CN110511972A - A kind of streptococcic culture medium of Sodium Hyaluronate and cultural method - Google Patents
A kind of streptococcic culture medium of Sodium Hyaluronate and cultural method Download PDFInfo
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- CN110511972A CN110511972A CN201910896073.6A CN201910896073A CN110511972A CN 110511972 A CN110511972 A CN 110511972A CN 201910896073 A CN201910896073 A CN 201910896073A CN 110511972 A CN110511972 A CN 110511972A
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- culture medium
- sodium hyaluronate
- streptococcic
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- sodium
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- 239000001963 growth medium Substances 0.000 title claims abstract description 53
- 229920002385 Sodium hyaluronate Polymers 0.000 title claims abstract description 42
- 229940010747 sodium hyaluronate Drugs 0.000 title claims abstract description 42
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 25
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims abstract description 30
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 15
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims abstract description 15
- 235000019341 magnesium sulphate Nutrition 0.000 claims abstract description 15
- 239000000843 powder Substances 0.000 claims abstract description 15
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 13
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims abstract description 13
- 235000019799 monosodium phosphate Nutrition 0.000 claims abstract description 13
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims abstract description 13
- 239000008103 glucose Substances 0.000 claims abstract description 12
- 241000194017 Streptococcus Species 0.000 claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229910001868 water Inorganic materials 0.000 claims abstract description 8
- 238000002255 vaccination Methods 0.000 claims abstract description 6
- 239000001110 calcium chloride Substances 0.000 claims description 10
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 10
- 239000011592 zinc chloride Substances 0.000 claims description 10
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 9
- 229910052927 chalcanthite Inorganic materials 0.000 claims description 7
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 7
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 7
- 229910052802 copper Inorganic materials 0.000 claims description 3
- 229910052748 manganese Inorganic materials 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 229910052725 zinc Inorganic materials 0.000 claims description 3
- GZCGUPFRVQAUEE-VANKVMQKSA-N aldehydo-L-glucose Chemical compound OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)C=O GZCGUPFRVQAUEE-VANKVMQKSA-N 0.000 claims description 2
- 229910052791 calcium Inorganic materials 0.000 claims description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 claims 1
- 241000894007 species Species 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 8
- 238000011218 seed culture Methods 0.000 abstract description 4
- 238000004904 shortening Methods 0.000 abstract 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 244000005700 microbiome Species 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 238000000855 fermentation Methods 0.000 description 6
- 230000004151 fermentation Effects 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 229910021645 metal ion Inorganic materials 0.000 description 5
- 150000002500 ions Chemical class 0.000 description 4
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 239000010949 copper Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 229920002674 hyaluronan Polymers 0.000 description 3
- 229960003160 hyaluronic acid Drugs 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 238000012549 training Methods 0.000 description 3
- 239000011701 zinc Substances 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 239000011572 manganese Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- QIVUCLWGARAQIO-OLIXTKCUSA-N (3s)-n-[(3s,5s,6r)-6-methyl-2-oxo-1-(2,2,2-trifluoroethyl)-5-(2,3,6-trifluorophenyl)piperidin-3-yl]-2-oxospiro[1h-pyrrolo[2,3-b]pyridine-3,6'-5,7-dihydrocyclopenta[b]pyridine]-3'-carboxamide Chemical compound C1([C@H]2[C@H](N(C(=O)[C@@H](NC(=O)C=3C=C4C[C@]5(CC4=NC=3)C3=CC=CN=C3NC5=O)C2)CC(F)(F)F)C)=C(F)C=CC(F)=C1F QIVUCLWGARAQIO-OLIXTKCUSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 1
- 230000037354 amino acid metabolism Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 229910001431 copper ion Inorganic materials 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940097043 glucuronic acid Drugs 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229910000765 intermetallic Inorganic materials 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000011177 media preparation Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000012846 protein folding Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- 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
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- 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
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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- Tropical Medicine & Parasitology (AREA)
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Abstract
The present invention relates to seed culture technical fields, and in particular to a kind of streptococcic culture medium of Sodium Hyaluronate and cultural method, culture medium include following components: glucose, yeast powder, magnesium sulfate, sodium dihydrogen phosphate, water and microelement；Thallus cultural method includes: to prepare culture medium；By Sodium Hyaluronate streptococcus vaccination into culture medium shaking table culture.Using culture medium provided by the invention and cultural method culture strain, the effect for improving cell concentration, the effect for shortening the seed culture period can reach, to promote the quality and yield of Sodium Hyaluronate.
