阅读说明：本技术 一种提高重组胶原蛋白生产水平的发酵工艺 (Fermentation process for improving production level of recombinant collagen ) 是由 周浩 郝东 王俊 魏文培 侯增淼 于 2021-08-28 设计创作，主要内容包括：本发明提供了一种提高重组胶原蛋白生产水平的发酵工艺,涉及基因重组工程菌发酵技术领域。一种提高重组胶原蛋白生产水平的发酵工艺,包括以下步骤：S1：将毕赤酵母工程菌接种于种子罐中培养,待溶氧大幅度回升后补加甘油和补料培养基,待湿重增至大于100g/L时,移种至发酵罐中开始发酵培养；S2：将发酵罐内的毕赤酵母工程菌培养至溶氧大幅度回升后,使用碳源培养基和补料培养基进行混合碳源补料,待物料湿重增至大于150g/L时,停止混合碳源补料；S3：碳源耗尽后,加入甲醇和补料培养基进行诱导表达至发酵结束。本发明可以提高重组胶原蛋白的表达量,缩短发酵周期,从而提高重组胶原蛋白的发酵生产水平,适用于稳定的工业化生产。(The invention provides a fermentation process for improving the production level of recombinant collagen, and relates to the technical field of fermentation of gene recombinant engineering bacteria. A fermentation process for improving the production level of recombinant collagen comprises the following steps: s1: inoculating pichia pastoris engineering bacteria into a seeding tank for culture, supplementing glycerol and a supplementing culture medium after dissolved oxygen is greatly increased back, and transferring the pichia pastoris engineering bacteria into a fermentation tank for starting fermentation culture when the wet weight is increased to be more than 100 g/L; s2: culturing pichia pastoris engineering bacteria in a fermentation tank until dissolved oxygen is greatly increased, then using a carbon source culture medium and a supplementing medium to supplement mixed carbon sources, and stopping supplementing the mixed carbon sources when the wet weight of the materials is increased to be more than 150 g/L; s3: after the carbon source is exhausted, adding methanol and a feed medium to perform induced expression until the end of fermentation. The invention can improve the expression quantity of the recombinant collagen and shorten the fermentation period, thereby improving the fermentation production level of the recombinant collagen and being suitable for stable industrial production.)

1. A fermentation process for improving the production level of recombinant collagen, which is characterized by comprising the following steps:

s1: inoculating pichia pastoris engineering bacteria into a seeding tank for culture, supplementing glycerol and a supplementing culture medium after dissolved oxygen is greatly increased back, and transferring the pichia pastoris engineering bacteria into a fermentation tank for starting fermentation culture when the wet weight is increased to be more than 100 g/L;

s2: culturing pichia pastoris engineering bacteria in a fermentation tank until dissolved oxygen is greatly increased, then using a carbon source culture medium and a supplementing medium to supplement mixed carbon sources, and stopping supplementing the mixed carbon sources when the wet weight of the materials is increased to be more than 150 g/L;

s3: after the carbon source is exhausted, adding methanol and a feed medium to perform induced expression until the end of fermentation.

2. The fermentation process for increasing the production level of recombinant collagen according to claim 1, wherein the fermentation medium of Pichia pastoris used in the seed tank and the fermentor in step S1 comprises the following components in concentration: CaSO₄·2H₂O 0.47～1.175g/L，K₂SO₄ 7.28～18.2g/L，MgSO₄·7H₂5.96-14.9 g/L of O, 1.625-4.13 g/L of KOH and 20.0-40.0 g/L of glycerol; 20.0-40.0 g/L of glucose; (NaPO)₃)₆6.5-13.0 g/L of white corn steep liquor dry powder and 3-7 g/L of white corn steep liquor dry powder.

3. The fermentation process for increasing the production level of recombinant collagen according to claim 2, wherein the fermentation medium of Pichia pastoris used in the seed tank and the fermentor in step S1 comprises the following components in concentration: CaSO₄·2H₂O 0.5875～0.8225g/L，K₂SO₄ 9.1～12.74g/L，MgSO₄·7H₂7.45-10.43 g/L of O, 2.07-2.891 g/L of KOH and 20.0-30.0 g/L of glycerol; 20.0-30.0 g/L of glucose; (NaPO)₃)₆6.5-9.1 g/L of white corn steep liquor dry powder and 3-5 g/L of white corn steep liquor dry powder.

4. The fermentation process for increasing the production level of recombinant collagen according to claim 1, wherein said feed medium comprises the following components in concentration: CaSO₄·2H₂O 0.1～0.705g/L；K₂SO₄ 0.15～10.92g/L；MgSO₄·7H₂0.25-8.94 g/L of O; 0.12-2.478 g/L of KOH; 0.3-4 g/L of white corn steep liquor dry powder.

5. The fermentation process for increasing the production level of recombinant collagen according to claim 4, wherein said feed medium comprisesThe following concentrations of the components: CaSO₄·2H₂O 0.25～0.5875g/L；K₂SO₄ 5.46～9.1g/L；MgSO₄·7H₂O4.47-7.45 g/L; KOH 0.75-2.065 g/L; 2.1-3.5 g/L white corn steep liquor dry powder.

