CN113564062A - Fermentation process for shortening culture time and improving collagen content - Google Patents
Fermentation process for shortening culture time and improving collagen content Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 90
- 230000004151 fermentation Effects 0.000 title claims abstract description 90
- 102000008186 Collagen Human genes 0.000 title claims abstract description 21
- 108010035532 Collagen Proteins 0.000 title claims abstract description 21
- 229920001436 collagen Polymers 0.000 title claims abstract description 21
- 238000004904 shortening Methods 0.000 title claims abstract description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 107
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 93
- 230000006698 induction Effects 0.000 claims abstract description 21
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 40
- 229910052760 oxygen Inorganic materials 0.000 claims description 40
- 239000001301 oxygen Substances 0.000 claims description 40
- 235000011187 glycerol Nutrition 0.000 claims description 34
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 17
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 17
- 238000009423 ventilation Methods 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 9
- 238000011081 inoculation Methods 0.000 claims description 8
- 101150051118 PTM1 gene Proteins 0.000 claims description 7
- 239000001963 growth medium Substances 0.000 claims description 4
- 239000000463 material Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 12
- 230000015572 biosynthetic process Effects 0.000 abstract description 5
- 238000003786 synthesis reaction Methods 0.000 abstract description 5
- 239000002028 Biomass Substances 0.000 abstract description 2
- 241001052560 Thallis Species 0.000 abstract description 2
- 239000006052 feed supplement Substances 0.000 abstract description 2
- 239000000047 product Substances 0.000 abstract description 2
- 238000007792 addition Methods 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 241000235058 Komagataella pastoris Species 0.000 description 6
- 238000005070 sampling Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 238000000034 method Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229910018890 NaMoO4 Inorganic materials 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 229910052927 chalcanthite Inorganic materials 0.000 description 1
- 230000037319 collagen production Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229910052602 gypsum Inorganic materials 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- -1 tissue engineering Substances 0.000 description 1
- 239000011592 zinc chloride Substances 0.000 description 1
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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Abstract
The invention discloses a fermentation process for shortening culture time and improving collagen content, which has the innovation points that the addition amount and the addition mode of glycerol are optimized, the feed supplement amount of the glycerol is increased, the biomass before induction is improved, simultaneously, the glycerol is mixed in methanol for induction in the methanol induction stage, the growth speed and the product synthesis speed of thalli are improved, the culture time is shortened, the fermentation level is improved by 39.50%, the production cost is saved, the fermentation efficiency is improved, the fermentation process is suitable for large-scale fermentation production, and the economic benefit is improved for production enterprises.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to a fermentation process for shortening culture time and improving collagen content.
Background
The collagen has the largest content and the most widely distributed functional protein in a human body, which accounts for 25-33% of the total amount of the protein, and has good biocompatibility, biodegradability and bioactivity, so that the collagen is widely applied to the fields of food, medicine, tissue engineering, cosmetics and the like, and the development of artificial synthesis of the collagen is a very significant matter.
The recombinant pichia pastoris exogenous expression collagen is a common collagen production mode at present, has the characteristics of high expression quantity, large-scale production, no animal-derived virus pollution risk and the like, and has long fermentation time, high energy consumption and complicated fermentation process in the fermentation process, so that the development of a method for ensuring the yield and shortening the culture time is particularly important.
Disclosure of Invention
The invention aims to provide a fermentation process for shortening the culture time and improving the content of collagen, which improves the growth rate of pichia pastoris, accelerates the synthesis of collagen, shortens the fermentation period and reduces the degradation level of collagen by optimizing the using amount of glycerol before and after induction.
The technical scheme of the invention is as follows:
step 1): inoculating the seed liquid into a 5L fermentation culture medium (10L fermentation tank) according to the inoculation amount of 10%, wherein the fermentation temperature is 28 ℃, the tank pressure is 0.05Mpa, the pH value of ammonia water is controlled to be 4.5-5.0, the rotating speed, the ventilation quantity and the feeding are adjusted to control the dissolved oxygen to be more than 35.0%, and when the dissolved oxygen rises suddenly, the glycerol feeding stage is carried out, 50% of glycerol is fed, and the feeding amount is 90-160 g/L.
Step 2): after the feeding is finished, when the dissolved oxygen is more than 80%, entering a methanol induction stage, adjusting the feeding speed of methanol (containing PTM 14.35mL/L) through dissolved oxygen feedback, adding 0-15% of glycerin into the methanol, and controlling the dissolved oxygen by 20%.
