1 solution. In addition to the above components, the Pichia yeast fermentation medium of the present invention also adds yeast extract, farnesol, and defoamer. Among them, the yeast extract contains various nutrients required for the growth of Pichia yeast, and is easily directly utilized by Pichia yeast, and has an obvious effect in the bacterial amplification stage; farnesol has anti-metabolic, antioxidant and antibacterial effects, and has a significant effect on the mycelial morphology and biological characteristics of Pichia yeast; the defoamer prevents the fermentation liquid from foaming, ensures the oxygen transfer efficiency, and the transfer and uniformity of the matrix. The Pichia yeast fermentation medium expressing bovine lactoferrin of the present invention optimizes the composition and content of the medium, and under the synergistic effect of each component and content, the subsequent Pichia yeast can be better fermented, and bovine lactoferrin is expressed in a more optimal growth environment, which improves its activity and stability, thereby improving the quality of the final product, and the bovine lactoferrin content can reach 6.3g / L.

[0028] In some embodiments, a feed medium is also included, and the composition of each liter of the feed medium is as follows: 400-600 g / L of glycerol, 10-14 ml / L of PTM1 solution, and the balance is water.

[0029] For example, in the feed medium, the concentration of glycerol can be 400 g / L, 420 g / L, 480 g / L, 500 g / L, 550 g / L, 570 g / L, and 600 g / L; the concentration of the PTM1 solution can be 10 ml / L, 11 ml / L, 12 ml / L, 12.5 ml / L, 13 ml / L, and 14 ml / L.

[0030] In some embodiments, an induction-feed medium is also included, and the composition of each liter of induction-feed medium is as follows: 900-1100 g / L methanol, 10-14 ml / L PTM1 solution, 2-6 mmol / L farnesol, 2-4 g / L glutamate, and the balance is water.

[0031] For example, the concentration of methanol in the induction-feed medium can be 900 g / L, 930 g / L, 1000 g / L, 1050 g / L, 1080 g / L, and 1100 g / L; the concentration of PTM1 solution can be 10 ml / L, 11 ml / L, 12 ml / L, 12.5 ml / L, 13 ml / L, and 14 ml / L; the concentration of farnesol can be 2 mmol / L, 3 mmol / L, 3.5 mmol / L, 4 mmol / L, 5 mmol / L, and 6 mmol / L; the concentration of glutamate can be 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, and 4 g / L.

[0032] The induction-feed medium of the present invention comprises farnesol and glutamic acid, and the content of each component is limited. Farnesol has a broad spectrum of antibacterial activity, can inhibit the growth of many Gram-positive and Gram-negative bacteria, help control bacterial contamination, can induce cell differentiation, has antioxidant properties, protects cells from damage in cell culture, which helps to maintain the healthy state of cells and prolong the survival time of cells; Glutamic acid can promote cell growth and division, provide energy for cells, partially neutralize the acid-base changes in the culture medium, maintain a stable pH environment, and is conducive to cell growth. The present invention introduces farnesol and glutamic acid as growth regulators into the fermentation process of bovine lactoferrin, selects a suitable concentration in conjunction with other components, obtains the induction-medium of the present invention, optimizes the fermentation conditions of bovine lactoferrin, promotes the growth rate and protein yield of cells, and significantly improves the yield and quality of the obtained bovine lactoferrin.

[0033] In some embodiments, the concentration of the phosphoric acid aqueous solution is 80-90wt%; the defoamer is a silicone or polyether defoamer. For example, the concentration of the phosphoric acid aqueous solution can be 80wt%, 82wt%, 85wt%, 88wt%, 90wt%. For example, the silicone defoamer can be SXP-101 fermentation defoamer, XJH-37G defoamer, and the polyether defoamer can be DF-803 polyether defoamer, HR polyether defoamer.

[0034] The second aspect of the present invention provides a Pichia yeast fermentation method for expressing bovine lactoferrin, using the above-mentioned culture medium, comprising the following steps:

[0035] S1: inoculating the working seeds of Pichia pastoris expressing bovine lactoferrin into the preculture medium and culturing to obtain seed liquid;

[0036] S2: Aseptically transfer the seed liquid into a fermentation tank containing Pichia yeast fermentation medium, and after the initial ventilation ratio, initial stirring speed, and initial relative tank pressure are stable, calibrate the dissolved oxygen DO to 100%, and start fermentation. During the process, the dissolved oxygen DO is controlled to be 30-50%, the pH value is controlled to be between 5.5-6.5, and the temperature is controlled to be 26-35°C;

[0037] S3: During the fermentation process, feed medium was added to the culture medium regularly to maintain bacterial growth and the expression of bovine lactoferrin. 600 When the value reaches 200-250, stop feeding and starve for 0.5-1h;

[0038] S4: After the starvation culture, the induction-feed medium is added, and the induction time is 24-36 hours;

[0039] After the fermentation, the cells were collected by centrifugation to measure the bovine lactoferrin content.