6. The fermentation process for increasing the production level of recombinant collagen according to claim 1, wherein the feeding rate of the feeding medium in step S2 is 5-8 mL/h/L, the feeding rate of the carbon source medium is 8-15 mL/h/L; the feeding rate of the fed-batch culture medium in the step S3 is 3-5 mL/h/L, and the feeding rate of the methanol is 3-7 mL/h/L.

7. The fermentation process for increasing the production level of recombinant collagen according to claim 1, wherein the feeding rate of the feeding medium in step S1 is 5-8 mL/h/L, and the feeding rate of glycerol is 10-18 mL/h/L.

8. The fermentation process for improving the production level of recombinant collagen according to claim 1, wherein the Pichia pastoris is cultured in step S1 until the wet weight is increased to 160-180 g/L, and is transferred to a fermentation tank; in the step S2, the Pichia pastoris engineering bacteria are cultured until the wet weight is increased to 180-200 g/L, and then the mixed carbon source feeding is stopped.

9. The fermentation process for increasing the production level of recombinant collagen according to claim 1, wherein said carbon source medium in step S2 is glycerol, glucose or a mixed medium of glycerol and glucose.

10. The fermentation process for increasing the production level of recombinant collagen according to claim 1, wherein said fermentation culture conditions are: the fermentation temperature is 28-32 ℃, the pH value is adjusted to 4.8-5.5 by ammonia water, the dissolved oxygen before induced expression is not less than 30%, the tank pressure is 0.04-0.06 MPa, the dissolved oxygen after induced expression is not less than 5%, and the tank pressure is 0.08-0.1 MPa.

Technical Field

The invention relates to the technical field of fermentation of gene recombination engineering bacteria, in particular to a fermentation process for improving the production level of recombinant collagen.

Background

Collagen (collagen) is a protein with the largest content in the body, is a main component of extracellular matrix (ECM), and plays an important role in maintaining normal physiological functions of cells, tissues and organs and repairing damage. Because of the unique structure, the collagen protein has excellent biocompatibility and low immunogenicity, and is widely applied to the industries of medicines, health care products and cosmetics.

In the prior art, the main source of collagen is animal tissue extraction, and with the development of biotechnology, the recombinant collagen obtained by a microbial fermentation method has achieved huge achievements by utilizing a gene recombination technology.

At present, pichia pastoris engineering bacteria are generally used for fermentation culture to produce recombinant collagen; however, most of the existing fermentation culture mediums mainly comprise a BSM culture medium provided by Invitrogen company, and only glycerol is used in a general carbon source feeding stage in a fermentation stage, and only methanol is used in an induced expression stage; such fermentation processes have some problems:

(1) the BSM culture medium is used for culturing seeds, the wet weight is 90-100g/L, the whole bacterial load is low, the lag phase is long after the seeds are transferred into the fermentation culture medium, and the whole production level is influenced;

(2) the BSM medium has too high concentration of salt ions, inhibits the growth of thalli, influences the activity of strains and is difficult to quickly reach the high-density fermentation level;

(3) the glycerin is viscous, and the excessively high proportion influences the oxygen mass transfer and the uniformity of the feed liquid;

(4) high salt of a BSM culture medium and the single use of glycerol and methanol easily cause the decay of thalli in the middle and later periods, the increase of protease secretion and the aggravation of protein degradation, thereby influencing the quality of the whole protein;

(5) the BSM culture medium contains high-concentration phosphoric acid, the initial pH of the culture medium is lower than 1.5, elements such as electrodes and the like are greatly damaged, and the service life of a fermentation tank is seriously influenced;

(6) BSM culture medium and supplemented glycerol and methanol contain PTM1, PTM1 are complex to prepare, cannot be sterilized at high temperature, need aseptic filtration and cause great trouble for industrial amplification;

(7) when dissolved oxygen is maintained by pichia pastoris engineering bacteria, pure oxygen needs to be introduced, so that the production cost is increased on one hand, and the pure oxygen belongs to flammable and explosive products and is easy to cause safety accidents on the other hand.

Disclosure of Invention

The invention aims to provide a fermentation process for improving the production level of recombinant collagen, which can improve the expression quantity of the recombinant collagen and shorten the fermentation period, thereby improving the fermentation production level of the recombinant collagen and being suitable for stable industrial production.

The embodiment of the invention is realized by the following technical scheme:

a fermentation process for improving the production level of recombinant collagen comprises the following steps:

s1: inoculating pichia pastoris engineering bacteria into a seeding tank for culture, supplementing glycerol and a supplementing culture medium after dissolved oxygen is greatly increased back, and transferring the pichia pastoris engineering bacteria into a fermentation tank for starting fermentation culture when the wet weight is increased to be more than 100 g/L;

s2: culturing pichia pastoris engineering bacteria in a fermentation tank until dissolved oxygen is greatly increased, then using a carbon source culture medium and a supplementing medium to supplement mixed carbon sources, and stopping supplementing the mixed carbon sources when the wet weight of the materials is increased to be more than 150 g/L;

s3: after the carbon source is exhausted, adding methanol and a feed medium to perform induced expression until the end of fermentation.

