CN113789358B - Fermentation process for improving production level of recombinant collagen - Google Patents
Fermentation process for improving production level of recombinant collagen Download PDFInfo
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Abstract
The invention provides a fermentation process for improving the production level of recombinant collagen, and relates to the technical field of fermentation of genetically recombinant engineering bacteria. A fermentation process for increasing the production level of recombinant collagen comprising the steps of: s1: inoculating Pichia pastoris engineering bacteria into a seed tank for culture, adding glycerol and a feed supplement culture medium after dissolved oxygen is greatly risen, and transferring the seeds into a fermentation tank for fermentation culture when the wet weight is increased to be more than 100 g/L; s2: culturing Pichia pastoris engineering bacteria in the fermentation tank until dissolved oxygen is greatly risen, and then using a carbon source culture medium and a feed supplement culture medium to carry out mixed carbon source feed supplement, and stopping mixed carbon source feed supplement when the wet weight of the material is increased to be more than 150 g/L; s3: after the carbon source is exhausted, methanol and a feed medium are added for induction expression until the fermentation is finished. 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 industrialized production.
Description
Technical Field
The invention relates to the technical field of fermentation of genetic recombinant engineering bacteria, in particular to a fermentation process for improving the production level of recombinant collagen.
Background
Collagen (collagen) is the most abundant protein in the body, the major component of the extracellular matrix (ECM), which plays an important role in maintaining normal physiological functions of cells, tissues, organs and repair of injury. Because of the unique structure, the collagen has determined excellent biocompatibility and low immunogenicity, and has been widely used in medicine, health care products and cosmetics industry.
In the prior art, the main source of the collagen is animal tissue extraction, and along with the development of biotechnology, the recombinant collagen obtained by a microbial fermentation method has great achievements by utilizing a gene recombination technology, and compared with natural collagen, the recombinant collagen produced by utilizing the technology solves the defect of hidden danger of viruses existing in the traditional extraction method, and simultaneously obviously improves the stability, hydrophilicity and biocompatibility of the collagen.
At present, pichia pastoris engineering bacteria are generally used for fermentation culture to produce recombinant collagen; however, the existing fermentation culture medium is mainly BSM culture medium provided by Invitrogen company, and only glycerol is used in the general carbon source feeding stage of the fermentation stage, and only methanol is used in the induction expression stage; such fermentation processes have the following problems:
(1) The BSM culture medium is used for culturing seeds, the wet weight is 90-100g/L, the total bacterial load is low, the delay period is long after the seeds are moved into the fermentation culture medium, and the total production level is affected;
(2) The salt ion concentration of the BSM culture medium is too high, so that the growth of thalli is inhibited, the activity of strains is influenced, and the high-density fermentation level is difficult to reach quickly;
(3) Glycerol is viscous, and the excessively high duty ratio influences oxygen mass transfer and feed liquid uniformity;
(4) The high salt of BSM culture medium and the independent use of glycerol and methanol are easy to cause the decay of the bacterial cells in the middle and later period and the increase of protease secretion, and the protein degradation is aggravated, 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, components such as electrodes and the like are greatly damaged, and the service life of a fermentation tank is seriously influenced;
(6) The BSM culture medium, the feed glycerol and the methanol contain PTM1, the PTM1 is complex in preparation and cannot be sterilized at high temperature, and sterile filtration is needed, so that great trouble is caused for industrial amplification;
(7) When the pichia pastoris engineering bacteria maintain dissolved oxygen, pure oxygen is required to be introduced, so that on one hand, the production cost is increased, and on the other hand, the pure oxygen belongs to inflammable and explosive products, and safety accidents are easy to cause.
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 increasing the production level of recombinant collagen comprising the steps of:
s1: inoculating Pichia pastoris engineering bacteria into a seed tank for culture, adding glycerol and a feed supplement culture medium after dissolved oxygen is greatly risen, and transferring the seeds into a fermentation tank for fermentation culture when the wet weight is increased to be more than 100 g/L;
s2: culturing Pichia pastoris engineering bacteria in the fermentation tank until dissolved oxygen is greatly risen, and then using a carbon source culture medium and a feed supplement culture medium to carry out mixed carbon source feed supplement, and stopping mixed carbon source feed supplement when the wet weight of the material is increased to be more than 150 g/L;
s3: after the carbon source is exhausted, methanol and a feed medium are added for induction expression until the fermentation is finished.
