CN106520869A - Fermentation method and fermentation medium for recombinant human III type collagen engineering bacteria - Google Patents
Fermentation method and fermentation medium for recombinant human III type collagen engineering bacteria Download PDFInfo
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- CN106520869A CN106520869A CN201611119122.8A CN201611119122A CN106520869A CN 106520869 A CN106520869 A CN 106520869A CN 201611119122 A CN201611119122 A CN 201611119122A CN 106520869 A CN106520869 A CN 106520869A
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- 238000000855 fermentation Methods 0.000 title claims abstract description 245
- 230000004151 fermentation Effects 0.000 title claims abstract description 245
- 241000894006 Bacteria Species 0.000 title claims abstract description 99
- 238000000034 method Methods 0.000 title abstract description 22
- 108010035532 Collagen Proteins 0.000 title abstract description 18
- 102000008186 Collagen Human genes 0.000 title abstract description 17
- 229920001436 collagen Polymers 0.000 title abstract description 15
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 104
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 14
- 229910052925 anhydrite Inorganic materials 0.000 claims abstract description 14
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 claims abstract description 14
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims abstract description 14
- 229910052939 potassium sulfate Inorganic materials 0.000 claims abstract description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 102
- 102000001187 Collagen Type III Human genes 0.000 claims description 100
- 108010069502 Collagen Type III Proteins 0.000 claims description 100
- 238000005215 recombination Methods 0.000 claims description 100
- 230000006798 recombination Effects 0.000 claims description 100
- 239000007788 liquid Substances 0.000 claims description 83
- 239000012531 culture fluid Substances 0.000 claims description 73
- 239000006052 feed supplement Substances 0.000 claims description 51
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 40
- 239000001301 oxygen Substances 0.000 claims description 40
- 229910052760 oxygen Inorganic materials 0.000 claims description 40
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims description 13
- 229910004619 Na2MoO4 Inorganic materials 0.000 claims description 13
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 13
- 229910052927 chalcanthite Inorganic materials 0.000 claims description 13
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 13
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 13
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 13
- 229910052603 melanterite Inorganic materials 0.000 claims description 13
- 239000011684 sodium molybdate Substances 0.000 claims description 13
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 13
- 239000011592 zinc chloride Substances 0.000 claims description 13
- 229910052564 epsomite Inorganic materials 0.000 claims description 12
- 230000000630 rising effect Effects 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 9
- 102000004169 proteins and genes Human genes 0.000 abstract description 8
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 7
- 230000008569 process Effects 0.000 abstract description 7
- 239000000463 material Substances 0.000 abstract description 4
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 abstract 1
- 235000011007 phosphoric acid Nutrition 0.000 abstract 1
- 238000007792 addition Methods 0.000 description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 18
- 230000001939 inductive effect Effects 0.000 description 17
- 230000000052 comparative effect Effects 0.000 description 15
- 239000002609 medium Substances 0.000 description 14
- 229910001868 water Inorganic materials 0.000 description 14
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 10
- 235000015097 nutrients Nutrition 0.000 description 10
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- 238000004659 sterilization and disinfection Methods 0.000 description 7
- 239000000306 component Substances 0.000 description 6
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- 229910021529 ammonia Inorganic materials 0.000 description 5
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- 241000235058 Komagataella pastoris Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000001186 cumulative effect Effects 0.000 description 4
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- 238000011081 inoculation Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
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- 239000000126 substance Substances 0.000 description 3
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- 108010000912 Egg Proteins Proteins 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
- 241001597008 Nomeidae Species 0.000 description 2
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- 229910052799 carbon Inorganic materials 0.000 description 2
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- 235000014103 egg white Nutrition 0.000 description 2
- 210000000969 egg white Anatomy 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
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- 238000010257 thawing Methods 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 108010046845 tryptones Proteins 0.000 description 2
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- 241000282412 Homo Species 0.000 description 1
- 241000235061 Pichia sp. Species 0.000 description 1
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- 230000006872 improvement Effects 0.000 description 1
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- 238000003259 recombinant expression Methods 0.000 description 1
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- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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Abstract
The invention discloses a fermentation method and fermentation medium for recombinant human III type collagen engineering bacteria and belongs to the technical field of engineering bacteria fermentation. The fermentation method comprises the following steps that the recombinant human III type collagen engineering bacteria are inoculated to a basic fermentation culture solution to conduct fermental cultivation, and the basic fermentation culture solution comprises, by liter, 25-27 ml of H3PO4, 0.8-1.0 g of CaSO4, 17-19 g of K2SO4, 13-15.5 g of MgSO4.7H2O, 3.8-4.3 g of KOH, 37-43 g of glycerinum and 3.5-4.2 ml of a first microelement solution; and in the fermental cultivation process, supplementary materials containing glycerinum and a second microelement solution are added into the basic fermentation culture solution. By optimizing the basic fermentation culture solution and the component content of the supplementary materials, the protein expressing quantity of the recombinant human III type collagen engineering bacteria is greatly improved by the fermentation method, and the method can be applicable to industrial large scale production.
Description
Technical field
The present invention relates to engineering bacterium fermentation technical field, in particular to a kind of recombination human source type III collagen protein work
The fermentation process and fermentation medium of journey bacterium.
Background technology
At present, the existing Escherichia coli fermentation method for producing human collagen (Human Collagen) is main
It is to be produced with inclusion bodies, although the method can form high yield, in terms of renaturing inclusion bodies and subsequent purification
Complicated loaded down with trivial details technological process can be formed, the large-scale production of target protein is limited.Also soluble expression systems use at present
In the human collagen for producing solubility, although, avoid the problem of inclusion body.But, target product human collagen
Yield is very low, is not enough to carry out large-scale production.
The content of the invention
It is an object of the invention to provide a kind of fermentation process of recombination human source type III collagen protein engineering bacteria, the fermentation
Method can improve the expression of the recombination human source type III collagen protein of recombination human source type III collagen protein engineering bacteria.