[0040] The preparation method of the present invention uses the above-mentioned Pichia yeast fermentation medium, feed medium, and induction-feed medium during the fermentation process, controls the dissolved oxygen content DO, pH, temperature and other parameter conditions at different stages during the fermentation process, realizes high-density fermentation through genetic engineering technology, replaces the traditional chemical synthesis method, reduces pollution to the environment, promotes green production, reduces the cumbersome steps of extracting bovine lactoferrin from cow's milk, and promotes industrial production; and most of the raw materials used in the fermentation process are inorganic salts, which reduces the cost of raw materials and operating expenses, thereby making the production of bovine lactoferrin more economically feasible. Using the preparation method of the present invention, Pichia yeast expresses bovine lactoferrin in a more optimal growth environment, improves its activity and stability, and improves the quality of the final product.

[0041] In some embodiments, in step S1, bovine lactoferrin is expressed intracellularly in Pichia pastoris, and the copy number of the bovine lactoferrin gene in the Pichia pastoris genome is 3-8, specifically 3, 4, 6, 7, or 8.

[0042] In some embodiments, in step S1, the preculture medium is BMGY medium or BMMY medium or YPG medium; the culture temperature when culturing the seed solution is 26-35°C, the stirring speed is 200-300 rpm, and the culture period is 24-36h; the OD of the seed solution is 600 When the value reaches 15-20, the seed liquid culture is ended.

[0043] For example, the composition of each liter of BMGY medium may be: 18-22 g / L peptone, 8-12 g / L yeast extract powder, 90-110 mmol / L phosphate buffer with a pH of 5.8-6.2, 1.25-1.35% yeast nitrogen base (YNB) by volume, 0.00004% biotin by volume, 8-12% glycerol by volume, and the balance is water;

[0044] The composition of each liter of BMMY medium can be specifically: 18-22g / L peptone, 8-12g / L yeast extract, 2.5-3.5g / L K 2 HPO 4 11-12g / L KH 2 PO 4 , 90-110 mL / L of 12-14% YNB solution, 1.5-2.5 mL / L of 0.02% biotin solution, 80-110 mL / L of 4-6% methanol aqueous solution, and the balance is water;

[0045] The composition of each liter of YPG medium may specifically be: 8-12 g / L yeast extract, 18-22 g / L peptone, 18-22 g / L galactose, and the remainder is water.

[0046] For example, the culture temperature when culturing the seed solution can be 26°C, 27°C, 30°C, 32°C, 34°C, or 35°C; the stirring speed can be 200rpm, 210rpm, 230rpm, 250rpm, 280rpm, 295rpm, or 300rpm; the culture period can be 24h, 27h, 28h, 30h, 33h, or 36h; the OD of the seed solution can be 600 , specifically it can be 15, 16, 18, 19, 20.

[0047] The Pichia pastoris expressing bovine lactoferrin is inoculated in the above-mentioned preculture medium, and the above-mentioned temperature, stirring speed and culture cycle are combined to maintain the stability of the intracellular environment, ensure the full growth of the bacteria and the accumulation of metabolites, promote the growth of the bacteria, and enable the Pichia pastoris expressing bovine lactoferrin to grow and reproduce well in the preculture medium.

[0048] In some embodiments, in step S2, the volume of the Pichia yeast fermentation medium in the fermenter accounts for 60-80% of the volume of the fermenter, and the volume of the seed liquid is 8-10% of the volume of the Pichia yeast fermentation medium in the fermenter. The initial ventilation rate is 2-4L / min, the initial stirring speed is 200-400rpm, and the initial relative tank pressure is 0.03-0.06MPa. After the initial ventilation ratio, initial stirring speed, and initial relative tank pressure reach the above-mentioned preset values ​​and stabilize, the dissolved oxygen DO value is corrected to 100%, and fermentation begins.