Further, the pichia pastoris engineering bacteria fermentation medium used in the seed tank and the fermentation tank in the step S1 comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 0.47～1.175g/L，K 2 SO 4 7.28～18.2g/L，MgSO 4 ·7H 2 5.96-14.9 g/L of O, 1.625-4.13 g/L of KOH and 20.0-40.0 g/L of glycerol; glucose 20.0-40.0 g/L; (NaPO) 3 ) 6 6.5-13.0 g/L, and 3-7 g/L of white corn steep liquor dry powder.
Further, the pichia pastoris engineering bacteria fermentation medium used in the seed tank and the fermentation tank in the step S1 comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 0.5875～0.8225g/L，K 2 SO 4 9.1～12.74g/L，MgSO 4 ·7H 2 7.45-10.43 g/L of O, 2.07-2.891 g/L of KOH and 20.0-30.0 g/L of glycerol; glucose 20.0-30.0 g/L; (NaPO) 3 ) 6 6.5-9.1 g/L, and 3-5 g/L of white corn steep liquor dry powder.
Further, the feed medium comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 0.1～0.705g/L；K 2 SO 4 0.15～10.92g/L；MgSO 4 ·7H 2 O0.25-8.94 g/L; KOH 0.12-2.478 g/L; white corn steep liquor dry powder 0.3-4 g/L.
Further, the feed medium comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 0.25～0.5875g/L；K 2 SO 4 5.46～9.1g/L；MgSO 4 ·7H 2 O4.47-7.45 g/L; KOH 0.75-2.065 g/L; white corn steep liquor dry powder 2.1-3.5 g/L.
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 feed 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, culturing the Pichia pastoris engineering bacteria until the wet weight is increased to 160-180 g/L, and transferring the Pichia pastoris engineering bacteria to a fermentation tank; and in the step S2, culturing the pichia pastoris engineering bacteria until the wet weight is increased to 180-200 g/L, and stopping mixing the carbon source feed supplement.
Further, the carbon source medium in the 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 regulated to 4.8-5.5 by ammonia water, the dissolved oxygen is not lower than 30% before the induced expression, the tank pressure is 0.04-0.06 MPa, the dissolved oxygen is not lower than 5% after the induced expression, and the tank pressure is 0.08-0.1 MPa.
Further, the Pichia pastoris engineering bacteria are Pichia pastoris, and are preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 20458 in the 31 th month of 2020.
The technical scheme of the embodiment of the invention has at least the following advantages and beneficial effects:
1. the invention uses the compound fermentation medium and adopts glycerin and glucose to compound, on one hand, the viscosity of the feed liquid can be reduced, and meanwhile, the generation of excessive organic acid can be avoided to inhibit the growth of strains; the salt ion concentration is reduced, and the inhibition on the growth of strains is reduced; the wet weight of the strain is increased to 160-200 g/L, and the delay period after the strain is transferred into a fermentation culture medium is shortened; thereby effectively improving the growth speed of the strain and shortening the fermentation period.
2. Before the strain is transferred to the fermentation tank, glycerol and a feed medium are used for feeding, and in the carbon source feed stage after the strain is transferred to the fermentation tank, the carbon source medium and the feed medium are also used for mixing the carbon source feed, and in the induction expression stage, methanol and the feed medium are used for mixing the feed for induction expression, so that the ionic strength and the trace elements are supplemented in the fermentation process are improved, the generation of collagenase is inhibited, and the expression quantity of the recombinant collagen is effectively improved.
3. According to the invention, no phosphoric acid is added into the compounded fermentation medium, so that the damage to elements such as electrodes and the like caused by the excessively low initial pH value can be avoided, and the influence on the service life of the fermentation tank is avoided.
4. The fermentation medium, the feed medium, the glycerol, the carbon source medium and the methanol used in the invention are not added with PTM1, and the method is simple and convenient to operate and is more suitable for industrial production.
5. According to the invention, in the induction stage, the tank pressure is increased, the induction pressure is increased to maintain a certain dissolved oxygen in the tank, 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 more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The fermentation process for improving the production level of recombinant collagen provided by the embodiment of the invention is specifically described below.
The Pichia engineering bacteria are Pichia pastoris and are preserved in China general microbiological culture Collection center (CGMCC) No.20458 in the year 2020, month 07 and 31.