Further, the method can be used for preparing a novel materialIn step S1, the fermentation medium of pichia pastoris engineering bacteria used in the seed tank and the fermentation tank comprises the following components in concentration: CaSO₄·2H₂O 0.47～1.175g/L，K₂SO₄ 7.28～18.2g/L，MgSO₄·7H₂5.96-14.9 g/L of O, 1.625-4.13 g/L of KOH and 20.0-40.0 g/L of glycerol; 20.0-40.0 g/L of glucose; (NaPO)₃)₆6.5-13.0 g/L of white corn steep liquor dry powder and 3-7 g/L of white corn steep liquor dry powder.

Further, the pichia pastoris engineering bacteria fermentation medium used in the seeding tank and the fermentation tank in the step S1 comprises the following components in concentration: CaSO₄·2H₂O 0.5875～0.8225g/L，K₂SO₄ 9.1～12.74g/L，MgSO₄·7H₂7.45-10.43 g/L of O, 2.07-2.891 g/L of KOH and 20.0-30.0 g/L of glycerol; 20.0-30.0 g/L of glucose; (NaPO)₃)₆6.5-9.1 g/L of white corn steep liquor dry powder and 3-5 g/L of white corn steep liquor dry powder.

Further, the feed medium comprises the following components in the following concentrations: CaSO₄·2H₂O 0.1～0.705g/L；K₂SO₄ 0.15～10.92g/L；MgSO₄·7H₂0.25-8.94 g/L of O; 0.12-2.478 g/L of KOH; 0.3-4 g/L of white corn steep liquor dry powder.

Further, the feed medium comprises the following components in the following concentrations: CaSO₄·2H₂O 0.25～0.5875g/L；K₂SO₄ 5.46～9.1g/L；MgSO₄·7H₂O4.47-7.45 g/L; KOH 0.75-2.065 g/L; 2.1-3.5 g/L white corn steep liquor dry powder.

Further, the feeding rate of the feed medium in the step S2 is 5-8 mL/h/L, and the feeding rate of the carbon source medium is 8-15 mL/h/L; the feeding rate of the fed-batch culture medium in the step S3 is 3-5 mL/h/L, and the feeding rate of the methanol is 3-7 mL/h/L.

Further, the feeding rate of the feed medium in the step S1 is 5-8 mL/h/L, and the feeding rate of the glycerol is 10-18 mL/h/L.

Further, in the step S1, when the wet weight of the engineering bacteria of the pichia pastoris is increased to 160-180 g/L, the engineering bacteria of the pichia pastoris are transferred to a fermentation tank; in the step S2, the Pichia pastoris engineering bacteria are cultured until the wet weight is increased to 180-200 g/L, and then the mixed carbon source feeding is stopped.

Further, the carbon source medium in step S2 is glycerol, glucose or a mixed medium of glycerol and glucose.

Further, the conditions of the fermentation culture are as follows: the fermentation temperature is 28-32 ℃, the pH value is adjusted to 4.8-5.5 by ammonia water, the dissolved oxygen before induced expression is not less than 30%, the tank pressure is 0.04-0.06 MPa, the dissolved oxygen after induced expression is not less than 5%, and the tank pressure is 0.08-0.1 MPa.

Furthermore, the Pichia pastoris engineering bacteria are Pichia pastoris, and are preserved in the common microorganism center of China general microbiological culture Collection center in 2020, 07 and 31 days, and the preservation number is CGMCC No. 20458.

The technical scheme of the embodiment of the invention at least has the following advantages and beneficial effects:

1. according to the invention, the compound fermentation medium is used, and the glycerol and the glucose are compounded, so that on one hand, the viscosity of the feed liquid can be reduced, and on the other hand, the phenomenon that the growth of strains is inhibited due to the generation of excessive organic acid can be avoided; the salt ion concentration is reduced, and the inhibition on the growth of strains is reduced; increasing the wet weight of the strain to 160-200 g/L, and shortening the lag phase after the strain is transferred into a fermentation medium; thereby effectively improving the growth speed of the strains and shortening the fermentation period.

2. According to the invention, before the strain is transferred to the fermentation tank, glycerol and a feeding culture medium are used for feeding, a carbon source culture medium and a feeding culture medium are also used for mixed carbon source feeding in a carbon source feeding stage after the strain is transferred into the fermentation tank, and methanol and the feeding culture medium are used for mixed feeding in an induction expression stage to induce expression, so that the ionic strength and the trace elements are supplemented in the fermentation process, the collagenase generation is inhibited, and the expression amount of the recombinant collagen is effectively increased.

3. According to the invention, the compound fermentation medium is not added with phosphoric acid, so that damage to elements such as electrodes and the like caused by too low initial pH can be avoided, and the service life of a fermentation tank is prevented from being influenced.

4. The fermentation medium, the feed medium, the glycerol, the carbon source medium and the methanol used in the invention are not added with the PTM1, the operation is simple and convenient, and the method is more suitable for industrial production.

5. In the induction stage, the pressure of the tank is increased to maintain certain dissolved oxygen in the tank, so that the aim of not using pure oxygen is fulfilled, the cost is saved, and the production operation is safer.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The following is a detailed description of a fermentation process for improving the production level of recombinant collagen according to the embodiments of the present invention.