[0049] The volume proportion of the Pichia yeast fermentation medium in the fermentation tank, the volume proportion of the seed liquid in the Pichia yeast fermentation medium, and the settings of the initial ventilation volume, initial stirring speed, and initial relative tank pressure provide good space and nutrients for the growth and reproduction of Pichia yeast, and provide a basis for the growth and reproduction of Pichia yeast.

[0050] In some embodiments, in step S3, the feed medium is added at a rate of 5-20 g / L per hour based on the volume of the Pichia yeast fermentation medium in the fermenter;

[0051] The conditions for adding feed medium to the culture medium at regular intervals are: when the pH reaches 7.5-9.0 or the dissolved oxygen DO reaches 55%-70%, start feeding; adjust the feed amount according to the dissolved oxygen DO value in the fermentation liquid, so that the dissolved oxygen DO is controlled at 20-40%, and the pH of the fermentation liquid is controlled between 5.5-6.5.

[0052] As the yeast grows and reproduces, it will continuously consume the nutrients and oxygen in the fermentation liquid. When the bacteria grow to a certain amount, the nutrients are consumed in large quantities, the growth rate of the bacteria slows down, and the consumption of dissolved oxygen decreases. When the pH of the fermentation liquid reaches 7.5-9.0 or the dissolved oxygen DO value reaches 55%-70%, the activity and reproduction capacity of the bacteria are still maintained at a good level. Feed medium is added to provide a nutritional basis for bacterial reproduction, and the dissolved oxygen DO value of the fermentation liquid is controlled between 20-40%, and the pH of the fermentation liquid is controlled between 5.5-6.5, so that the yeast continues to grow and reproduce well and improves the synthesis efficiency of the fermentation product. When the OD value of the fermentation liquid reaches 7.5-9.0 or the dissolved oxygen DO value reaches 55%-70%, the activity and reproduction capacity of the bacteria are still maintained at a good level. Feed medium is added to provide a nutritional basis for bacterial reproduction, and the dissolved oxygen DO value of the fermentation liquid is controlled between 20-40%, and the pH of the fermentation liquid is controlled between 5.5-6.5. 600 When the value reaches 200-250, the yeast metabolites have reached a certain accumulation, and feeding is stopped to avoid excessive accumulation of metabolites and feedback inhibition, which is beneficial to product separation and purification; starvation for 0.5-1h is beneficial to cell cycle synchronization, reducing the generation of cell impurities and improving the purity of the final bovine lactoferrin.

[0053] In some embodiments, in step S4, the rate of adding the induction-feed medium is 1-20 g / L per hour based on the volume of the Pichia fermentation medium in the fermenter;

[0054] During the induction period, the pH was controlled at 6.0±0.2, the temperature was controlled at 22±0.5°C, the dissolved oxygen content was controlled at 10-30%, and the tank pressure was controlled at 0.03-0.06MPa.

[0055] In the above technical scheme, by controlling the pH, temperature, dissolved oxygen content, tank pressure of the fermentation broth and the rate of addition of the induction-feed medium, the Pichia pastoris in the fermentation broth maintains good activity, the Pichia pastoris is induced to express bovine lactoferrin, and the prepared bovine lactoferrin maintains good activity and yield.

[0056] In some embodiments, in steps S2 to S4, the stirring speed is 200-1000 rpm, and the relative tank pressure is 0.02-1 MPa;

[0057] The dissolved oxygen content in step S2-step S4 is controlled by the stirring speed and the ventilation volume. When the rotation speed is 350-450 rpm, the ventilation volume is adjusted to 2.8-3.2 L / min; when the rotation speed is 450-550 rpm, the ventilation volume is adjusted to 3.3-3.7 L / min; when the rotation speed is 550-650 rpm, the ventilation volume is adjusted to 3.8-4.2 L / min; when the rotation speed is 650-750 rpm, the ventilation volume is adjusted to 4.3-4.7 L / min; when the rotation speed is 750-850 rpm, the ventilation volume is adjusted to 4.8-5.2 L / min.

[0058] The present invention will be specifically described below by way of examples. The content of the present invention is not limited to the following examples.

[0059] Example 1

[0060] A Pichia yeast fermentation method for expressing bovine lactoferrin comprises the following steps:

[0061] S1: The working seeds of Pichia pastoris expressing bovine lactoferrin were inoculated into BMGY pre-culture medium and cultured at 26°C and 250 rpm for 30 h to obtain seed solution. The OD of the seed solution was 600 is 16.