Description
Technical field
The present invention relates to seed culture technical fields, and in particular to a kind of streptococcic culture medium of Sodium Hyaluronate and culture
Method.
Background technique
Hyaluronic acid is that the straight chain that the disaccharide repetitive unit of glucuronic acid and N-acetylglucosamine connects and composes is high
Molecule mucopolysaccharide is widely present in cytoplasm, vitreum, umbilical cord, skin, knuckle synovia and rooster comb of people and animal etc.
In soft connective tissue.Commodity hyaluronic acid refers generally to the sodium salt of hyaluronic acid, i.e. Sodium Hyaluronate.
The preparation method of Sodium Hyaluronate mainly includes extraction method and two kinds of fermentation method, and wherein extraction method is referred to from animal
It is directly extracted in body tissue, fermentation method is referred to through microorganism fungus kind fermenting and producing Sodium Hyaluronate.Due to animal body raw material
It is limited, be difficult to collection processing, at high price and separation process it is complicated, with the continuous expansion of Sodium Hyaluronate application range, mention
The Sodium Hyaluronate for following the example of acquisition is no longer satisfied the production requirement of the industries such as medicine, skin care.
Although fermentation method overcomes the defect that extraction method raw material is limited, at high cost, separation process is complicated, but it is high to want preparation
There is still a need for control the condition in fermentation process for the Sodium Hyaluronate of quality.Seed liquor cultivation stage is Sodium Hyaluronate hair
One important link of ferment production, the cell concentration in seed liquor will have a direct impact on the product quality of Sodium Hyaluronate.Meet
The seed liquor that concentration requires can significantly shorten fermentation production process, effectively evade fermenting and producing risk, to promote the output value；One
Cell concentration in denier seed liquor is below standard, then needs to extend cultivation cycle, and so the activity of strain will reduce, seriously
Influence the yield of Sodium Hyaluronate.
Summary of the invention
For the above-mentioned deficiency of the prior art, the present invention provides a kind of streptococcic culture medium of Sodium Hyaluronate and culture side
Method.Using culture medium provided by the invention and cultural method culture strain, it can reach and improve the effect of cell concentration, shorten seed
The effect of cultivation cycle, to promote the quality and yield of Sodium Hyaluronate.
On the one hand, the present invention provides a kind of streptococcic culture medium of Sodium Hyaluronate, culture medium includes following components: Portugal
Grape sugar, yeast powder, magnesium sulfate, sodium dihydrogen phosphate, water and microelement.
The streptococcic culture medium of Sodium Hyaluronate provided by the invention simplifies the complexity of formula, reduces impurity and draws
Enter possibility, strain is with good stability in the medium, by adding microelement into culture medium, reaches and improves bacterium
The effect of bulk concentration.
Further, the culture medium includes glucose 50-100g/L, yeast powder 20-40g/L, magnesium sulfate 1-5g/L, phosphorus
Acid dihydride sodium 1-4g/L, microelement 4 × 10-3-5×10-3g/L。
Further, the microelement includes the combination of any one of Cu, Mn, Zn, Ca or at least two.
Further, the microelement exists in the form of an ion.
Microorganism has a suction-operated to metal ion, suitable metal ion in conjunction with microorganism after growth to microorganism
Metabolic process has very important effect, plays a part of catalyst in the biochemical reaction process of microorganism, can stablize
Protein structure, cell wall and the balance for maintaining osmotic pressure, more conducively thallus are survived.Such as: suitable zinc ion is for hammer
Bacterium has protein-bonded effect, is mainly manifested in answering pressure, protein folding and transhipment, amino acid metabolism etc..In right amount
Copper ion be streptococcus metabolism coenzyme, participate in the respiration of microorganism and to intracellular free radical have cleaning make
With.By adding these trace element ions into culture medium, the survival rate of thallus can be effectively promoted, to make thallus shorter
Time in reach required concentration.