Preferably, in step 1), 50% glycerol is added in a stream, and 4.35mL/L of PTM1 is added. Through a large number of experiments, the applicant finds that the growth rate of pichia pastoris can be increased and collagen synthesis can be accelerated by adding the PTM1 when 50% of glycerin is fed in the previous step.
For example, chinese patent application CN111733200A discloses a fermentation process for improving the production level of recombinant collagen by adjusting the addition of PTM1, which comprises the steps of inoculating pichia pastoris strain liquid into a sterilized fermentation culture medium, after fermentation culture for 14-18 hours, beginning to supplement methanol for induction expression, and simultaneously adding PTM1 into the fermentation culture medium in a flowing manner, which is different from the present invention in that the PTM1 is added in the methanol induction stage.
Preferably, in step 1), the optimal glycerol addition amount is 120 g/L.
Preferably, in the step 2) methanol induction stage, the ammonia water is controlled to have a pH of 6.0.
Preferably, in the step 2) methanol induction stage, the addition amount of glycerol is 10%.
The pichia pastoris is GS115 pichia pastoris expression strain, is purchased from Beijing Baiohobowei biotechnology limited company and is a public strain.
The fermentation medium refers to an operation manual of Invitrogen company, and the specific formula is as follows: 85% H3PO426.7mL/L;CaSO4·2H2O 1.175g/L;K2SO418.2g/L;MgSO4·7H2O14.9 g/L; KOH 4.13 g/L; 40.0g/L of glycerol; PTM 14.35mL/L.
The PTM1 refers to an operation manual of Invitrogen company, and the specific formula is as follows: CuSO4·5H2O 6.0g/L;NaI 0.08g/L;MnSO4·H2O 3.0g/L;NaMoO4·2H2O 0.2g/L;H3BO30.02g/L;CoCl20.5g/L;ZnCl2 20.0g/L;FeSO47H2O 65.0.0 g/L; biotin 0.2 g/L; h2SO45.0mL/L, filter sterilized with a 0.22 μm filter and stored at 4 ℃.
Compared with the prior art, the invention has the following advantages:
in the fermentation process, the fermentation process for shortening the culture time and improving the collagen content is obtained by optimizing the addition amount and the addition mode of the glycerol. The method has the innovation points that the feed supplement amount of glycerol is increased, the biomass before induction is improved, and meanwhile, in the methanol induction stage, the glycerol is mixed in the methanol for induction, so that the growth speed and the product synthesis speed of the thalli are improved, the culture time is shortened, the fermentation level is improved by 39.50%, the production cost is saved, the fermentation efficiency is improved, the method is suitable for large-scale fermentation production, and the economic value is created for enterprises.
Detailed Description
The invention provides the following 5 examples and 1 comparative example, which are different in addition amount and addition mode of glycerol and are compared with fermentation results of various modes by setting different fermentation processes.
Example 1
Inoculating the seed liquid into a 5L fermentation medium (10L fermentation tank) according to the inoculation amount of 10%, wherein the fermentation temperature is 28 ℃, the tank pressure is 0.05Mpa, the pH value of ammonia water is controlled to be 4.5-5.0, the rotating speed, the ventilation quantity and the feeding are adjusted to control the dissolved oxygen to be more than 35.0%, when the dissolved oxygen is suddenly increased, the glycerol fed-batch stage is carried out, 600g of 50% glycerol is fed-batch, and the fermentation time is 19 h.
After the feeding is finished, when the dissolved oxygen is more than 80%, entering a methanol induction stage, controlling the fermentation temperature to be 28 ℃, the tank pressure to be 0.06Mpa, controlling the pH value of ammonia water to be 6.0, keeping the rotating speed and the ventilation volume unchanged, adjusting the feeding speed of methanol (containing PTM 14.35mL/L) by dissolved oxygen feedback, controlling the dissolved oxygen to be 20%,
sampling and detecting every 1h at the end of fermentation until OD is reached600And (5) stopping fermentation without increasing, and obtaining supernatant.
HPLC detection shows that the final collagen concentration is 14.20g/L, the fermentation period is 108h, and the fermentation production level is 0.131 g/L.h, which is 10.01% higher than that of comparative example 1.