[0062] S2: Aseptically transfer the seed liquid into a fermentation tank containing Pichia yeast fermentation medium. After the initial ventilation volume, initial stirring speed and initial relative tank pressure are stable, calibrate the dissolved oxygen DO to 100% and start fermentation. Control the dissolved oxygen content during the process by stirring speed and ventilation volume. The dissolved oxygen DO is 35%, and the process pH is controlled to 5.7 and the temperature is 28°C.

[0063] The specific components and their proportions in the aqueous solution of Pichia yeast fermentation medium are: 4mmol / L farnesol, 8g / L yeast extract, 4ml / L PTM1 solution, 1.2g / L calcium sulfate, 19g / L potassium sulfate, 6g / L magnesium sulfate, 3.8g / L potassium hydroxide, 29g / L phosphoric acid aqueous solution, 30g / L glycerol and 0.5ml / L HR polyether defoamer, and the balance is water. The method for preparing the aqueous solution of Pichia yeast fermentation medium is: weigh or measure the yeast extract, potassium phosphate salt and other raw materials according to the formula, add appropriate amount of distilled water, heat and stir evenly until completely dissolved; then adjust the pH value with an acid-base regulator, transfer to a volumetric flask to a fixed volume; finally sterilize, and after sterilization, seal and place in a 4°C refrigerator for standby use.

[0064] The volume of the Pichia yeast fermentation medium in the fermentation tank accounts for 60% of the volume of the fermentation tank, and the volume of the seed liquid is 8% of the volume of the Pichia yeast fermentation medium in the fermentation tank. The initial ventilation volume is controlled to be 3L / min, the initial stirring speed is 250rpm, and the initial relative tank pressure is 0.04MPa; the correction of the dissolved oxygen content DO100% is to define the DO value at that time as DO100%, and then control the dissolved oxygen content in the process to be DO35% through the stirring speed and ventilation volume; sodium hydroxide is used to control the process pH to 5.7 and the temperature to 28°C.

[0065] S3: During the fermentation process, feed medium was regularly added to the culture medium at a rate of 10 g / L per hour to maintain bacterial growth and the expression of bovine lactoferrin.

[0066] When the pH of the fermentation broth reaches 7.5 or the dissolved oxygen DO reaches 60%, start adding feed medium; adjust the feed amount according to the dissolved oxygen curve, and then control the dissolved oxygen DO at 20% by adjusting the stirring speed and ventilation volume; take samples regularly to detect the OD of the fermentation broth600 When the OD value of the fermentation broth 600 When the pH reaches 240, stop feeding and starve for 0.8 h.

[0067] The specific components and their proportions of the feed medium are: 400 g / L glycerol, 12 ml / L PTM1 solution, and the balance is water. The method for preparing the feed medium is: weigh or measure glycerol and PTM1 solution according to the formula, add distilled water to dissolve the glycerol, add PTM1 solution and continue stirring, adjust the pH with acid or alkali, transfer to a volumetric flask to a fixed volume, sterilize at 121°C for 15-20 minutes, and store in a refrigerator at 4°C for future use after sterile cooling.

[0068] S4: When the starvation culture is over, the induction-feed medium is added, and the induction time is 36 hours. During the induction period, the pH is controlled at 6.0±0.2, the temperature is controlled at 22±0.5℃, the dissolved oxygen DO is controlled at 10%, and the tank pressure is controlled at 0.03MPa.

[0069] The specific components and proportions of the induction-feed medium are: methanol 1100 g / L, PTM1 solution 10 ml / L, farnesol 4 mmol / L, glutamic acid 2 g / L, and the balance is water. The method for preparing the induction-feed medium is: weigh or measure methanol, PTM1 solution, farnesol, and glutamic acid according to the formula, wherein the methanol operation must be carried out in a fume hood and with good protection, methanol is added to about 80% of the total volume of water and stirred, and then PTM1 solution, farnesol, and glutamic acid are added in sequence and stirred to dissolve, transferred to a container and fixed to volume with water, and sterilized and stored for later use.

[0070] After the fermentation was completed, the bacteria were collected by centrifugation and the bovine lactoferrin content was measured by high performance liquid chromatography to be 6.3 g / L.

[0071] Example 2

[0072] A Pichia yeast fermentation method for expressing bovine lactoferrin comprises the following steps:

[0073] S1: The Pichia yeast working seeds expressing bovine lactoferrin were inoculated into the BMMY pre-culture medium and cultured at a temperature of 30°C and a stirring speed of 200 rpm for 28 h to obtain the seed solution. The OD of the seed solution was 600 is 18.