Another object of the present invention is to provide a kind of fermentation medium, the fermentation medium can be applied to recombination human source
The fermentation of type III collagen protein engineering bacteria, it is possible to increase improve the recombination human source of recombination human source type III collagen protein engineering bacteria
The expression of type III collagen protein.
What the present invention was realized in：
A kind of fermentation process of recombination human source type III collagen protein engineering bacteria, which includes：By recombination human source type III collagen
Protein engineering bacterium is seeded in basal fermentation culture fluid, carries out fermentation culture, and basal fermentation culture fluid includes based on per liter：H3PO4
25-27ml、CaSO4 0.8-1.0g、K2SO4 17-19g、MgSO4·7H2O 13-15.5g, KOH 3.8-4.3g, glycerol 37-
43g and the first liquid microelement 3.5-4.2ml；
During fermentation culture, feed supplement is added toward basal fermentation culture fluid, feed supplement includes glycerol and second micro
Element liquid；
Wherein, the first liquid microelement and the second liquid microelement include based on per liter：CuSO4·5H2O 5-7g、NaI
0.06-0.09g、MnSO4·H2O 2.5-3.2g、Na2MoO4·2H2O 0.1-0.3g、H3BO3 0.01-0.03g、CoCl20.4-
0.6g、ZnCl218-22g、FeSO4·7H2O 60-68g、VH0.1-0.3g and H2SO44-6ml。
A kind of fermentation medium, which includes basal fermentation culture fluid and feed supplement, and basal fermentation culture fluid includes based on per liter：
H3PO4 25-27ml、CaSO4 0.8-1.0g、K2SO4 17-19g、MgSO4·7H2It is O 13-15.5g, KOH 3.8-4.3g, sweet
Oily 37-43g and the first liquid microelement 3.5-4.2ml, feed supplement include glycerol and the second liquid microelement；
First liquid microelement and the second liquid microelement include based on per liter：CuSO4·5H2O 5-7g、NaI
0.06-0.09g、MnSO4·H2O 2.5-3.2g、Na2MoO4·2H2O 0.1-0.3g、H3BO3 0.01-0.03g、CoCl20.4-
0.6g、ZnCl218-22g、FeSO4·7H2O 60-68g、VH0.1-0.3g and H2SO44-6ml。
Compared with prior art, the invention has the beneficial effects as follows：
The fermentation process of the recombination human source type III collagen protein engineering bacteria of the present invention, according to recombination human source type III collagen egg
The growth characteristics of white engineering bacteria, the Ingredient Amount for optimizing basal fermentation culture fluid is to include based on per liter：H3PO4 25-27ml、
CaSO4 0.8-1.0g、K2SO4 17-19g、MgSO4·7H2O 13-15.5g, KOH 3.8-4.3g, glycerol 37-43g and
One liquid microelement 3.5-4.2ml, which provides best nutritional basis for the growth of recombination human source type III collagen protein engineering bacteria,
Meanwhile, according to the consumption of nutrient substance during fermentation culture, in time add the benefit containing glycerol and the second liquid microelement
Material, it is ensured that the growth vigor of recombination human source type III collagen protein engineering bacteria.
Wherein, and to the first liquid microelement and the second liquid microelement it is optimized, the two includes based on per liter：
CuSO4·5H2O 5-7g、NaI 0.06-0.09g、MnSO4·H2O2.5-3.2g、Na2MoO4·2H2O 0.1-0.3g、H3BO3
0.01-0.03g、CoCl20.4-0.6g、ZnCl218-22g、FeSO4·7H2O 60-68g、VH0.1-0.3g and H2SO44-
6ml, the growth for recombination human source type III collagen protein engineering bacteria provide the trace element of abundance, and then can be with the table of high yield
Reach recombination human source type III collagen protein, with suitable for large-scale production.
Description of the drawings
In order to be illustrated more clearly that the technical scheme of the embodiment of the present invention, below by to be used attached needed for embodiment
Figure is briefly described, it will be appreciated that the following drawings illustrate only certain embodiments of the present invention, thus be not construed as it is right
The restriction of scope, for those of ordinary skill in the art, on the premise of not paying creative work, can be with according to this
A little accompanying drawings obtain other related accompanying drawings.
Fig. 1 is the biological deposits information of recombination human source type III collagen protein engineering bacteria provided in an embodiment of the present invention；
The gel electrophoresis figure of the fermented liquid supernatant that Fig. 2 is obtained for embodiment 1-3 that the present invention is provided.
Specific embodiment
To make purpose, technical scheme and the advantage of the embodiment of the present invention clearer, below will be in the embodiment of the present invention
Technical scheme be clearly and completely described.In embodiment, unreceipted actual conditions person, builds according to normal condition or manufacturer
The condition of view is carried out.Agents useful for same or the unreceipted production firm person of instrument, are the conventional product that can pass through that commercially available purchase is obtained
Product.
Fermentation process and fermentation medium to the recombination human source type III collagen protein engineering bacteria of the embodiment of the present invention below
It is specifically described.
The fermentation process of recombination human source type III collagen protein engineering bacteria provided in an embodiment of the present invention, including：
Recombination human source type III collagen protein engineering bacteria is seeded in basal fermentation culture fluid, fermentation culture is carried out.
Basal fermentation culture fluid includes based on per liter：H3PO4 25-27ml、CaSO4 0.8-1.0g、K2SO4 17-19g、
MgSO4·7H2O 13-15.5g, KOH 3.8-4.3g, glycerol 37-43g and the first liquid microelement 3.5-4.2ml.
During fermentation culture, feed supplement is added toward basal fermentation culture fluid, feed supplement includes glycerol and second micro
Element liquid.
Wherein, the first liquid microelement and the second liquid microelement include based on per liter：CuSO4·5H2O 5-7g、NaI
0.06-0.09g、MnSO4·H2O 2.5-3.2g、Na2MoO4·2H2O 0.1-0.3g、H3BO3 0.01-0.03g、CoCl20.4-
0.6g、ZnCl218-22g、FeSO4·7H2O 60-68g、VH0.1-0.3g and H2SO44-6ml。
Preferably, adopting deposit number is carried out for the recombination human source type III collagen protein engineering bacteria of CGMCC No.12491
Fermentation.