Example 1
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 seed tank containing 70L fermentation medium according to 10% of inoculation amount for culture, wherein the temperature is 28 ℃, the pH is regulated to 4.8 by ammonia water, the tank pressure is 0.06MPa, the dissolved oxygen is not lower than 30%, 50% of glycerol and a feed supplement medium are added after the dissolved oxygen is greatly risen, the feeding rate of the 50% of glycerol is 10mL/h/L, and the feeding rate of the feed supplement medium is 5mL/h/L; when the wet weight is increased to 160g/L, transferring the seeds to a 1500L fermentation tank containing 700L fermentation medium for fermentation culture, regulating the pH to 4.8 by ammonia water at 28 ℃, and controlling the tank pressure to be 0.06MPa and the dissolved oxygen to be not lower than 30%;
s2: culturing Pichia pastoris engineering bacteria in a fermentation tank until dissolved oxygen is greatly risen, and then using 50% glycerol and a feed medium to carry out mixed carbon source feed, wherein the feeding rate of the 50% glycerol is 8mL/h/L, and the feeding rate of the feed medium is 5mL/h/L; stopping mixing the carbon source feeding when the wet weight of the material is increased to 185 g/L;
s3: after glycerol is exhausted, methanol and a feed culture medium are added for induction expression until fermentation is finished, the methanol is dynamically regulated and fed in a range of 3-7 mL/h/L of feeding rate, the feeding rate of the feed culture medium is 3mL/h/L, the temperature is 28 ℃, the pH is regulated to 4.8 by ammonia water, the tank pressure is 0.08MPa, the dissolved oxygen is not lower than 5%, and the whole fermentation process is finished when the wet bacterial weight of pichia pastoris or the protein expression quantity is not increased any more.
The pichia pastoris engineering bacteria fermentation medium used in the seed tank and the fermentation tank comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 0.5875g/L，K 2 SO 4 8.05g/L，MgSO 4 ·7H 2 7.45g/L of O, 3.59g/L of KOH and 20.0g/L of glycerol; glucose 30.0g/L; (NaPO) 3 ) 6 7.1g/L, and 3g/L of white corn steep liquor dry powder.
The feed medium comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 0.1g/L；K 2 SO 4 5.46g/L；MgSO 4 ·7H 2 O7.45 g/L; KOH 0.23g/L; white corn steep liquor dry powder 3.5g/L.
Example 2
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 seed tank containing 50L fermentation medium according to an inoculum size of 6% for culture, wherein the temperature is 30 ℃, the pH is regulated to 5.5 by ammonia water, the tank pressure is 0.06MPa, the dissolved oxygen is not lower than 30%, 50% glycerol and a feed supplement medium are added after the dissolved oxygen is greatly risen, the feeding rate of the 50% glycerol is 16mL/h/L, and the feeding rate of the feed supplement medium is 6mL/h/L; when the wet weight is increased to 180g/L, transferring seeds to a 1500L fermentation tank containing 600L fermentation medium for fermentation culture, regulating the pH to 5.5 by ammonia water at 30 ℃, and controlling the tank pressure to be 0.06MPa and the dissolved oxygen to be not lower than 30%;
s2: culturing Pichia pastoris engineering bacteria in a fermentation tank until dissolved oxygen is greatly risen, and then using glucose and a feed culture medium to carry out mixed carbon source feed, wherein the feeding rate of the glucose is 9mL/h/L, and the feeding rate of the feed culture medium is 8mL/h/L; stopping mixing the carbon source feeding when the wet weight of the material is increased to 200 g/L;
s3: after the glucose is exhausted, methanol and a feed culture medium are added for induced expression until the fermentation is finished, the methanol is dynamically regulated and fed in a range of 3-7 mL/h/L of feeding rate, the feeding rate of the feed culture medium is 5mL/h/L, the temperature is 30 ℃, the pH is regulated 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 wet bacterial weight of pichia pastoris or the protein expression quantity is not increased any more.
The pichia pastoris engineering bacteria fermentation medium used in the seed tank and the fermentation tank comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 0.47g/L，K 2 SO 4 18.2g/L，MgSO 4 ·7H 2 O6.21 g/L, KOH 2.07g/L, glycerol 21.0g/L; glucose 22.0g/L; (NaPO) 3 ) 6 6.5g/L, and 3.5g/L of white corn steep liquor dry powder.