[0074] S2: Aseptically transfer the seed liquid into a fermentation tank containing Pichia yeast fermentation medium. After the initial ventilation volume, initial stirring speed and initial relative tank pressure are stable, calibrate the dissolved oxygen DO to 100% and start fermentation. Control the dissolved oxygen content during the process by stirring speed and ventilation volume. The dissolved oxygen DO is 45%, and the process pH is controlled to 5.5 and the temperature is 33°C.

[0075] The specific components and their proportions in the aqueous solution of Pichia yeast fermentation medium are: 3 mmol / L of farnesol, 10 g / L of yeast extract, 3 ml / L of PTM1 solution, 0.9 g / L of calcium sulfate, 18 g / L of potassium sulfate, 7 g / L of magnesium sulfate, 4.2 g / L of potassium hydroxide, 25 g / L of phosphoric acid aqueous solution, 35 g / L of glycerol and 1.5 ml / L of SXP-101 fermentation type defoamer, and the balance is water. The preparation method of the aqueous solution of Pichia yeast fermentation medium is the same as that in Example 1.

[0076] The volume of the Pichia yeast fermentation medium in the fermentation tank accounts for 75% of the volume of the fermentation tank, and the volume of the seed liquid is 9% of the volume of the Pichia yeast fermentation medium in the fermentation tank. The initial ventilation volume is controlled to be 2L / min, the initial stirring speed is 200rpm, and the initial relative tank pressure is 0.05MPa; the correction of the dissolved oxygen DO100% is to define the DO value at that time as DO100%, and then control the dissolved oxygen content in the process to be DO45% through the stirring speed and ventilation volume; sodium hydroxide is used to control the process pH to 5.5 and the temperature to 33°C.

[0077] S3: During the fermentation process, feed medium was regularly added to the culture medium at a rate of 16 g / L per hour to maintain bacterial growth and the expression of bovine lactoferrin.

[0078] When the pH of the fermentation broth reaches 8.0 or the dissolved oxygen DO reaches 70%, start adding feed medium; adjust the feed amount according to the dissolved oxygen curve, and then control the dissolved oxygen DO at 28% by adjusting the stirring speed and ventilation volume; take samples regularly to detect the OD of the fermentation broth 600 When the OD value of the fermentation broth 600 When the pH reaches 200, stop feeding and starve the animals for 0.5 h.

[0079] The specific components and their proportions of the feed medium are: 500 g / L glycerol, 10 ml / L PTM1 solution, and the balance is water. The preparation method of the feed medium is the same as that of Example 1.

[0080] S4: When the starvation culture is over, the induction-feed medium is added, and the induction time is 32 hours. During the induction period, the pH is controlled at 6.0±0.2, the temperature is controlled at 22±0.5℃, the dissolved oxygen DO is controlled at 30%, and the tank pressure is controlled at 0.05MPa.

[0081] The specific components and their proportions of the induction-feed medium are: 900 g / L methanol, 11 ml / L PTM1 solution, 6 mmol / L farnesol, 3 g / L glutamic acid, and the balance is water. The preparation method of the induction-feed medium is the same as that of Example 1.

[0082] After the fermentation was completed, the bacteria were collected by centrifugation and the bovine lactoferrin content was measured by high performance liquid chromatography to be 6.2 g / L.

[0083] Example 3

[0084] A Pichia yeast fermentation method for expressing bovine lactoferrin comprises the following steps:

[0085] S1: The Pichia pastoris working seeds expressing bovine lactoferrin were inoculated into the YPG pre-culture medium and cultured at a temperature of 35°C and a stirring speed of 280 rpm for 24 h to obtain the seed solution. The OD of the seed solution was 600 is 15.

[0086] S2: Aseptically transfer the seed liquid into a fermentation tank containing Pichia yeast fermentation medium. After the initial ventilation volume, initial stirring speed and initial relative tank pressure are stable, calibrate the dissolved oxygen DO to 100% and start fermentation. Control the dissolved oxygen content during the process by stirring speed and ventilation volume. The dissolved oxygen DO is 30%, and the process pH is controlled to 6.5 and the temperature is 35°C.