The Pichia pastoris engineering bacteria are Pichia pastoris, are preserved in China general microbiological culture Collection center (CGMCC) at 2020, 07, 31 and have a preservation number of CGMCC No. 20458.

Example 1

The embodiment provides a fermentation process for improving the production level of recombinant collagen, which comprises the following steps:

s1: inoculating the pichia pastoris engineering bacteria into a 100L seeding tank containing 70L fermentation medium according to the inoculation amount of 10% to culture, adjusting the pH value to be 4.8 by ammonia water at the temperature of 28 ℃, adjusting the tank pressure to be 0.06MPa, and adjusting the dissolved oxygen to be not less than 30%, supplementing 50% of glycerol and a supplemented medium after the dissolved oxygen is greatly increased, wherein the feeding rate of the 50% of glycerol is 10mL/h/L, and the feeding rate of the supplemented medium is 5 mL/h/L; when the wet weight is increased to 160g/L, transferring the seeds to a 1500L fermentation tank containing 700L fermentation medium, starting fermentation culture, adjusting the pH to 4.8 by ammonia water at 28 ℃, adjusting the tank pressure to 0.06MPa, and enabling the dissolved oxygen to be not less than 30%;

s2: culturing pichia pastoris engineering bacteria in a fermentation tank until dissolved oxygen is greatly increased, and then using 50% glycerol and a feed supplement culture medium to supplement mixed carbon sources, wherein the feeding rate of the 50% glycerol is 8mL/h/L, and the feeding rate of the feed supplement culture medium is 5 mL/h/L; stopping the mixed carbon source feeding when the wet weight of the material is increased to 185 g/L;

s3: after the glycerol is exhausted, adding methanol and a feed supplement culture medium for induction expression until the fermentation is finished, dynamically adjusting the feed supplement by the methanol within the range of the feed supplement rate of 3-7 mL/h/L, wherein the feed supplement culture medium has the feed supplement rate of 3mL/h/L, the temperature of 28 ℃, the ammonia water for adjusting the pH value to 4.8, the tank pressure of 0.08MPa and the dissolved oxygen content of not less than 5 percent, and finishing the whole fermentation process when the wet bacteria weight or the protein expression amount of the pichia pastoris is not increased any more.

The pichia pastoris engineering bacteria fermentation culture medium used in the seeding tank and the fermentation tank comprises the following components in concentration: CaSO₄·2H₂O 0.5875g/L，K₂SO₄ 8.05g/L，MgSO₄·7H₂O7.45 g/L, KOH 3.59g/L, glycerol 20.0 g/L; glucose is 30.0 g/L; (NaPO)₃)₆7.1g/L and 3g/L of white corn steep liquor dry powder.

The feed medium included the following components at the following concentrations: CaSO₄·2H₂O 0.1g/L；K₂SO₄ 5.46g/L；MgSO₄·7H₂O7.45 g/L; KOH 0.23 g/L; white corn steep liquor dry powder 3.5 g/L.

Example 2

The embodiment provides a fermentation process for improving the production level of recombinant collagen, which comprises the following steps:

s1: inoculating the pichia pastoris engineering bacteria into a 100L seeding tank containing 50L fermentation medium according to the inoculation amount of 6% to culture, adjusting the temperature to 30 ℃, adjusting the pH to 5.5 by ammonia water, adjusting the tank pressure to 0.06MPa, and adjusting the dissolved oxygen content to be not less than 30%, supplementing 50% of glycerol and a supplemented medium after the dissolved oxygen content is greatly increased, wherein the fed-batch rate of the 50% of glycerol is 16mL/h/L, and the fed-batch rate of the supplemented medium is 6 mL/h/L; when the wet weight is increased to 180g/L, transferring the seeds to a 1500L fermentation tank containing 600L fermentation medium, starting fermentation culture, adjusting the pH to 5.5 by ammonia water at 30 ℃, adjusting the tank pressure to 0.06MPa, and enabling the dissolved oxygen to be not less than 30%;

s2: culturing pichia pastoris engineering bacteria in a fermentation tank until dissolved oxygen is greatly increased, and then using glucose and a feed supplement culture medium to supplement a mixed carbon source, wherein the feeding rate of the glucose is 9mL/h/L, and the feeding rate of the feed supplement culture medium is 8 mL/h/L; stopping the mixed carbon source feeding when the wet weight of the material is increased to 200 g/L;

s3: after glucose is exhausted, adding methanol and a feed supplement culture medium for induction expression until fermentation is finished, dynamically adjusting the feed supplement by the methanol within the range of the feed supplement speed of 3-7 mL/h/L, wherein the feed supplement speed of the feed supplement culture medium is 5mL/h/L, the temperature is 30 ℃, the pH value is adjusted to 5.5 by ammonia water, the tank pressure is 0.09MPa, the dissolved oxygen is not lower than 5%, and the whole fermentation process is finished when the weight of the wet pichia pastoris or the protein expression amount is not increased any more.