The feed medium comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 0.6217g/L；K 2 SO 4 0.15g/L；MgSO 4 ·7H 2 O6.55 g/L; KOH 0.12g/L; white corn steep liquor dry powder 2.1g/L.
Example 3
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 seed tank containing 60L fermentation medium according to an inoculum size of 8% for culture, wherein the temperature is 32 ℃, the pH is regulated to 5.0 by ammonia water, the tank pressure is 0.05MPa, the dissolved oxygen is not lower than 30%, 50% glycerol and a feed supplement medium are added after the dissolved oxygen is greatly risen, the feeding rate of the 50% glycerol is 13mL/h/L, and the feeding rate of the feed supplement medium is 6mL/h/L; when the wet weight is increased to 180g/L, transferring the seeds to a 1500L fermentation tank containing 650L fermentation medium for fermentation culture, regulating the pH to 5.0 by ammonia water at the temperature of 32 ℃, and controlling the tank pressure to be 0.05MPa and the dissolved oxygen to be not lower than 30%;
s2: culturing Pichia pastoris engineering bacteria in a fermentation tank until dissolved oxygen is greatly risen, and then using 50% glycerol and a feed medium to carry out mixed carbon source feed, wherein the feeding rate of the 50% glycerol is 15mL/h/L, and the feeding rate of the feed medium is 7mL/h/L; stopping mixing the carbon source feeding when the wet weight of the material is increased to 190 g/L;
s3: after glycerol is exhausted, methanol and a feed culture medium are added for induction expression until fermentation is finished, the methanol is dynamically regulated and fed in a range of 3-7 mL/h/L of feeding rate, the feeding rate of the feed culture medium is 4mL/h/L, the temperature is 32 ℃, the pH is regulated 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 wet bacterial weight of pichia pastoris or the protein expression quantity is not increased any more.
The pichia pastoris engineering bacteria fermentation medium used in the seed tank and the fermentation tank comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 1.175g/L，K 2 SO 4 16.15g/L，MgSO 4 ·7H 2 O5.96 g/L, KOH 3.92g/L and glycerol 30.0g/L; glucose 20.0g/L; (NaPO) 3 ) 6 8.8g/L, and 5g/L of white corn steep liquor dry powder.
The feed medium comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 0.19g/L；K 2 SO 4 8.56g/L；MgSO 4 ·7H 2 O0.25 g/L; KOH 2.065g/L; white corn steep liquor dry powder 0.4g/L.
Example 4
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 seed tank containing 70L fermentation medium according to 10% of inoculation amount for culture, wherein the temperature is 28 ℃, the pH is regulated to 5.2 by ammonia water, the tank pressure is 0.06MPa, the dissolved oxygen is not lower than 30%, 50% of glycerol and a feed supplement medium are added after the dissolved oxygen is greatly risen, the feeding rate of the 50% of glycerol is 18mL/h/L, and the feeding rate of the feed supplement medium is 7.5mL/h/L; when the wet weight is increased to 175g/L, transferring the seeds into a 1500L fermentation tank containing 700L fermentation medium to start fermentation culture, regulating the pH to 5.5 by ammonia water at 30 ℃, and controlling the tank pressure to be 0.05MPa and the dissolved oxygen to be not lower than 30%;
s2: culturing Pichia pastoris engineering bacteria in a fermentation tank until dissolved oxygen is greatly raised, and then using a mixed culture medium and a feeding culture medium of 50% glycerol and glucose according to a ratio of 1:1 to carry out mixed carbon source feeding, wherein the feeding rate of the 50% glycerol and glucose is 12mL/h/L, and the feeding rate of the feeding culture medium is 6.5mL/h/L; stopping mixing the carbon source feeding when the wet weight of the material is increased to 200 g/L;
s3: after glycerol and glucose are exhausted, methanol and a feed culture medium are added for induced expression until fermentation is finished, the methanol is dynamically regulated and fed in a range of 3-7 mL/h/L of feeding rate, the feeding rate of the feed culture medium is 4mL/h/L, the temperature is 32 ℃, the pH is regulated to 4.9 by ammonia water, the tank pressure is 0.09MPa, dissolved oxygen is not lower than 5%, and the whole fermentation process is finished when the wet bacterial weight of pichia pastoris or the protein expression quantity is not increased any more.