[0087] The specific components and their proportions in the aqueous solution of Pichia yeast fermentation medium are: 5 mmol / L of farnesol, 8 g / L of yeast extract, 5 ml / L of PTM1 solution, 0.8 g / L of calcium sulfate, 17 g / L of potassium sulfate, 8 g / L of magnesium sulfate, 3.5 g / L of potassium hydroxide, 30 g / L of phosphoric acid aqueous solution, 32 g / L of glycerol and 1.2 ml / L of DF-803 polyether defoamer, and the balance is water. The preparation method of the aqueous solution of Pichia yeast fermentation medium is the same as that in Example 1.

[0088] The volume of the Pichia yeast fermentation medium in the fermentation tank accounts for 70% of the volume of the fermentation tank, and the volume of the seed liquid is 10% of the volume of the Pichia yeast fermentation medium in the fermentation tank. The initial ventilation volume is controlled to be 4L / min, the initial stirring speed is 360rpm, and the initial relative tank pressure is 0.06MPa; the correction of the dissolved oxygen DO100% is to define the DO value at that time as DO100%, and then control the dissolved oxygen content in the process to be DO30% through the stirring speed and ventilation volume; sodium hydroxide is used to control the process pH6.5 and the temperature to be 35°C.

[0089] S3: During the fermentation process, feed medium was regularly added to the culture medium at a rate of 5 g / L per hour to maintain bacterial growth and the expression of bovine lactoferrin.

[0090] When the pH of the fermentation broth reaches 8.4 or the dissolved oxygen DO reaches 67%, start adding feed medium; adjust the feed amount according to the dissolved oxygen curve, and then control the dissolved oxygen DO at 37% by adjusting the stirring speed and ventilation volume; take samples regularly to detect the OD of the fermentation broth 600 When the OD value of the fermentation broth 600 When the temperature reaches 215, stop feeding and starve for 1 hour.

[0091] The specific components and their proportions of the feed medium are: 550 g / L glycerol, 14 ml / L PTM1 solution, and the balance is water. The preparation method of the feed medium is the same as that of Example 1.

[0092] S4 When the starvation culture is over, the induction-feed medium is added, and the induction time is 28 hours. During the induction period, the pH is controlled at 6.0±0.2, the temperature is controlled at 22±0.5℃, the dissolved oxygen DO is controlled at 25%, and the tank pressure is controlled at 0.06MPa.

[0093] The specific components and proportions of the induction-feed medium are: 1000 g / L methanol, 14 ml / L PTM1 solution, 5 mmol / L farnesol, 4 g / L glutamic acid, and the balance is water. The preparation method of the induction-feed medium is the same as that of Example 1.

[0094] After the fermentation was completed, the bacteria were collected by centrifugation and the bovine lactoferrin content was measured by high performance liquid chromatography to be 6.1 g / L.

[0095] Example 4

[0096] A Pichia yeast fermentation method for expressing bovine lactoferrin comprises the following steps:

[0097] S1: The Pichia yeast working seeds expressing bovine lactoferrin were inoculated into the BMMY pre-culture medium and cultured at a temperature of 32°C and a stirring speed of 300 rpm for 36 h to obtain the seed solution. The OD of the seed solution was 600 is 20.

[0098] S2: Aseptically transfer the seed liquid to a fermentation tank containing Pichia yeast fermentation medium. After the initial ventilation volume, initial stirring speed and initial relative tank pressure are stable, calibrate the dissolved oxygen DO to 100% and start fermentation. Control the dissolved oxygen content during the process by stirring speed and ventilation volume. The dissolved oxygen DO is 50%, and the process pH is controlled to 6.0 and the temperature is 26°C.

[0099] The specific components and their proportions in the aqueous solution of Pichia yeast fermentation medium are: 6 mmol / L of farnesol, 9 g / L of yeast extract, 6 ml / L of PTM1 solution, 1.1 g / L of calcium sulfate, 17.6 g / L of potassium sulfate, 7.5 g / L of magnesium sulfate, 4.5 g / L of potassium hydroxide, 27 g / L of phosphoric acid aqueous solution, 34 g / L of glycerol and 0.8 ml / L of XJH-37G defoamer, and the balance is water. The preparation method of the aqueous solution of Pichia yeast fermentation medium is the same as that in Example 1.

[0100] The volume of the Pichia yeast fermentation medium in the fermenter accounts for 80% of the volume of the fermenter, and the volume of the seed liquid is 8.8% of the volume of the Pichia yeast fermentation medium in the fermenter. The initial ventilation volume is controlled to be 3.5L / min, the initial stirring speed is 400rpm, and the initial relative tank pressure is 0.04MPa; the correction of the dissolved oxygen DO100% is to define the DO value at that time as DO100%, and then control the dissolved oxygen content in the process to be DO50% through the stirring speed and ventilation volume; sodium hydroxide is used to control the process pH to 6.0 and the temperature to 26°C.