Example 2
Inoculating the seed liquid into a 5L fermentation medium (10L fermentation tank) according to the inoculation amount of 10%, wherein the fermentation temperature is 28 ℃, the tank pressure is 0.05Mpa, the pH value of ammonia water is controlled to be 4.5-5.0, the rotating speed, the ventilation quantity and the feeding are adjusted to control the dissolved oxygen to be more than 35.0%, when the dissolved oxygen is suddenly increased, the glycerol feeding stage is carried out, 800g of 50% glycerol is fed, and the fermentation time is 21 h.
After the feeding is finished, when the dissolved oxygen is more than 80%, entering a methanol induction stage, controlling the fermentation temperature to be 28 ℃, the tank pressure to be 0.06MPa, controlling the pH value of ammonia water to be 6.0, keeping the rotating speed and the ventilation quantity unchanged, adjusting the feeding speed of methanol (containing PTM 14.35mL/L) through dissolved oxygen feedback, and controlling the dissolved oxygen to be 20%.
Sampling and detecting every 1h at the end of fermentation until OD is reached600And (5) stopping fermentation without increasing, and obtaining supernatant.
HPLC detection shows that the final collagen concentration is 13.91g/L, the fermentation period is 112h, and the fermentation production level is 0.124 g/L.h, which is improved by 4.20% compared with the comparative example 1.
Example 3
Inoculating the seed liquid into a 5L fermentation medium (10L fermentation tank) according to the inoculation amount of 10%, wherein the fermentation temperature is 28 ℃, the tank pressure is 0.05Mpa, the pH value of ammonia water is controlled to be 4.5-5.0, the rotating speed, the ventilation quantity and the feeding are adjusted to control the dissolved oxygen to be more than 35.0%, when the dissolved oxygen is suddenly increased, the glycerol fed-batch stage is carried out, 600g of 50% glycerol is fed-batch, and the fermentation time is 19 h.
After the feeding is finished, when the dissolved oxygen is more than 80%, entering a methanol induction stage, controlling the fermentation temperature to be 28 ℃, the tank pressure to be 0.06MPa, controlling the pH value of ammonia water to be 6.0, keeping the rotating speed and the ventilation quantity unchanged, adjusting the feeding speed of methanol (containing PTM 14.35mL/L) through dissolved oxygen feedback, adding 5% of glycerol into the methanol, and controlling the dissolved oxygen to be 20%.
Sampling and detecting every 1h at the end of fermentation until OD is reached600And (5) stopping fermentation without increasing, and obtaining supernatant.
HPLC detection shows that the final collagen concentration is 15.22g/L, the fermentation period is 107h, and the fermentation production level is 0.142 g/L.h, which is improved by 19.33% compared with the comparative example 1.
Example 4
Inoculating the seed liquid into a 5L fermentation medium (10L fermentation tank) according to the inoculation amount of 10%, wherein the fermentation temperature is 28 ℃, the tank pressure is 0.05Mpa, the pH value of ammonia water is controlled to be 4.5-5.0, the rotating speed, the ventilation quantity and the feeding are adjusted to control the dissolved oxygen to be more than 35.0%, when the dissolved oxygen is suddenly increased, the glycerol fed-batch stage is carried out, 600g of 50% glycerol is fed-batch, and the fermentation time is 19 h.
After the feeding is finished, when the dissolved oxygen is more than 80%, entering a methanol induction stage, controlling the fermentation temperature to be 28 ℃, the tank pressure to be 0.06MPa, controlling the pH value of ammonia water to be 6.0, keeping the rotating speed and the ventilation quantity unchanged, adjusting the feeding speed of methanol (containing PTM 14.35mL/L) through dissolved oxygen feedback, adding 10% of glycerol into the methanol, and controlling the dissolved oxygen to be 20%.
Sampling and detecting every 1h at the end of fermentation until OD is reached600And (5) stopping fermentation without increasing, and obtaining supernatant.
HPLC detection shows that the final collagen concentration is 16.91g/L, the fermentation period is 102h, and the fermentation production level is 0.166 g/L.h, which is 39.50% higher than that of comparative example 1.
Example 5
Inoculating the seed liquid into a 5L fermentation medium (10L fermentation tank) according to the inoculation amount of 10%, wherein the fermentation temperature is 28 ℃, the tank pressure is 0.05Mpa, the pH value of ammonia water is controlled to be 4.5-5.0, the rotating speed, the ventilation quantity and the feeding are adjusted to control the dissolved oxygen to be more than 35.0%, when the dissolved oxygen is suddenly increased, the glycerol fed-batch stage is carried out, 600g of 50% glycerol is fed-batch, and the fermentation time is 19 h.