The pichia pastoris engineering bacteria fermentation medium used in the seed tank and the fermentation tank comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 0.8225g/L，K 2 SO 4 9.1g/L，MgSO 4 ·7H 2 9.15g/L of O, 4.13g/L of KOH and 26.0g/L of glycerol; glucose 23.0g/L; (NaPO) 3 ) 6 10.2g/L, white corn steep liquor dry powder 7g/L.
The feed medium comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 0.25g/L；K 2 SO 4 10.92g/L；MgSO 4 ·7H 2 O4.47 g/L; KOH 0.52g/L; white corn steep liquor dry powder 2.7g/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 seed tank containing 65L fermentation medium according to 9% of inoculum size, culturing at 28 ℃, regulating pH to 4.9 by ammonia water, carrying out tank pressure to 0.05MPa, adding 50% glycerol and a feed supplement medium after dissolved oxygen is greatly raised, wherein the feeding rate of the 50% glycerol is 12mL/h/L, and the feeding rate of the feed supplement medium is 8mL/h/L; when the wet weight is increased to 170g/L, transferring seeds into a 1500L fermentation tank containing 650L fermentation medium for fermentation culture, regulating the pH to 5.2 by ammonia water at 30 ℃, and controlling the tank pressure to 0.04MPa and the dissolved oxygen to be not lower than 30%;
s2: culturing Pichia pastoris engineering bacteria in a fermentation tank until dissolved oxygen is greatly risen, and then using 50% glycerol and a feed medium to carry out mixed carbon source feed, wherein the feeding rate of the 50% glycerol is 10mL/h/L, and the feeding rate of the feed medium is 7.5mL/h/L; stopping mixing the carbon source feeding when the wet weight of the material is increased to 185 g/L;
s3: after glycerol is exhausted, methanol and a feed culture medium are added for induction expression until fermentation is finished, the methanol is dynamically regulated and fed in a range of 3-7 mL/h/L of feeding rate, the feeding rate of the feed culture medium is 4.5mL/h/L, the temperature is 28 ℃, the pH is regulated to 5.2 by ammonia water, the tank pressure is 0.08MPa, dissolved oxygen is not lower than 5%, and the whole fermentation process is finished when the wet bacterial weight of pichia pastoris or the protein expression quantity is not increased any more.
The pichia pastoris engineering bacteria fermentation medium used in the seed tank and the fermentation tank comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 0.6521g/L，K 2 SO 4 7.28g/L，MgSO 4 ·7H 2 14.9g/L of O, 2.34g/L of KOH and 25.0g/L of glycerol; glucose 24.0g/L; (NaPO) 3 ) 6 13g/L, white corn steep liquor dry powder 6.5g/L.
The feed medium comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 0.4622g/L；K 2 SO 4 0.34g/L；MgSO 4 ·7H 2 O2.18 g/L; 2.478g/L KOH; white corn steep liquor dry powder 4g/L.
Example 6
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 seed tank containing 70L fermentation medium according to 10% of inoculation amount for culture, wherein the temperature is 30 ℃, the pH is regulated to 4.9 by ammonia water, the tank pressure is 0.05MPa, the dissolved oxygen is not lower than 30%, 50% of glycerol and a feed supplement medium are added after the dissolved oxygen is greatly risen, the feeding rate of the 50% of glycerol is 15mL/h/L, and the feeding rate of the feed supplement medium is 8mL/h/L; when the wet weight is increased to 165g/L, transferring seeds into a 1500L fermentation tank containing 650L fermentation medium to start fermentation culture, regulating pH to 5 by ammonia water at 30 ℃, and controlling the tank pressure to be 0.04MPa and the dissolved oxygen to be not lower than 30%;
s2: culturing Pichia pastoris engineering bacteria in a fermentation tank until dissolved oxygen is greatly risen, and then using 50% glycerol and a feed medium to carry out mixed carbon source feed, wherein the feeding rate of the 50% glycerol is 14mL/h/L, and the feeding rate of the feed medium is 6.5mL/h/L; stopping mixing the carbon source feeding when the wet weight of the material is increased to 180 g/L;
s3: after glycerol is exhausted, methanol and a feed culture medium are added for induction expression until fermentation is finished, the methanol is dynamically regulated and fed in a range of 3-7 mL/h/L of feeding rate, the feeding rate of the feed culture medium is 3.5mL/h/L, the temperature is 28 ℃, the pH is regulated to 5 by ammonia water, the tank pressure is 0.08MPa, the dissolved oxygen is not lower than 5%, and the whole fermentation process is finished when the wet bacterial weight of pichia pastoris or the protein expression quantity is not increased any more.