The pichia pastoris engineering bacteria fermentation culture medium used in the seeding tank and the fermentation tank comprises the following components in concentration: CaSO₄·2H₂O 0.47g/L，K₂SO₄ 18.2g/L，MgSO₄·7H₂O6.21 g/L, KOH 2.07g/L, glycerol 21.0 g/L; 22.0g/L of glucose; (NaPO)₃)₆6.5g/L and 3.5g/L white corn steep liquor dry powder.

The feed medium included the following components at the following concentrations: CaSO₄·2H₂O 0.6217g/L；K₂SO₄ 0.15g/L；MgSO₄·7H₂O6.55 g/L; KOH 0.12 g/L; white corn steep liquor dry powder 2.1 g/L.

Example 3

The embodiment provides a fermentation process for improving the production level of recombinant collagen, which comprises the following steps:

s1: inoculating the pichia pastoris engineering bacteria into a 100L seeding tank containing 60L fermentation medium according to the inoculation amount of 8% to culture, adjusting the temperature to 32 ℃, adjusting the pH to 5.0 by ammonia water, adjusting the tank pressure to 0.05MPa, and adding 50% of glycerol and a supplemented medium after the dissolved oxygen is greatly increased, wherein the feeding rate of the 50% of glycerol is 13mL/h/L, and the feeding rate of the supplemented medium is 6 mL/h/L; when the wet weight is increased to 180g/L, transferring the seeds to a 1500L fermentation tank containing 650L of fermentation medium, starting fermentation culture, adjusting the pH to 5.0 by ammonia water at 32 ℃, adjusting the tank pressure to 0.05MPa, and enabling the dissolved oxygen to be not less than 30%;

s2: culturing pichia pastoris engineering bacteria in a fermentation tank until dissolved oxygen is greatly increased, and then using 50% glycerol and a feed supplement culture medium to supplement mixed carbon sources, wherein the feeding rate of the 50% glycerol is 15mL/h/L, and the feeding rate of the feed supplement culture medium is 7 mL/h/L; stopping feeding the mixed carbon source when the wet weight of the material is increased to 190 g/L;

s3: after the glycerol is exhausted, adding methanol and a feed-supplement culture medium for induction expression until the fermentation is finished, dynamically adjusting the feed-supplement by the methanol within the range of the feed-supplement rate of 3-7 mL/h/L, wherein the feed-supplement rate of the feed-supplement culture medium is 4mL/h/L, the temperature is 32 ℃, the pH value is adjusted to 5.0 by ammonia water, the tank pressure is 0.1MPa, the dissolved oxygen is not lower than 5%, and the whole fermentation process is finished when the weight of the wet bacteria of the pichia pastoris or the protein expression amount is not increased any more.

The pichia pastoris engineering bacteria fermentation culture medium used in the seeding tank and the fermentation tank comprises the following components in concentration: CaSO₄·2H₂O 1.175g/L，K₂SO₄ 16.15g/L，MgSO₄·7H₂O5.96 g/L, KOH 3.92g/L, glycerol 30.0 g/L; glucose 20.0 g/L; (NaPO)₃)₆8.8g/L and 5g/L of white corn steep liquor dry powder.

The feed medium included the following components at the following concentrations: CaSO₄·2H₂O 0.19g/L；K₂SO₄ 8.56g/L；MgSO₄·7H₂O0.25 g/L; KOH 2.065 g/L; white corn steep liquor dry powder 0.4 g/L.

Example 4

The embodiment provides a fermentation process for improving the production level of recombinant collagen, which comprises the following steps:

s1: inoculating the pichia pastoris engineering bacteria into a 100L seeding tank containing 70L fermentation medium according to the inoculation amount of 10% to culture, adjusting the pH to be 5.2 by ammonia water at the temperature of 28 ℃, adjusting the tank pressure to be 0.06MPa, and adjusting the dissolved oxygen to be not less than 30%, supplementing 50% of glycerol and a supplemented medium after the dissolved oxygen is greatly increased, wherein the feeding rate of the 50% of glycerol is 18mL/h/L, and the feeding rate of the supplemented medium is 7.5 mL/h/L; when the wet weight is increased to 175g/L, transferring the seeds to a 1500L fermentation tank containing 700L fermentation medium, starting fermentation culture, adjusting the pH to 5.5 with ammonia water at 30 ℃, adjusting the tank pressure to 0.05MPa, and adjusting the dissolved oxygen content to not less than 30%;

s2: culturing pichia pastoris engineering bacteria in a fermentation tank until dissolved oxygen is greatly increased, and then mixing a culture medium and a feeding culture medium by using 50% of glycerol and glucose according to a ratio of 1:1 to feed a mixed carbon source, wherein the feeding rate of the 50% of glycerol and glucose is 12mL/h/L, and the feeding rate of the feeding culture medium is 6.5 mL/h/L; stopping the mixed carbon source feeding when the wet weight of the material is increased to 200 g/L;

s3: after the glycerol and the glucose are exhausted, adding methanol and a feed-supplement culture medium for induction expression until the fermentation is finished, dynamically adjusting the fed-batch by the methanol within the range of the fed-batch rate of 3-7 mL/h/L, adjusting the pH to 4.9 by ammonia water at the temperature of 32 ℃ and the tank pressure to 0.09MPa, wherein the dissolved oxygen is not less than 5%, and finishing the whole fermentation process when the wet bacteria weight or the protein expression amount of the pichia pastoris is not increased any more.