The recombination human source type III collagen protein engineering bacterium biological preservation information：Classification And Nomenclature is pichia pastoris phaff
(Pichia Pastoris), on May 23rd, 2016 positioned at the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences
China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation of institute of microbiology, deposit number is CGMCC
No.12491.The biological deposits information of recombination human source type III collagen protein engineering bacteria used by the present embodiment and survival proof can
Referring to Fig. 1.
It should be noted that deposit number is adopted in embodiments of the invention for the recombination human source of CGMCC No.12491
Type III collagen protein engineering bacteria carries out fermentation culture, in other examples, also can be to carry out sending out using other strains
Ferment culture, the selection of strain are not limited to the recombination human source type III collagen protein engineering that deposit number is CGMCC No.12491
Bacterium.As long as the recombination human source type III collagen protein engineering bacteria used of the present invention its have and give expression to recombination human source type III collagen
The ability of albumen.
Preferably, during fermentation culture, when the dissolved oxygen of basal fermentation culture fluid rises to 50%-60%, addition
Feed supplement.When dissolved oxygen rises to 50%-60%, the carbon source in basal fermentation culture fluid is that glycerol is consumed completely, and now addition contains
The feed supplement of glycerol and liquid microelement, constantly can provide carbon source for recombination human source type III collagen protein engineering bacteria, it is ensured that its
Growth vigor.
Preferably, feed supplement includes based on per liter：Second liquid microelement of the glycerol and 4-6ml of 460-520g.
Preferably, based on every liter of basal fermentation culture fluid, the volume for adding feed supplement is 72-90ml.
Preferably, feed supplement is added by the flow velocity of 10-12ml/min.
Further, during fermentation culture, after addition feed supplement, add methanol toward basal fermentation culture fluid,
Carry out inducing culture.
Preferably, when the dissolved oxygen in basal fermentation culture fluid rises to 80%-95%, methanol is added with the first flow velocity；Then
When the dissolved oxygen of basal fermentation culture fluid is down to 40%-45%, methanol is added with second flow speed and basal fermentation culture fluid is controlled
Dissolved oxygen be 10%-40%.Wherein, the first flow velocity is less than second flow speed.
It is different according to dissolved oxygen, methanol is added with different in flow rate, it is first slow rear fast, basal fermentation culture fluid can be caused
Recombination human source type III collagen protein engineering bacteria can better adapt to culture environment, it is to avoid methanol adds the flow velocity mistake during initial stage
High recombination human source type III collagen protein engineering bacteria brings discomfort.
It is highly preferred that the first flow velocity is 1.8-2.2ml/h L, second flow speed is 4-6ml/h L.Arranged in pairs or groups using the flow velocity
Mode add methanol and may insure that recombination human source type III collagen protein engineering bacteria is not added the culture environment brought by methanol
The impact of change.Can ensure that the group human collagen expression highest of its inducing recombinant human source type III collagen protein engineering bacteria.
It should be noted that the first flow velocity is referred to based on every liter of basal fermentation culture fluid per hour for 1.8-2.2ml/h L
The methanol volume being added in basal fermentation culture fluid is 1.8-2.2ml.Second flow speed is referred to by every liter of base for 4-6ml/h L
Plinth fermentation culture meter is added to the methanol volume in basal fermentation culture fluid per hour for 4-6ml.
Further, during fermentation culture, fermentation temperature is controlled for 27 DEG C -29 DEG C, control basal fermentation culture fluid
PH be 4.8-5.2.The state of the temperature and pH value of yeasting in relative constancy is kept, is conducive to recombination human source type III
The growth and breeding of collagen protein engineering bacteria.
Preferably, recombination human source type III collagen protein engineering bacteria is seeded in basal fermentation culture fluid and is referred to：Will restructuring
People source type III collagen protein engineering bacteria seed liquor presses 1:The volume ratio of 6-12 is seeded in basal fermentation culture fluid, recombination human source
Type III collagen protein engineering bacteria seed liquor is by obtaining after the activated culture of recombination human source type III collagen protein engineering bacteria.Through work
The vigor for changing the recombination human source type III collagen protein engineering bacteria of culture is higher.
It is highly preferred that carrying out activation culture as follows：Take the recombination human source type III collagen protein engineering of freezen protective
Recombination human source type III collagen protein engineering bacteria streak inoculation to YPD solid mediums, culture, after thawing, are obtained single bacterium by bacterium
Fall.Picking single bacterium colony is seeded in YPD fluid mediums, culture, obtains activation culture liquid.Activation culture liquid is seeded to
In 2nd YPD fluid mediums, culture obtains above-mentioned recombination human source type III collagen protein engineering bacteria seed liquor.
Explanation is needed, in other examples, it is also possible to which the step of not carrying out step, the activation culture is optional step
Suddenly, can be selected according to the activity intensity of recombination human source type III collagen protein engineering bacteria.For example, during primary stage of inoculation, recombined human
The activity of source type III collagen protein engineering bacteria is just stronger, it is possible to which in being directly inoculated with, basal fermentation medium carries out sending out
Ferment culture.Or, needs are used the long-term recombination human source type III collagen protein engineering bacteria in -80 DEG C of preservations and carry out fermentation culture,
It is then rotatable first to carry out activation culture, obtain the stronger recombination human source type III collagen protein engineering bacteria seed liquor of vigor, Ran Hou
Carry out fermentation culture.
In addition, a kind of fermentation medium that the present invention is provided, which includes basal fermentation culture fluid and feed supplement.Basal fermentation is trained
Nutrient solution includes based on per liter：H3PO4 25-27ml、CaSO4 0.8-1.0g、K2SO4 17-19g、MgSO4·7H2O 13-
15.5g, KOH 3.8-4.3g, glycerol 37-43g and the first liquid microelement 3.5-4.2ml.Feed supplement includes glycerol and second micro-
Secondary element liquid.