The pichia pastoris engineering bacteria fermentation medium used in the seed tank and the fermentation tank comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 0.4912g/L，K 2 SO 4 10.55g/L，MgSO 4 ·7H 2 12.42g/L of O, 1.625g/L of KOH and 27.0g/L of glycerol; glucose 21.0g/L; (NaPO) 3 ) 6 11.5g/L, white corn steep liquor dry powder 5g/L.
The feed medium comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 0.705g/L；K 2 SO 4 2.75g/L；MgSO 4 ·7H 2 O8.94 g/L; KOH 1.83g/L; white corn steep liquor dry powder 1.6g/L.
Example 7
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 seed tank containing 60L fermentation medium according to an inoculum size of 8% for culture, wherein the temperature is 29 ℃, the pH is regulated to 4.8 by ammonia water, the tank pressure is 0.05MPa, the dissolved oxygen is not lower than 30%, 50% glycerol and a feed supplement medium are added after the dissolved oxygen is greatly risen, the feeding rate of the 50% glycerol is 14mL/h/L, and the feeding rate of the feed supplement medium is 7mL/h/L; when the wet weight is increased to 175g/L, transferring the seeds into a 1500L fermentation tank containing 650L fermentation medium to start fermentation culture, regulating the pH to 4.9 by ammonia water at 28 ℃, and controlling the tank pressure to be 0.04MPa and the dissolved oxygen to be not lower than 30%;
s2: culturing Pichia pastoris engineering bacteria in a fermentation tank until dissolved oxygen is greatly risen, and then using 50% glycerol and a feed medium to carry out mixed carbon source feed, wherein the feeding rate of the 50% glycerol is 11mL/h/L, and the feeding rate of the feed medium is 8mL/h/L; stopping mixing the carbon source feeding when the wet weight of the material is increased to 190 g/L;
s3: after glycerol is exhausted, methanol and a feed culture medium are added for induction expression until fermentation is finished, the methanol is dynamically regulated and fed in a range of 3-7 mL/h/L of feeding rate, the feeding rate of the feed culture medium is 3mL/h/L, the temperature is 28 ℃, the pH is regulated to 4.9 by ammonia water, the tank pressure is 0.08MPa, the dissolved oxygen is not lower than 5%, and the whole fermentation process is finished when the wet bacterial weight of pichia pastoris or the protein expression quantity is not increased any more.
The pichia pastoris engineering bacteria fermentation medium used in the seed tank and the fermentation tank comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 0.5105g/L，K 2 SO 4 12.74g/L，MgSO 4 ·7H 2 13.6g/L of O, 2.891g/L of KOH and 22.0g/L of glycerol; glucose 26.0g/L; (NaPO) 3 ) 6 12.3g/L, and 6g/L of white corn steep liquor dry powder.
The feed medium comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 0.3521g/L；K 2 SO 4 9.1g/L；MgSO 4 ·7H 2 O0.51 g/L; KOH 1.15g/L; white corn steep liquor dry powder 3g/L.
Example 8
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 seed tank containing 75L fermentation medium according to an inoculum size of 8% for culture, wherein the temperature is 30 ℃, the pH is adjusted to 5 by ammonia water, the tank pressure is 0.05MPa, the dissolved oxygen is not lower than 30%, 50% glycerol and a feed supplement medium are added after the dissolved oxygen is greatly risen, the feeding rate of the 50% glycerol is 12mL/h/L, and the feeding rate of the feed supplement medium is 6.5mL/h/L; when the wet weight is increased to 170g/L, transferring seeds into a 1500L fermentation tank containing 650L fermentation medium to start fermentation culture, regulating pH to 5 with ammonia water at 30 ℃, and controlling the tank pressure to 0.04MPa and the dissolved oxygen to be not lower than 30%;
s2: culturing Pichia pastoris engineering bacteria in a fermentation tank until dissolved oxygen is greatly risen, and then using 50% glycerol and a feed medium to carry out mixed carbon source feed, wherein the feeding rate of the 50% glycerol is 15mL/h/L, and the feeding rate of the feed medium is 5.5mL/h/L; stopping mixing the carbon source feeding when the wet weight of the material is increased to 195 g/L;
s3: after glycerol is exhausted, methanol and a feed culture medium are added for induction expression until fermentation is finished, the methanol is dynamically regulated and fed in a range of 3-7 mL/h/L of feeding rate, the feeding rate of the feed culture medium is 4mL/h/L, the temperature is 29 ℃, the pH is regulated to 5.2 by ammonia water, the tank pressure is 0.08MPa, the dissolved oxygen is not lower than 5%, and the whole fermentation process is finished when the wet bacterial weight of pichia pastoris or the protein expression quantity is not increased any more.