[0101] S3: During the fermentation process, feed medium was regularly added to the culture medium at a rate of 20 g / L per hour to maintain bacterial growth and the expression of bovine lactoferrin.

[0102] When the pH of the fermentation broth reaches 9.0 or the dissolved oxygen DO reaches 55%, start adding feed medium; adjust the feed amount according to the dissolved oxygen curve, and then control the dissolved oxygen DO at 40% by adjusting the stirring speed and ventilation volume; take samples regularly to detect the OD of the fermentation broth 600 When the OD value of the fermentation broth 600 When the pH reaches 250, stop feeding and starve for 0.6 h.

[0103] The specific components and their proportions of the feed medium are: 600 g / L glycerol, 13 ml / L PTM1 solution, and the balance is water. The preparation method of the feed medium is the same as that of Example 1.

[0104] S4: When the starvation culture is over, the induction-feed medium is added, and the induction time is 24 hours. During the induction period, the pH is controlled at 6.0±0.2, the temperature is controlled at 22±0.5℃, the dissolved oxygen DO is controlled at 17%, and the tank pressure is controlled at 0.04MPa.

[0105] The specific components and their proportions of the induction-feed medium are: 1050 g / L methanol, 12 ml / L PTM1 solution, 2 mmol / L farnesol, 2.5 g / L glutamic acid, and the balance is water. The preparation method of the induction-feed medium is the same as that of Example 1.

[0106] After the fermentation was completed, the cells were collected by centrifugation and the bovine lactoferrin content was measured by high performance liquid chromatography to be 6.0 g / L.

[0107] Comparative Example 1

[0108] Bovine lactoferrin was prepared according to the method of Example 1, except that the specific components and their proportions in the aqueous solution of Pichia yeast fermentation medium were as follows: 8 g / L yeast extract, 4 ml / L PTM1 solution, 1.2 g / L calcium sulfate, 19 g / L potassium sulfate, 6 g / L magnesium sulfate, 3.8 g / L potassium hydroxide, 29 g / L phosphoric acid aqueous solution, 30 g / L glycerol and 0.5 ml / L HR polyether defoamer, with the balance being water.

[0109] The content of bovine lactoferrin finally prepared was 4.3 g / L.

[0110] Comparative Example 2

[0111] Bovine lactoferrin was prepared according to the method of Example 1, except that the specific components and proportions of the induction-feed medium used were: 1100 g / L methanol, 10 ml / L PTM1 solution, 2 g / L glutamic acid, and the balance was water.

[0112] The content of bovine lactoferrin finally prepared was 4.5 g / L.

[0113] Comparative Example 3

[0114] Bovine lactoferrin was prepared according to the method of Example 1, except that the specific components and proportions of the induction-feed medium used were: 1100 g / L methanol, 10 ml / L PTM1 solution, 4 mmol / L farnesol, and the balance was water.

[0115] The content of bovine lactoferrin finally prepared was 5.3 g / L.

[0116] It can be seen from Examples 1 to 4 and Comparative Examples 1 to 3 that, under the combined action of the Pichia yeast fermentation medium and the fermentation method of the present invention, the Pichia yeast expressing bovine lactoferrin can be better fermented, and bovine lactoferrin is expressed in a more optimal growth environment, thereby improving its activity and stability, thereby improving the quality of the final bovine lactoferrin product, and the bovine lactoferrin content can be as high as 6.3 g / L.

[0117] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art who is familiar with the present technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A Pichia yeast fermentation medium for expressing bovine lactoferrin, characterized in that: The composition of the culture medium per liter is as follows: 2-6 mmol / L of farnesol, 8-12 g / L of yeast extract, 2-6 ml / L of PTM1 solution, 0.8-1.2 g / L of calcium sulfate, 17-19 g / L of potassium sulfate, 6-8 g / L of magnesium sulfate, 3.5-4.5 g / L of potassium hydroxide, 25-30 g / L of phosphoric acid aqueous solution, 30-35 g / L of glycerol and 0.5-1.5 ml / L of defoaming agent, and the balance is water.

2. The culture medium according to claim 1, characterized in that The invention also comprises a feed medium, wherein each liter of the feed medium has the following composition: 400-600 g / L of glycerol, 10-14 ml / L of PTM1 solution, and the balance is water.