First liquid microelement and the second liquid microelement include based on per liter：CuSO4·5H2O 5-7g、NaI
0.06-0.09g、MnSO4·H2O 2.5-3.2g、Na2MoO4·2H2O 0.1-0.3g、H3BO3 0.01-0.03g、CoCl20.4-
0.6g、ZnCl218-22g、FeSO4·7H2O 60-68g、VH0.1-0.3g and H2SO44-6ml。
The fermentation medium is applicable to above-mentioned fermentation process and carries out fermentation culture, it is also possible to suitable for other fermentation process
Fermentation culture.As long as carrying out fermentation culture using above-mentioned fermentation medium falls into protection scope of the present invention.
To sum up, the fermentation process of recombination human source type III collagen protein engineering bacteria of the invention, according to recombination human source type III
The growth characteristics of collagen protein engineering bacteria, the Ingredient Amount for optimizing basal fermentation culture fluid is to include based on per liter：H3PO4 25-
27ml、CaSO4 0.8-1.0g、K2SO4 17-19g、MgSO4·7H2O 13-15.5g, KOH 3.8-4.3g, glycerol 37-43g with
And the first liquid microelement 3.5-4.2ml, which provides best nutritional base for the growth of recombination human source type III collagen protein engineering bacteria
Plinth, meanwhile, according to the consumption of nutrient substance during fermentation culture again, in time add containing glycerol and the second liquid microelement
Feed supplement, it is ensured that the growth vigor of recombination human source type III collagen protein engineering bacteria.
Wherein, and to the first liquid microelement and the second liquid microelement it is optimized, the two includes based on per liter：
CuSO4·5H2O 5-7g、NaI 0.06-0.09g、MnSO4·H2O 2.5-3.2g、Na2MoO4·2H2O 0.1-0.3g、H3BO3
0.01-0.03g、CoCl20.4-0.6g、ZnCl218-22g、FeSO4·7H2O 60-68g、VH0.1-0.3g and H2SO44-
6ml, the growth for recombination human source type III collagen protein engineering bacteria provide the trace element of abundance, and then can be with the table of high yield
Reach recombination human source type III collagen protein, with suitable for large-scale production.
To sum up, the fermentation process and fermentation medium of the recombination human source type III collagen protein engineering bacteria that the present invention is provided
By the optimization to medium component content and yeasting system, and feed supplement and derivant are addition time and the stream of methanol
Isoparametric optimization of speed etc. so that the fermentation process of the present invention greatly provides recombination human source type III collagen protein engineering bacteria
Expressing quantity, the present invention provide fermentation process and fermentation medium can be applied to industrialized large-scale production.
With reference to embodiments the feature and performance of the present invention are described in further detail.
Embodiment 1
The operating procedure of the fermentation process of the recombination human source type III collagen protein engineering bacteria that the present embodiment is provided is as follows.
1 activated spawn
The 1.1 recombination human source type III collagen protein engineering bacterias for taking freezen protective, after thawing, by recombination human source type III collagen
The streak inoculation of protein engineering bacterium to YPD solid mediums, in 28 DEG C, growth 48 hours to a diameter of 3mm of single bacterium colony or so.
Wherein, the recombination human source type III collagen protein engineering bacteria used by the present embodiment is with recombinant expression carrier pPICZaA
Conversion is finished redYeastObtain after (Pichia Pastoris), wherein, pPICZaA carries the people's collagen of coding human collagen
Protein gene (Col3-3), the restriction enzyme site at the gene 5 ' end is XhoI, and the 3 ' restriction enzyme sites held are SacII.
Recombination human source type III collagen protein engineering bacterium biological preservation information used by the present embodiment：Classification And Nomenclature is Bath
Moral Pichia sp. (Pichia Pastoris), on May 23rd, 2016 positioned at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation of Institute of Microorganism, Academia Sinica, preservation are compiled
Number be CGMCC No.12491.The biological deposits information of recombination human source type III collagen protein engineering bacteria used by the present embodiment and
Survival proof can be found in Fig. 1.
Wherein, the following method that refers to of the YPD solid mediums used by the present embodiment makes.Prepare the YPD solids of 1L
Culture medium：Take 20g antibacterial culturing tryptones, 10g Bacto-yeast extracts, 20g NaCl, 20g antibacterial culturing
With agar, it is added in container, adds deionized water to be completely dissolved solute, deionized water is settled to 1L, in 15lbf/in2
(1.034×105Pa) steam sterilization 20 minutes under high pressure.
1.2 picking single bacterium colonies are seeded in YPD fluid mediums of 200ml, under the conditions of 28 DEG C, 200r/min,
Activation culture 24 or so, obtains activation culture liquid.
Wherein, the following method that refers to of the YPD fluid mediums used by the present embodiment makes.Prepare 200ml's
First YPD fluid mediums：4g antibacterial culturing tryptones, 2g Bacto-yeast extracts, 4g NaCl are taken, plus
Enter in container, add deionized water to be completely dissolved solute and be settled to 200ml, in 15lbf/in2 (1.034 × 105Pa) high pressure
Lower steam sterilization 20 minutes, can add heat labile material when culture medium temperature is down to 50 DEG C.
1.3 press 1:20 volume ratio, activation culture liquid is seeded in the 2nd YPD fluid mediums of 200ml, in 28
DEG C, 200r/min, culture 24h or so, obtain recombination human source type III collagen protein engineering bacteria seed liquor.
Wherein, the manufacture method of the 2nd YPD fluid mediums is identical with the YPD fluid mediums in step 1.2.
2 fermentation culture
2.1 by the recombination human source type III collagen protein engineering bacteria seed liquor for obtaining with 1:12 volume ratio be seeded to containing
Fermentation tank (Shanghai bolune, the model of 40L basal fermentation culture fluid：BLBIO-100STA-UIP, total fermentation capacity 70L).This
Fermentation tank can show the parameters such as dissolved oxygen, pH, temperature, speed of agitator, and can carry out pH with peristaltic pump and automatically control and automatic feeding.