The pichia pastoris engineering bacteria fermentation medium used in the seed tank and the fermentation tank comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 0.9815g/L，K 2 SO 4 14.1g/L，MgSO 4 ·7H 2 10.43g/L O, 1.91g/L KOH and 28.0g/L glycerol; glucose 20.0g/L; (NaPO) 3 ) 6 9.1g/L, and 4g/L of white corn steep liquor dry powder.
The feed medium comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 0.5875g/L；K 2 SO 4 6.52g/L；MgSO 4 ·7H 2 O8.11 g/L; KOH 0.75g/L; white corn steep liquor dry powder 3.2g/L.
Comparative example
The comparative example provides a fermentation process of recombinant collagen, comprising the following steps:
s1: inoculating Pichia pastoris engineering bacteria into a 100L seed tank containing 70L fermentation medium according to 10% of inoculum size, culturing at 30 ℃, regulating pH to 4.6 by ammonia water, carrying out tank pressure to 0.03MPa, and after dissolved oxygen is greatly raised, transferring the Pichia pastoris engineering bacteria into a 1500L fermentation tank containing 700L fermentation medium for fermentation culture, wherein the temperature is 30 ℃, the pH is regulated to 4.6 by ammonia water, the tank pressure is 0.03MPa, and the dissolved oxygen is not lower than 30%;
s2: culturing Pichia pastoris engineering bacteria in a fermentation tank until dissolved oxygen is greatly risen, and then using 50% glycerol for carbon source feeding, wherein the feeding rate of the 50% glycerol is 6mL/h/L; stopping mixing the 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 fermentation is finished, wherein the flow rate of the methanol is 8mL/h/L, the temperature is 30 ℃, the pH is regulated to 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%, and finishing the whole fermentation process when the wet bacterial weight of pichia pastoris or the protein expression quantity is not increased any more.
The pichia pastoris engineering bacteria fermentation medium used in the seed tank and the fermentation tank comprises the following components in concentration: 85% H 3 PO 4 26.7mL/L；CaSO 4 ·2H 2 O 1.175g/L；K 2 SO 4 18.2g/L；MgSO 4 ·7H 2 O14.9 g/L; KOH 4.13g/L; glycerol 40.0g/L; PTM 1.35 mL/L.
Wherein, PTM1 comprises the following concentration components: cuSO 4 ·5H 2 O 6.0g/L；NaI 0.08g/L；MnSO 4 ·H 2 O 3.0g/L；NaMoO 4 ·2H 2 O 0.2g/L；H 3 BO 3 0.02g/L；CoCl 2 0.5g/L；ZnCl 2 20.0g/L；FeSO 4 ·7H 2 O65.0 g/L; biotin 0.2g/L; h 2 SO 4 5.0mL/L. The solution was sterilized by filtration through a 0.22 μm filter membrane and stored at room temperature.
Experimental example
Taking fermentation liquids obtained after fermentation of examples 1-8 and comparative example, detecting the concentration of recombinant collagen by HPLC, recording fermentation periods of examples 1-8 and comparative example, and calculating 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, the fermentation method used in the present invention, examples 1 to 8, compared with the comparative example, produced recombinant collagen at a higher concentration and a shorter fermentation period, resulted in a higher fermentation production level of recombinant collagen.