The pichia pastoris engineering bacteria fermentation culture medium used in the seeding tank and the fermentation tank comprises the following components in concentration: CaSO₄·2H₂O 0.8225g/L，K₂SO₄ 9.1g/L，MgSO₄·7H₂O9.15 g/L, KOH 4.13g/L and glycerol 26.0 g/L; glucose 23.0 g/L; (NaPO)₃)₆10.2g/L and 7g/L of white corn steep liquor dry powder.

The feed medium included the following components at the following concentrations: CaSO₄·2H₂O 0.25g/L；K₂SO₄ 10.92g/L；MgSO₄·7H₂O4.47 g/L; KOH 0.52 g/L; white corn steep liquor dry powder 2.7 g/L.

Example 5

The embodiment provides a fermentation process for improving the production level of recombinant collagen, which comprises the following steps:

s1: inoculating pichia pastoris engineering bacteria into a 100L seeding tank containing 65L fermentation medium according to the inoculation amount of 9 percent for culture, adjusting the pH value to be 4.9 by ammonia water at the temperature of 28 ℃, adjusting the tank pressure to be 0.05MPa, and adding 50 percent of glycerol and a supplemented medium after the dissolved oxygen is greatly increased, wherein the feeding rate of the 50 percent of glycerol is 12mL/h/L, and the feeding rate of the supplemented medium is 8 mL/h/L; when the wet weight is increased to 170g/L, transferring the seeds to a 1500L fermentation tank containing 650L of fermentation medium, starting fermentation culture, adjusting the pH to 5.2 by ammonia water at 30 ℃, adjusting the tank pressure to 0.04MPa, and adjusting the dissolved oxygen content to not less than 30%;

s2: culturing pichia pastoris engineering bacteria in a fermentation tank until dissolved oxygen is greatly increased, and then using 50% glycerol and a feed medium to feed a mixed carbon source, wherein the feeding rate of the 50% glycerol is 10mL/h/L, and the feeding rate of the feed medium is 7.5 mL/h/L; stopping the mixed carbon source feeding when the wet weight of the material is increased to 185 g/L;

s3: after the glycerol is exhausted, adding methanol and a feed supplement culture medium for induction expression until the fermentation is finished, dynamically adjusting the feed supplement by the methanol within the range of the feed supplement rate of 3-7 mL/h/L, wherein the feed supplement culture medium has the feed supplement rate of 4.5mL/h/L, the temperature of 28 ℃, the pH value of ammonia water regulated to 5.2, the tank pressure of 0.08MPa and the dissolved oxygen content of not less than 5 percent, and finishing the whole fermentation process when the wet bacteria weight or the protein expression amount of the pichia pastoris is not increased any more.

The pichia pastoris engineering bacteria fermentation culture medium used in the seeding tank and the fermentation tank comprises the following components in concentration: CaSO₄·2H₂O 0.6521g/L，K₂SO₄ 7.28g/L，MgSO₄·7H₂14.9g/L of O, 2.34g/L of KOH and 25.0g/L of glycerol; 24.0g/L glucose; (NaPO)₃)₆13g/L and 6.5g/L white corn steep liquor dry powder.

The feed medium included the following components at the following concentrations: CaSO₄·2H₂O 0.4622g/L；K₂SO₄ 0.34g/L；MgSO₄·7H₂O2.18 g/L; 2.478g/L KOH; white corn steep liquor dry powder 4 g/L.

Example 6

The embodiment provides a fermentation process for improving the production level of recombinant collagen, which comprises the following steps:

s1: inoculating the pichia pastoris engineering bacteria into a 100L seeding tank containing 70L fermentation medium according to the inoculation amount of 10% to culture, adjusting the temperature to 30 ℃, adjusting the pH to 4.9 by ammonia water, adjusting the tank pressure to 0.05MPa, and adjusting the dissolved oxygen content to be not less than 30%, supplementing 50% of glycerol and a supplemented medium after the dissolved oxygen content is greatly increased, wherein the fed-batch rate of the 50% of glycerol is 15mL/h/L, and the fed-batch rate of the supplemented medium is 8 mL/h/L; when the wet weight is increased to 165g/L, transferring the seeds to a 1500L fermentation tank containing 650L of fermentation medium, starting fermentation culture, adjusting the temperature to 30 ℃, adjusting the pH value to 5 by ammonia water, adjusting the tank pressure to 0.04MPa, and adjusting the dissolved oxygen content to not less than 30%;

s2: culturing pichia pastoris engineering bacteria in a fermentation tank until dissolved oxygen is greatly increased, and then using 50% glycerol and a feeding culture medium to feed a mixed carbon source, wherein the feeding rate of the 50% glycerol is 14mL/h/L, and the feeding rate of the feeding culture medium is 6.5 mL/h/L; stopping the mixed carbon source feeding when the wet weight of the material is increased to 180 g/L;

s3: after the glycerol is exhausted, adding methanol and a feed supplement culture medium for induction expression until the fermentation is finished, dynamically adjusting the feed supplement by the methanol within the range of the feed supplement rate of 3-7 mL/h/L, wherein the feed supplement culture medium has the feed supplement rate of 3.5mL/h/L, the temperature of 28 ℃, the pH value of ammonia water adjusted to 5, the tank pressure of 0.08MPa and the dissolved oxygen content of not less than 5 percent, and finishing the whole fermentation process when the wet bacteria weight or the protein expression amount of the pichia pastoris is not increased any more.