Wherein, the basal fermentation culture fluid used by the present embodiment includes based on per liter：H3PO4 26.7ml、CaSO4
0.93g、K2SO4 18.2g、MgSO4·7H2O 14.9g, KOH 4.13g, glycerol 40g and liquid microelement 4ml.
Wherein, the liquid microelement used by the present embodiment includes based on per liter：：CuSO4·5H2O 6g、NaI 0.08g、
MnSO4·H2O 3g、Na2MoO4·2H2O 0.2g、H3BO3 0.02g、CoCl20.5g、ZnCl220g、FeSO4·7H2O 65g、
VH0.2g and H2SO45ml.Liquid microelement refers to following method and makes：Each component, deionized water are taken by aforementioned formula
1L is settled to, after being sufficiently stirred for, filtration sterilization is carried out with 0.22 μm of filter membrane, preserve in 4 DEG C.
2.2 adjust fermentation tank parameter, to control fermentation condition, carry out fermentation culture.
Wherein, the fermentation condition of the present embodiment is specially：28 DEG C of fermentation temperature of control, controls the pH of basal fermentation culture fluid
For 5.0, basal fermentation culture fluid pH is adjusted using 7mol/L ammonia.
2.3 addition feed supplements.Treat that the glycerol in fermentation tank runs out of, when dissolved oxygen rises to 50% or so, add toward fermentation tank
(based on every liter of basal fermentation culture fluid, the volume for adding feed supplement is 72ml to enter the feed supplement that cumulative volume is 2880ml.), the 2880ml
Feed supplement add to basal fermentation culture fluid by the flow velocity of flow velocity 12ml/min.After adding feed supplement, in basal fermentation culture fluid
Dissolved oxygen declines, and continues culture.
Wherein, the feed supplement used by the present embodiment includes based on per liter：Glycerol 500g and liquid microelement 4ml.
2.4 addition methanol, inducing culture.When dissolved oxygen rises to 80%, hungry culture 30 minutes, then train toward basal fermentation
Methanol is added in nutrient solution, inducing culture is carried out.Specifically, methanol is added with the first flow velocity 2ml/h L；Then in basal fermentation
When the dissolved oxygen of culture fluid is down to 40%, methanol is continued with second flow speed 4ml/h L additions, and controls basal fermentation culture fluid
Dissolved oxygen is 30% or so.After inducing culture 72h or so, whole fermentation culture process can be terminated.Containing recombination human source type III collagen
Albumen obtains fermentation liquid.
After 2.5 stop fermentation, using existing extraction process such as solid-liquid isolation method, ion exchange etc., can be from fermentation liquid
In purify out human collagen.
Embodiment 2
The operating procedure of the fermentation process of the recombination human source type III collagen protein engineering bacteria that the present embodiment is provided is as follows.
1 activated spawn
The concrete steps of the activated spawn in the present embodiment are substantially the same manner as Example 1, to obtain recombination human source type III glue
Former protein engineering bacterium seed liquor.
2 fermentation culture
2.1 by the recombination human source type III collagen protein engineering bacteria seed liquor for obtaining with 1:10 volume ratio be seeded to containing
Fermentation tank (Shanghai bolune, the model of 40L basal fermentation culture fluid：BLBIO-100STA-UIP, total capacity 60L).The fermentation tank
The parameters such as dissolved oxygen, pH, temperature, speed of agitator can be shown, and pH can be carried out with peristaltic pump and be automatically controlled and automatic feeding.
Wherein, the basal fermentation culture fluid used by the present embodiment includes based on per liter：H3PO4 25ml、CaSO4 0.8g、
K2SO4 19g、MgSO4·7H2O 15.5g, KOH 4.3g, glycerol 37g and liquid microelement 4.2ml.
Wherein, the liquid microelement used by the present embodiment includes based on per liter：：CuSO4·5H2O 7g、NaI 0.09g、
MnSO4·H2O 2.5g、Na2MoO4·2H2O 0.1g、H3BO3 0.01g、CoCl20.6g、ZnCl222g、FeSO4·7H2O
68g、VH0.3g and H2SO46ml.Liquid microelement refers to following method and makes：Each component is taken by aforementioned formula, spend from
Sub- water is settled to 1L, after being sufficiently stirred for, carries out filtration sterilization with 0.22 μm of filter membrane, preserves in 4 DEG C.
2.2 adjust fermentation tank parameter, to control fermentation condition, carry out fermentation culture.
Wherein, the fermentation condition of the present embodiment is specially：27 DEG C of fermentation temperature of control, controls the pH of basal fermentation culture fluid
For 5.2, basal fermentation culture fluid pH is adjusted using 7mol/L ammonia.
2.3 addition feed supplements.Treat that the glycerol in fermentation tank runs out of, when dissolved oxygen rises to 55% or so, add toward fermentation tank
(based on every liter of basal fermentation culture fluid, the volume for adding feed supplement is 90ml to enter the feed supplement that cumulative volume is 3600ml.), the 3600ml
Feed supplement add to basal fermentation culture fluid by the flow velocity of flow velocity 10ml/min.After adding feed supplement, in basal fermentation culture fluid
Dissolved oxygen declines, and continues culture.
Wherein, the feed supplement used by the present embodiment includes based on per liter：Glycerol 520g and liquid microelement 6ml.
2.4 addition methanol, inducing culture.When dissolved oxygen rises to 85%, hungry culture 30 minutes, then train toward basal fermentation
Nutrient solution presses middle addition methanol, carries out inducing culture.Specifically, methanol is added with the first flow velocity 1.8ml/h；Then in basal fermentation
When the dissolved oxygen of culture fluid is down to 45%, methanol is continued with second flow speed 6ml/h L additions, and controls basal fermentation culture fluid
Dissolved oxygen is 30% or so.After inducing culture 72h or so, whole fermentation culture process can be terminated.Containing recombination human source type III collagen
Albumen obtains fermentation liquid
After 2.5 stop fermentation, using existing extraction process such as solid-liquid isolation method, ion exchange etc., can be from fermentation liquid
In purify out human collagen.