After the feeding is finished, when the dissolved oxygen is more than 80%, entering a methanol induction stage, controlling the fermentation temperature to be 28 ℃, the tank pressure to be 0.06MPa, controlling the pH value of ammonia water to be 6.0, keeping the rotating speed and the ventilation quantity unchanged, adjusting the feeding speed of methanol (containing PTM 14.35mL/L) through dissolved oxygen feedback, adding 15% of glycerin into the methanol, and controlling the dissolved oxygen to be 20%.
Sampling and detecting every 1h at the end of fermentation until OD is reached600And (4) stopping fermentation without increasing, obtaining supernatant, and detecting by HPLC (high performance liquid chromatography), wherein the final collagen concentration is 14.74g/L, the fermentation period is 102h, and the fermentation production level is 0.145 g/L.h, which is 21.85% higher than that of the comparative example 1.
Comparative example 1
Inoculating the seed liquid into a 5L fermentation medium (10L fermentation tank) according to the inoculation amount of 10%, wherein the fermentation temperature is 28 ℃, the tank pressure is 0.05Mpa, the pH value of ammonia water is controlled to be 4.5-5.0, the rotating speed, the ventilation quantity and the feeding are adjusted to control the dissolved oxygen to be more than 35.0%, when the dissolved oxygen is suddenly increased, the glycerol fed-batch stage is carried out, 400g of 50% glycerol is fed-batch, and the fermentation time is 17 h.
After the feeding is finished, when the dissolved oxygen is more than 80%, entering a methanol induction stage, controlling the fermentation temperature to be 28 ℃, the tank pressure to be 0.06MPa, controlling the pH value of ammonia water to be 6.0, keeping the rotating speed and the ventilation quantity unchanged, adjusting the feeding speed of methanol (containing PTM 14.35mL/L) through dissolved oxygen feedback, and controlling the dissolved oxygen to be 20%.
Sampling and detecting every 1h at the end of fermentation until OD is reached600And (5) stopping fermentation without increasing, and obtaining supernatant.
HPLC detection shows that the final collagen concentration is 13.40g/L, the fermentation period is 113h, and the fermentation production level is 0.119 g/L.h.
It is to be understood that the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and those skilled in the art should understand that they can make various changes, modifications, additions and substitutions within the spirit and scope of the present invention.
Claims (5)
1. A fermentation process for shortening culture time and improving collagen content is characterized by comprising the following specific steps:
step 1): inoculating the seed liquid into a 5L fermentation culture medium according to the inoculation amount of 10%, wherein the fermentation temperature is 28 ℃, the tank pressure is 0.05Mpa, the pH value of ammonia water is controlled to be 4.5-5.0, the dissolved oxygen is controlled to be more than 35.0% by adjusting the rotating speed, the ventilation quantity and the feeding material, and when the dissolved oxygen rises suddenly, the glycerol feeding stage is carried out, 50% of glycerol is fed, and the feeding amount is 90-160 g/L;
step 2): after the feeding is finished, when the dissolved oxygen is more than 80%, entering a methanol induction stage, controlling the fermentation temperature to be 28 ℃, the tank pressure to be 0.06MPa, controlling the pH value of ammonia water to be 5.5-6.0, keeping the rotating speed and the ventilation quantity unchanged, adjusting the feeding speed of methanol (containing PTM 14.35mL/L) through dissolved oxygen feedback, adding 0-15% of glycerin into the methanol, and controlling the dissolved oxygen to be 20%.
2. The fermentation process according to claim 1, wherein in step 1), 50% glycerol is fed-batch, and 4.35mL/L of PTM1 is added.
3. The fermentation process of claim 1, wherein in step 1), the optimal glycerol addition is 120 g/L.
4. The fermentation process of claim 1, wherein the ammonia water is controlled to have a pH of 6.0 during the methanol induction step.
5. The fermentation process of claim 1, wherein the glycerol is added in an amount of 10% during the methanol induction phase.
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CN114045321A (en) * | 2021-12-24 | 2022-02-15 | 江苏江山聚源生物技术有限公司 | Application of glutathione in fermentation production of recombinant human collagen |
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