The pichia pastoris engineering bacteria fermentation culture medium used in the seeding tank and the fermentation tank comprises the following components in concentration: CaSO₄·2H₂O 0.4912g/L，K₂SO₄ 10.55g/L，MgSO₄·7H₂O12.42 g/L, KOH 1.625g/L, glycerol 27.0 g/L; glucose 21.0 g/L; (NaPO)₃)₆11.5g/L and 5g/L of white corn steep liquor dry powder.

The feed medium included the following components at the following concentrations: CaSO₄·2H₂O 0.705g/L；K₂SO₄ 2.75g/L；MgSO₄·7H₂O8.94 g/L; KOH 1.83 g/L; white corn steep liquor dry powder is 1.6 g/L.

Example 7

The embodiment provides a fermentation process for improving the production level of recombinant collagen, which comprises the following steps:

s1: inoculating the pichia pastoris engineering bacteria into a 100L seeding tank containing 60L fermentation medium according to the inoculation amount of 8% to culture, adjusting the pH to be 4.8 by ammonia water at the temperature of 29 ℃, adjusting the tank pressure to be 0.05MPa, and adding 50% of glycerol and a supplemented medium after the dissolved oxygen is greatly increased, wherein the feeding rate of the 50% of glycerol is 14mL/h/L, and the feeding rate of the supplemented medium is 7 mL/h/L; when the wet weight is increased to 175g/L, transferring the seeds to a 1500L fermentation tank containing 650L of fermentation medium, starting fermentation culture, adjusting the pH to 4.9 by ammonia water at 28 ℃, adjusting the tank pressure to 0.04MPa, and adjusting the dissolved oxygen content to not less than 30%;

s2: culturing pichia pastoris engineering bacteria in a fermentation tank until dissolved oxygen is greatly increased, and then using 50% glycerol and a feed medium to feed a mixed carbon source, wherein the feeding rate of the 50% glycerol is 11mL/h/L, and the feeding rate of the feed medium is 8 mL/h/L; stopping feeding the mixed carbon source when the wet weight of the material is increased to 190 g/L;

s3: after the glycerol is exhausted, adding methanol and a feed supplement culture medium for induction expression until the fermentation is finished, dynamically adjusting the feed supplement by the methanol within the range of the feed supplement rate of 3-7 mL/h/L, wherein the feed supplement culture medium has the feed supplement rate of 3mL/h/L, the temperature of 28 ℃, the ammonia water for adjusting the pH value to 4.9, the tank pressure of 0.08MPa and the dissolved oxygen content of not less than 5 percent, and finishing the whole fermentation process when the wet bacteria weight or the protein expression amount of the pichia pastoris is not increased any more.

The pichia pastoris engineering bacteria fermentation culture medium used in the seeding tank and the fermentation tank comprises the following components in concentration: CaSO₄·2H₂O 0.5105g/L，K₂SO₄ 12.74g/L，MgSO₄·7H₂O13.6 g/L, KOH 2.891g/L, glycerol 22.0 g/L; 26.0g/L glucose; (NaPO)₃)₆12.3g/L and 6g/L of white corn steep liquor dry powder.

The feed medium included the following components at the following concentrations: CaSO₄·2H₂O 0.3521g/L；K₂SO₄ 9.1g/L；MgSO₄·7H₂O0.51 g/L; KOH 1.15 g/L; 3g/L white corn steep liquor dry powder.

Example 8

The embodiment provides a fermentation process for improving the production level of recombinant collagen, which comprises the following steps:

s1: inoculating the pichia pastoris engineering bacteria into a 100L seeding tank containing 75L fermentation medium according to the inoculation amount of 8% to culture, adjusting the temperature to 30 ℃, adjusting the pH to 5 by ammonia water, adjusting the tank pressure to 0.05MPa, and adding 50% of glycerol and a supplemented medium after the dissolved oxygen is greatly increased, wherein the feeding rate of the 50% of glycerol is 12mL/h/L, and the feeding rate of the supplemented medium is 6.5 mL/h/L; when the wet weight is increased to 170g/L, transferring the seeds to a 1500L fermentation tank containing 650L of fermentation medium, starting fermentation culture, adjusting the temperature to 30 ℃, adjusting the pH value to 5 by ammonia water, adjusting the tank pressure to 0.04MPa, and adjusting the dissolved oxygen content to not less than 30%;

s2: culturing pichia pastoris engineering bacteria in a fermentation tank until dissolved oxygen is greatly increased, and then using 50% glycerol and a feed supplement culture medium to supplement mixed carbon sources, wherein the feeding rate of the 50% glycerol is 15mL/h/L, and the feeding rate of the feed supplement culture medium is 5.5 mL/h/L; stopping feeding the mixed carbon source when the wet weight of the material is increased to 195 g/L;

s3: after the glycerol is exhausted, adding methanol and a feed supplement culture medium for induction expression until the fermentation is finished, dynamically adjusting the feed supplement by the methanol within the range of the feed supplement rate of 3-7 mL/h/L, wherein the feed supplement culture medium has the feed supplement rate of 4mL/h/L, the temperature of 29 ℃, the ammonia water for adjusting the pH value to 5.2, the tank pressure of 0.08MPa and the dissolved oxygen content of not less than 5 percent, and finishing the whole fermentation process when the wet bacteria weight or the protein expression amount of the pichia pastoris is not increased any more.