Embodiment 3
The operating procedure of the fermentation process of the recombination human source type III collagen protein engineering bacteria that the present embodiment is provided is as follows.
1 activated spawn
The concrete steps of the activated spawn in the present embodiment are substantially the same manner as Example 1, to obtain recombination human source type III glue
Former protein engineering bacterium seed liquor.
2 fermentation culture
2.1 by the recombination human source type III collagen protein engineering bacteria seed liquor for obtaining with 1:8 volume ratio be seeded to containing
Fermentation tank (Shanghai bolune, the model of 40L basal fermentation culture fluid：BLBIO-100STA-UIP, total fermentation capacity 70L).This
Fermentation tank can show the parameters such as dissolved oxygen, pH, temperature, speed of agitator, and can carry out pH with peristaltic pump and automatically control and automatic feeding.
Wherein, the basal fermentation culture fluid used by the present embodiment includes based on per liter：H3PO4 27ml、CaSO4 1.0g、
K2SO4 17g、MgSO4·7H2O 13g, KOH 3.8g, glycerol 43g and liquid microelement 3.5ml.
Wherein, the liquid microelement used by the present embodiment includes based on per liter：：CuSO4·5H2O 5g、NaI 0.06g、
MnSO4·H2O 3.2g、Na2MoO4·2H2O 0.3g、H3BO3 0.03g、CoCl20.4g、ZnCl218g、FeSO4·7H2O
60g、VH0.1g and H2SO44ml.Liquid microelement refers to following method and makes：Each component is taken by aforementioned formula, spend from
Sub- water is settled to 1L, after being sufficiently stirred for, carries out filtration sterilization with 0.22 μm of filter membrane, preserves in 4 DEG C.
2.2 adjust fermentation tank parameter, to control fermentation condition, carry out fermentation culture.
Wherein, the fermentation condition of the present embodiment is specially：29 DEG C of fermentation temperature of control, controls the pH of basal fermentation culture fluid
For 4.8, basal fermentation culture fluid pH is adjusted using 7mol/L ammonia.
2.3 addition feed supplements.Treat that the glycerol in fermentation tank runs out of, when dissolved oxygen rises to 60% or so, by toward fermentation tank
The feed supplement that cumulative volume is 2880ml, the feed supplement of the 2880ml is added to add to basal fermentation culture by the flow velocity of flow velocity 11ml/min
Liquid.After adding feed supplement, the dissolved oxygen in basal fermentation culture fluid declines, and continues culture.
Wherein, the feed supplement used by the present embodiment includes based on per liter：Glycerol 460g and liquid microelement 4ml.
2.4 addition methanol, inducing culture.When dissolved oxygen rises to 95%, hungry culture 30 minutes, then train toward basal fermentation
Nutrient solution presses middle addition methanol, carries out inducing culture.Specifically, methanol is added with the first flow velocity 2.2ml/h；Then in basal fermentation
When the dissolved oxygen of culture fluid is down to 40%, methanol is continued with second flow speed 4ml/h L additions, and controls basal fermentation culture fluid
Dissolved oxygen is 30% or so.After inducing culture 72h or so, whole fermentation culture process can be terminated.Containing recombination human source type III collagen
Albumen obtains fermentation liquid.
After 2.5 stop fermentation, using existing extraction process such as solid-liquid isolation method, ion exchange etc., can be from fermentation liquid
In purify out human collagen.
Embodiment 4
The operating procedure of the fermentation process of the recombination human source type III collagen protein engineering bacteria that the present embodiment is provided is as follows.
1 activated spawn
The concrete steps of the activated spawn in the present embodiment are substantially the same manner as Example 1, to obtain recombination human source type III glue
Former protein engineering bacterium seed liquor.
2 fermentation culture
2.1 by the recombination human source type III collagen protein engineering bacteria seed liquor for obtaining with 1:6 volume ratio be seeded to containing
Fermentation tank (Shanghai bolune, the model of 40L basal fermentation culture fluid：BLBIO-100STA-UIP, total fermentation capacity 70L).This
Fermentation tank can show the parameters such as dissolved oxygen, pH, temperature, speed of agitator, and can carry out pH with peristaltic pump and automatically control and automatic feeding.
Wherein, the basal fermentation culture fluid used by the present embodiment includes based on per liter：H3PO4 26.7ml、CaSO4
0.93g、K2SO4 17g、MgSO4·7H2O 15.5g, KOH 4.3g, glycerol 40g and liquid microelement 4ml.
Wherein, the liquid microelement used by the present embodiment includes based on per liter：：CuSO4·5H2O 5g、NaI 0.08g、
MnSO4·H2O 3.2g、Na2MoO4·2H2O 0.1g、H3BO3 0.01g、CoCl20.5g、ZnCl220g、FeSO4·7H2O
65g、VH0.3g and H2SO45ml.Liquid microelement refers to following method and makes：Each component is taken by aforementioned formula, spend from
Sub- water is settled to 1L, after being sufficiently stirred for, carries out filtration sterilization with 0.22 μm of filter membrane, preserves in 4 DEG C.
2.2 adjust fermentation tank parameter, to control fermentation condition, carry out fermentation culture.
Wherein, the fermentation condition of the present embodiment is specially：28 DEG C of fermentation temperature of control, controls the pH of basal fermentation culture fluid
For 5.0, basal fermentation culture fluid pH is adjusted using 7mol/L ammonia.
2.3 addition feed supplements.Treat that the glycerol in fermentation tank runs out of, when dissolved oxygen rises to 50% or so, add toward fermentation tank
Enter the feed supplement that cumulative volume is 3600ml, the feed supplement of the 3600ml is added to basal fermentation culture by the flow velocity of flow velocity 12ml/min
Liquid.After adding feed supplement, the dissolved oxygen in basal fermentation culture fluid declines, and continues culture.
Wherein, the feed supplement used by the present embodiment includes based on per liter：Glycerol 500g and liquid microelement 4ml.