In summary, the fermentation process for improving the production level of the recombinant collagen provided by the application uses a fermentation medium which is improved relative to a BSM medium used in the prior art, and in the fermentation process, before a strain is transferred to a fermentation tank, glycerol and a feed supplement medium are used for supplementing materials, in a carbon source feed supplement stage after the strain is transferred to the fermentation tank, a carbon source culture medium and a feed supplement medium are also used for carrying out mixed carbon source feed supplement, methanol and the feed supplement medium are used for carrying out mixed feed supplement induction expression in an induction expression stage, so that the expression level 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 of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A fermentation process for increasing the production level of recombinant collagen comprising the steps of:
s1: inoculating Pichia pastoris engineering bacteria into a seed tank for culture, adding glycerol and a feed supplement culture medium after dissolved oxygen is greatly risen, and transferring the seeds into a fermentation tank for fermentation culture when the wet weight is increased to be more than 100 g/L;
s2: culturing Pichia pastoris engineering bacteria in the fermentation tank until dissolved oxygen is greatly risen, and then using a carbon source culture medium and a feed supplement culture medium to carry out mixed carbon source feed supplement, and stopping mixed carbon source feed supplement when the wet weight of the material is increased to be more than 150 g/L;
s3: after the carbon source is exhausted, methanol and a feed medium are added for induction expression until fermentation is finished;
pichia pastoris engineering bacteria with the preservation number of CGMCC No.20458;
the pichia pastoris engineering bacteria fermentation medium used in the seed tank and the fermentation tank in the step S1 comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 0.47~1.175g/L，K 2 SO 4 7.28~18.2g/L，MgSO 4 ·7H 2 5.96-14.9 g/L of O, 1.625-4.13 g/L of KOH and 20.0-40.0 g/L of glycerol; glucose 20.0-40.0 g/L; (NaPO) 3 ) 6 6.5-13.0 g/L, and 3-7 g/L of white corn steep liquor dry powder;
the feed medium comprises the following components in concentration: caSO (Caso-like conductor) 4 ·2H 2 O 0.1~0.705g/L；K 2 SO 4 0.15~10.92g/L；MgSO 4 ·7H 2 O is 0.25-8.94 g/L; 0.12-2.478 g/L of KOH; 0.3-4 g/L of white corn steep liquor dry powder;
the carbon source culture medium in the step S2 is glycerol, glucose or a glycerol and glucose mixed culture medium;
the conditions of the fermentation culture are as follows: the fermentation temperature is 28-32 ℃, the pH is regulated to 4.8-5.5 by ammonia water, the dissolved oxygen is not lower than 30% before the induced expression, the tank pressure is 0.04-0.06 MPa, the dissolved oxygen is not lower than 5% after the induced expression, and the tank pressure is 0.08-0.1 MPa.
2. The fermentation method for increasing the production level of recombinant collagen according to claim 1, wherein the pichia pastoris engineered fermentation medium used in the seed tank and the fermentor in step S1 comprises the following components in the following concentrations: caSO (Caso-like conductor) 4 ·2H 2 O 0.5875~0.8225g/L，K 2 SO 4 9.1~12.74g/L，MgSO 4 ·7H 2 7.45-10.43 g/L of O, 2.07-2.891 g/L of KOH and 20.0-30.0 g/L of glycerol; glucose 20.0-30.0 g/L; (NaPO) 3 ) 6 6.5-9.1 g/L, and 3-5 g/L of white corn steep liquor dry powder.
3. The method of claim 1, wherein the feed medium comprises the following concentrations of components: caSO (Caso-like conductor) 4 ·2H 2 O 0.25~0.5875g/L；K 2 SO 4 5.46~9.1g/L；MgSO 4 ·7H 2 O4.47-7.45 g/L; KOH 0.75-2.065 g/L; white corn steep liquor dry powder 2.1-3.5 g/L.
4. The fermentation method for improving the production level of the recombinant collagen according to claim 1, wherein 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 feed medium in the step S3 is 3-5 mL/h/L, and the feeding rate of the methanol is 3-7 mL/h/L.
5. The fermentation method for improving the production level of the recombinant collagen according to claim 1, wherein 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.
6. The fermentation method for improving the production level of the recombinant collagen according to claim 1, wherein in the step S1, the pichia pastoris engineering bacteria are cultivated until the wet weight is increased to 160-180 g/L, and the pichia pastoris engineering bacteria are transplanted to a fermentation tank; and in the step S2, culturing the pichia pastoris engineering bacteria until the wet weight is increased to 180-200 g/L, and stopping mixing the carbon source feed supplement.
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