The pichia pastoris engineering bacteria fermentation culture medium used in the seeding tank and the fermentation tank comprises the following components in concentration: CaSO₄·2H₂O 0.9815g/L，K₂SO₄ 14.1g/L，MgSO₄·7H₂O10.43 g/L, KOH 1.91g/L, glycerol 28.0 g/L; glucose 20.0 g/L; (NaPO)₃)₆9.1g/L and 4g/L of white corn steep liquor dry powder.

The feed medium included the following components at the following concentrations: CaSO₄·2H₂O 0.5875g/L；K₂SO₄ 6.52g/L；MgSO₄·7H₂O8.11 g/L; KOH 0.75 g/L; white corn steep liquor dry powder 3.2 g/L.

Comparative example

The comparative example provides a fermentation process of recombinant collagen, comprising the following steps:

s1: inoculating the pichia pastoris engineering bacteria into a 100L seeding tank containing 70L fermentation medium according to the inoculation amount of 10% to culture, adjusting the temperature to 30 ℃, adjusting the pH value to 4.6 by ammonia water, adjusting the tank pressure to 0.03MPa, and adjusting the dissolved oxygen content to not less than 30%, after the dissolved oxygen content is greatly increased, transferring the pichia pastoris engineering bacteria into a 1500L fermentation tank containing 700L fermentation medium to start fermentation culture, adjusting the temperature to 30 ℃, adjusting the pH value to 4.6 by ammonia water, adjusting the tank pressure to 0.03MPa, and adjusting the dissolved oxygen content to not less than 30%;

s2: culturing pichia pastoris engineering bacteria in a fermentation tank until dissolved oxygen is greatly increased, and then using 50% glycerol to supplement a carbon source, wherein the feeding rate of the 50% glycerol is 6 mL/h/L; stopping the mixed carbon source feeding when the wet weight of the material is increased to 120 g/L;

s3: after the carbon source is exhausted, adding methanol for induction expression until the fermentation is finished, wherein the feeding rate of the methanol is 8mL/h/L, the temperature is 30 ℃, the pH value is adjusted to be 4.6 by ammonia water, the tank pressure is 0.03MPa, adding liquid oxygen to ensure that the dissolved oxygen is not less than 20 percent, and finishing the whole fermentation process when the wet bacteria weight or the protein expression amount of the pichia pastoris is not increased any more.

The pichia pastoris engineering bacteria fermentation culture medium used in the seeding tank and the fermentation tank comprises the following components in concentration: 85% H₃PO₄ 26.7mL/L；CaSO₄·2H₂O 1.175g/L；K₂SO₄ 18.2g/L；MgSO₄·7H₂O14.9 g/L; KOH 4.13 g/L; 40.0g/L of glycerol; PTM 14.35mL/L.

Wherein, the PTM1 comprises the following components in concentration: CuSO₄·5H₂O 6.0g/L；NaI 0.08g/L；MnSO₄·H₂O 3.0g/L；NaMoO₄·2H₂O 0.2g/L；H₃BO₃ 0.02g/L；CoCl₂ 0.5g/L；ZnCl₂ 20.0g/L；FeSO₄·7H₂O65.0 g/L; biotin 0.2 g/L; h₂SO₄5.0 mL/L. Filtering and sterilizing with 0.22 μm filter membrane, and storing at room temperature.

Examples of the experiments

Respectively taking fermentation liquor obtained after fermentation of the examples 1-8 and the comparative example, detecting the concentration of the recombinant collagen through HPLC, recording the fermentation periods of the examples 1-8 and the comparative example, and calculating the fermentation production level; the results are shown in Table 1.

Fermentation production level (g/L. h) ═ concentration of recombinant collagen (g/L)/fermentation period (h)

TABLE 1 fermentation production level

As can be seen from Table 1, in the fermentation method used in the present invention, compared with the comparative examples 1-8, the concentration of the obtained recombinant collagen is higher, the fermentation period is shorter, and the fermentation production level of the recombinant collagen is higher.

In summary, the fermentation process for improving the production level of the recombinant collagen provided by the present application uses a fermentation medium which is improved relative to a BSM medium used in the prior art, and in the fermentation process, before the strain is transferred to the fermentation tank, glycerol and a supplement medium are used for supplementing, in the carbon source supplement stage after the strain is transferred to the fermentation tank, a carbon source medium and a supplement medium are also used for supplementing a mixed carbon source, in the induction expression stage, methanol and the supplement medium are used for mixed supplement induction expression, so that the expression quantity of the recombinant collagen is effectively improved, the fermentation period is shortened, the fermentation production level of the recombinant collagen is improved, and the fermentation process is suitable for stable industrial production.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.