2.4 addition methanol, inducing culture.When dissolved oxygen rises to 80%, hungry culture 30 minutes, then train toward basal fermentation
Nutrient solution presses middle addition methanol, carries out inducing culture.Specifically, methanol is added with the first flow velocity 2ml/h；Then train in basal fermentation
When the dissolved oxygen of nutrient solution is down to 40%, methanol is continued with second flow speed 6ml/h L additions, and controls the molten of basal fermentation culture fluid
Oxygen is 30% or so.After inducing culture 72h or so, whole fermentation culture process can be terminated.Containing recombination human source type III collagen egg
White obtains fermentation liquid.
After 2.5 stop fermentation, using existing extraction process such as solid-liquid isolation method, ion exchange etc., can be from fermentation liquid
In purify out human collagen.
Comparative example 1
The operating procedure of the fermentation process of the recombination human source type III collagen protein engineering bacteria that this comparative example is provided is as follows.Need
It is noted that each parameter value of fermentation process of the recombination human source type III collagen protein engineering bacteria in this comparative example is not disposed on
In protection scope of the present invention.
1 activated spawn
The concrete steps of the activated spawn in this comparative example are substantially the same manner as Example 1, to obtain recombination human source type III glue
Former protein engineering bacterium seed liquor.
2 fermentation culture
2.1 by the recombination human source type III collagen protein engineering bacteria seed liquor for obtaining with 1:4 volume ratio be seeded to containing
Fermentation tank (Shanghai bolune, the model of 40L basal fermentation culture fluid：BLBIO-100STA-UIP, total capacity 70L).The fermentation tank
The parameters such as dissolved oxygen, pH, temperature, speed of agitator can be shown, and pH can be carried out with peristaltic pump and be automatically controlled and automatic feeding.
Wherein, the basal fermentation culture fluid used by this comparative example includes based on per liter：H3PO4 20ml、CaSO4 0.7g、
K2SO4 20g、MgSO4·7H2O 11g, KOH 5g, glycerol 35g and liquid microelement 3ml.
Wherein, the liquid microelement used by this comparative example includes based on per liter：：CuSO4·5H2O 8g、NaI 0.1g、
MnSO4·H2O 3.5g、Na2MoO4·2H2O 0.5g、H3BO3 0.05g、CoCl20.2g、ZnCl216g、FeSO4·7H2O
60g、VH0.4g and H2SO42ml.Liquid microelement refers to following method and makes：Each component is taken by aforementioned formula, spend from
Sub- water is settled to 1L, after being sufficiently stirred for, carries out filtration sterilization with 0.22 μm of filter membrane, preserves in 4 DEG C.
2.2 adjust fermentation tank parameter, to control fermentation condition, carry out fermentation culture.
Wherein, the fermentation condition of this comparative example is specially：28 DEG C of fermentation temperature of control, controls the pH of basal fermentation culture fluid
For 5.0, basal fermentation culture fluid pH is adjusted using 7mol/L ammonia.
2.3 addition feed supplements.Treat that the glycerol in fermentation tank runs out of, when dissolved oxygen rises to 70% or so, add toward fermentation tank
Enter feed supplement, after adding feed supplement, the dissolved oxygen in basal fermentation culture fluid declines, and continues culture.
Wherein, the feed supplement used by this comparative example includes based on per liter：Glycerol 350g and liquid microelement 7ml.
2.4 addition methanol, inducing culture.When dissolved oxygen rises to 70%, hungry culture 30 minutes, then train toward basal fermentation
Nutrient solution presses middle addition methanol, carries out inducing culture.Specifically, methanol is added with the first flow velocity 4ml/h；Then train in basal fermentation
When the dissolved oxygen of nutrient solution is down to 50%, methanol is continued with second flow speed 7ml/h L additions, and controls the molten of basal fermentation culture fluid
Oxygen is 30% or so.After inducing culture 72h or so, whole fermentation culture process can be terminated.Containing recombination human source type III collagen egg
White obtains fermentation liquid.
After 2.5 stop fermentation, using existing extraction process such as solid-liquid isolation method, ion exchange etc., can be from fermentation liquid
In purify out human collagen.
Experimental example 1
According to a conventional method, the fermented liquid supernatant that Example 1, embodiment 2, embodiment 3 and comparative example 1 are obtained, is carried out
Proteins gel electrophoresis are tested, and detect the expression of recombination human source type III collagen protein, as a result as shown in Figure 2.
As shown in Figure 2 (in figure：M is albumen Maker, and 1,2,3,4 respectively containing 0.25 μ g, 0.5 μ g, 1 μ g, 2 μ g recombined humans
The standard substance of source type III collagen protein, 5,6,7,8 is that volume is comparative example 1, comparative example 3, comparative example 2, comparative example 1 respectively
Fermented liquid supernatant, loading volume are 5 μ l, are the recombination human source type III collagen protein that molecular weight is 130Kd at arrow indication),
In the position of 130Kd, the band color of embodiment 1,2 and 3 is deeper, and the band color of comparative example 1 is relative to embodiment 1,2 and 3
It is shallower, illustrate the recombination human source type III collagen content in the fermented liquid supernatant that embodiment 1,2 and 3 is obtained than comparative example 1
Content it is high；It is compared to each other in embodiment 1,2 and 3, it is known that, band color of the embodiment 1 in 130Kd positions is most deep,
The recombination human source type III collagen content highest for obtaining fermented liquid supernatant of embodiment 1 is illustrated, this also indicates that embodiment 1
Parameters are optimized designs, and which has unforeseeable effect.
To sum up, the fermentation process of the recombination human source type III collagen protein engineering bacteria for being provided using the present invention can improve weight
The expression of group people source type III collagen protein, and improve effect substantially, also illustrate simultaneously, the fermentation medium that the present invention is provided
The recombination human source type III collagen protein expression of recombination human source type III collagen protein engineering bacteria can be improved.