3. The culture medium according to claim 1, characterized in that Also included is an induction-feed medium, wherein the composition of the induction-feed medium per liter is as follows: 900-1100 g / L methanol, 10-14 ml / L PTM1 solution, 2-6 mmol / L farnesol, 2-4 g / L glutamic acid, and the balance is water; and / or, The concentration of the phosphoric acid aqueous solution is 80-90wt%; the defoaming agent is an organosilicon or polyether defoaming agent.

4. A Pichia yeast fermentation method for expressing bovine lactoferrin, characterized in that: The method uses the culture medium according to any one of claims 1 to 3, comprising the following steps: Step S1: inoculating the working seeds of Pichia pastoris expressing bovine lactoferrin into the preculture medium, and culturing to obtain a seed solution; Step S2: aseptically introducing the seed liquid into a fermentation tank containing a Pichia yeast fermentation medium, and after the initial ventilation volume, the initial stirring speed, and the initial relative tank pressure are stable, the dissolved oxygen DO value is corrected to 100%, and the fermentation is started. The dissolved oxygen DO value during the control process is controlled to be 30-50%, the pH value during the control process is controlled to be between 5.5-6.5, and the temperature is controlled to be 26-35° C.; Step S3: During the fermentation process, feed medium is added to the culture medium regularly to maintain bacterial growth and the expression of bovine lactoferrin. 600 When the value reaches 200-250, stop feeding and starve for 0.5-1h; Step S4: After the starvation culture is completed, the induction-feed medium is added, and the induction time is 24-36 hours; After the fermentation, the cells were collected by centrifugation to measure the bovine lactoferrin content.

5. The method according to claim 4, characterized in that In the step S1, the bovine lactoferrin is expressed intracellularly in the Pichia pastoris, and the copy number of the bovine lactoferrin gene in the genome of the Pichia pastoris is 3-8.

6. The method according to claim 4, characterized in that In step S1, the pre-culture medium is BMGY culture medium or BMMY culture medium or YPG culture medium; and / or, The seed solution is cultured at a temperature of 26-35° C., a stirring speed of 200-300 rpm, and a culture period of 24-36 h; and / or, The OD of the seed solution 600 The value is 15-20.

7. The method according to claim 4, characterized in that In step S2, the volume of the Pichia yeast fermentation medium in the fermenter accounts for 60-80% of the volume of the fermenter, and the volume of the seed liquid accounts for 8-10% of the volume of the Pichia yeast fermentation medium in the fermenter; and / or, The initial ventilation volume is 2-4 L / min, the initial stirring speed is 200-400 rpm, and the initial relative tank pressure is 0.03-0.06 MPa.

8. The method according to claim 4, characterized in that In the step S3, the feed medium is added at a rate of 5-20 g / L per hour based on the volume of the Pichia yeast fermentation medium in the fermenter; The conditions for adding feed medium to the culture medium at regular intervals are: when the pH reaches 7.5-9.0 or the dissolved oxygen DO value reaches 55%-70%, start feeding; adjust the feed amount according to the dissolved oxygen DO value in the fermentation liquid, so that the dissolved oxygen DO value is controlled at 20-40%, and control the pH of the fermentation liquid between 5.5-6.

5.

9. The method according to claim 4, characterized in that In the step S4, the rate of adding the induction-feed medium is 1-20 g / L per hour based on the volume of the Pichia yeast fermentation medium in the fermenter; During the induction period, the pH is controlled at 6.0±0.2, the temperature is controlled at 22±0.5°C, the dissolved oxygen DO value is controlled at 10-30%, and the tank pressure is controlled at 0.03-0.06MPa.

10. The method according to claim 4, characterized in that In the steps S2 to S4, the stirring speed is 200-1000 rpm, and the relative tank pressure is 0.02-1 MPa; The dissolved oxygen DO value in the step S2 to the step S4 is controlled by the stirring speed and the ventilation volume. When the speed is 350-450 rpm, the ventilation volume is adjusted to 2.8-3.2 L / min; when the speed is 450-550 rpm, the ventilation volume is adjusted to 3.3-3.7 L / min; when the speed is 550-650 rpm, the ventilation volume is adjusted to 3.8-4.2 L / min; when the speed is 650-750 rpm, the ventilation volume is adjusted to 4.3-4.7 L / min; when the speed is 750-850 rpm, the ventilation volume is adjusted to 4.8-5.2 L / min.