In a word, the fermentation process and fermentation medium of the recombination human source type III collagen protein engineering bacteria that the present invention is provided,
By the optimization of component content to basal fermentation culture fluid and feed supplement and the optimization of yeasting system, and feed supplement and derivant
That is the addition opportunity of methanol and the isoparametric optimization of flow velocity etc., greatly increase recombination human source type III collagen protein engineering bacteria
Expressing quantity, the fermentation process and fermentation medium can be applied to industrialized large-scale production, contribute to reducing Jing
Ji cost, improve production efficiency.
The preferred embodiments of the present invention are the foregoing is only, the present invention is not limited to, for the skill of this area
For art personnel, the present invention can have various modifications and variations.It is all within the spirit and principles in the present invention, made any repair
Change, equivalent, improvement etc., should be included within the scope of the present invention.
Claims (10)
1. a kind of fermentation process of recombination human source type III collagen protein engineering bacteria, it is characterised in which includes：By recombination human source
Type III collagen protein engineering bacteria is seeded in basal fermentation culture fluid, carries out fermentation culture, and the basal fermentation culture fluid is by every
Rising meter includes：H3PO4 25-27ml、CaSO4 0.8-1.0g、K2SO4 17-19g、MgSO4·7H2O 13-15.5g、KOH
3.8-4.3g, glycerol 37-43g and the first liquid microelement 3.5-4.2ml；
During the fermentation culture, add feed supplement toward the basal fermentation culture fluid, the feed supplement include glycerol and
Second liquid microelement；
Wherein, first liquid microelement and second liquid microelement include based on per liter：CuSO4·5H2O 5-
7g、NaI 0.06-0.09g、MnSO4·H2O 2.5-3.2g、Na2MoO4·2H2O 0.1-0.3g、H3BO3 0.01-0.03g、
CoCl20.4-0.6g、ZnCl218-22g、FeSO4·7H2O 60-68g、VH0.1-0.3g and H2SO44-6ml。
2. the fermentation process of recombination human source type III collagen protein engineering bacteria according to claim 1, it is characterised in that institute
State the recombination human source type III collagen egg that recombination human source type III collagen protein engineering bacteria is that deposit number is CGMCC No.12491
White engineering bacteria.
3. the fermentation process of recombination human source type III collagen protein engineering bacteria according to claim 2, it is characterised in that
During the fermentation culture, after the feed supplement is added, add methanol toward the basal fermentation culture fluid, induced
Culture.
4. the fermentation process of recombination human source type III collagen protein engineering bacteria according to claim 3, it is characterised in that in
When the dissolved oxygen of the basal fermentation culture fluid rises to 80%-95%, the methanol is added with the first flow velocity, then on the basis
When the dissolved oxygen of fermentation culture is down to 40%-45%, the methanol is added with second flow speed and the basal fermentation culture is controlled
The dissolved oxygen of liquid is 10%-40%；Wherein, first flow velocity is less than the second flow speed.
5. the fermentation process of recombination human source type III collagen protein engineering bacteria according to claim 4, it is characterised in that institute
It is 1.8-2.2ml/h L to state the first flow velocity, and the second flow speed is 4-6ml/h L.
6. the fermentation process of the recombination human source type III collagen protein engineering bacteria according to any one of claim 1-5, which is special
Levy and be, during the fermentation culture, control fermentation temperature and be 27 DEG C -29 DEG C, control the basal fermentation culture fluid
PH is 4.8-5.2.
7. the fermentation process of the recombination human source type III collagen protein engineering bacteria according to any one of claim 1-5, which is special
Levy and be, the feed supplement includes based on per liter：Second liquid microelement of the glycerol and 4-6ml of 460-520g.
8. the fermentation process of the recombination human source type III collagen protein engineering bacteria according to any one of claim 1-5, which is special
Levy and be, during the fermentation culture, when the dissolved oxygen of the basal fermentation culture fluid rises to 50%-60%, addition
The feed supplement.
9. the fermentation process of the recombination human source type III collagen protein engineering bacteria according to any one of claim 1-5, which is special
Levy and be, the recombination human source type III collagen protein engineering bacteria is seeded in the basal fermentation culture fluid and is referred to：Will restructuring
People source type III collagen protein engineering bacteria seed liquor presses 1:The volume ratio of 6-12 is seeded in the basal fermentation culture fluid, described
Recombination human source type III collagen protein engineering bacteria seed liquor is by the activated culture of the recombination human source type III collagen protein engineering bacteria
After obtain.
10. a kind of fermentation medium, it is characterised in which includes basal fermentation culture fluid and feed supplement, the basal fermentation culture
Liquid includes based on per liter：H3PO4 25-27ml、CaSO4 0.8-1.0g、K2SO4 17-19g、MgSO4·7H2O 13-15.5g、
KOH 3.8-4.3g, glycerol 37-43g and the first liquid microelement 3.5-4.2ml, the feed supplement include glycerol and second micro
Element liquid；
First liquid microelement and second liquid microelement include based on per liter：CuSO4·5H2O 5-7g、NaI
0.06-0.09g、MnSO4·H2O 2.5-3.2g、Na2MoO4·2H2O 0.1-0.3g、H3BO3 0.01-0.03g、CoCl20.4-
0.6g、ZnCl218-22g、FeSO4·7H2O 60-68g、VH0.1-0.3g and H2SO44-6ml。
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| CN107988294A (en) * | 2018-01-18 | 2018-05-04 | 江苏江山聚源生物技术有限公司 | Adjust the zymotechnique that temperature improves recombination human source collagen production level |
| CN112626074A (en) * | 2021-01-11 | 2021-04-09 | 肽源（广州）生物科技有限公司 | Hydroxyproline-modified recombinant human III-type collagen mature peptide and preparation method and application thereof |
| CN113564062A (en) * | 2021-09-10 | 2021-10-29 | 汉肽生物医药集团有限公司 | Fermentation process for shortening culture time and improving collagen content |
| CN114457136A (en) * | 2022-01-27 | 2022-05-10 | 美尔健（深圳）生物科技有限公司 | Fermentation liquid based on rose fermentation recombinant collagen and application thereof |
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