Further, the Cu is by CuSO4·5H2O is provided, and Mn is by MnSO4It provides, Zn is by ZnCl2It provides, Ca is by CaCl2
It provides.
CuSO4·5H2O、MnSO4、ZnCl2、CaCl2It is common metallic compound, has and easily obtain, is cheap
Advantage.
Further, the culture medium includes glucose 70g/L, yeast powder 32.5g/L, magnesium sulfate 3g/L, biphosphate
Sodium 2.5g/L, CuSO4·5H2O 5.12×10-5G/L, MnSO4 4.785×10-4G/L, ZnCl21.235×10-4G/L, CaCl2
3.8×10-3g/L。
Further, the pH value of culture medium is 7.0-7.5.
On the other hand, the present invention provides a kind of Sodium Hyaluronate streptococcus cultural method using the culture medium, sides
Method includes:
(1) culture medium is prepared；
(2) by Sodium Hyaluronate streptococcus vaccination into culture medium shaking table culture, condition of culture are as follows: 36-38 DEG C, shaking table turn
16-18h is cultivated under fast 200rpm.
Further, culture medium preparation the following steps are included:
(1) glucose, yeast powder are added to the water, the stirring at 25 DEG C is until obtain uniform mixed liquor；
(2) magnesium sulfate, sodium dihydrogen phosphate and microelement are added into mixed liquor, stirring is until obtain uniform mixing
Liquid obtains the culture medium.
The beneficial effects of the present invention are,
The streptococcic culture medium of Sodium Hyaluronate provided by the invention has formula is simple, supplementary material is common to be easy to get, impurity
The characteristics of content is low, is suitable for industrialization large-scale production.By adding metal trace element ion into culture medium, hammer is utilized
Bacterium, to the important function of microorganism growth metabolism, promotes cell concentration to the suction-operated of metal ion and metal ion.Existing rank
Section the Sodium Hyaluronate streptococcic seed culture period be generally 20h even it is longer, the present invention shorten cell concentration reach production
It is required that time, largely reduce fermentation production process, can effectively evade fermenting and producing risk, thus reach improve the output value mesh
's.
Specific embodiment
Technical solution in order to enable those skilled in the art to better understand the present invention below will be implemented the present invention
Technical solution in example is clearly and completely described, it is clear that described embodiment is only that present invention a part is implemented
Example, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making creativeness
Every other embodiment obtained, should fall within the scope of the present invention under the premise of labour.
Embodiment 1
A kind of streptococcic culture medium of Sodium Hyaluronate, every 1L culture medium are composed of the following components: glucose 60g, yeast
Powder 24g, magnesium sulfate 1.5g, sodium dihydrogen phosphate 1.5g, CuSO4·5H2O 4.48×10-5G, MnSO4 4.4×10-4G, ZnCl2
1.95×10-4G, CaCl2 4×10-5g。
Cultural method: glucose, yeast powder are added to the water, and the stirring at 25 DEG C is until obtain uniform mixed liquor；
Magnesium sulfate, sodium dihydrogen phosphate and microelement are added into mixed liquor, stirring obtains the training until the uniform mixed liquor of acquisition
Support base；By Sodium Hyaluronate streptococcus vaccination into culture medium shaking table culture, condition of culture are as follows: 37.5 DEG C, shaking speed
Seed liquor reaches defined 1.5 or more absorbance after cultivating 17.5h under 200rpm.
Embodiment 2
A kind of streptococcic culture medium of Sodium Hyaluronate, every 1L culture medium are composed of the following components: glucose 78g, yeast
Powder 35g, magnesium sulfate 4.2g, sodium dihydrogen phosphate 3.7g, CuSO4·5H2O 6.4×10-5G, MnSO4 4.95×10-4G, ZnCl2
6.63×10-5G, CaCl2 3.52×10-5g。
Cultural method: glucose, yeast powder are added to the water, and the stirring at 25 DEG C is until obtain uniform mixed liquor；
Magnesium sulfate, sodium dihydrogen phosphate and microelement are added into mixed liquor, stirring obtains the training until the uniform mixed liquor of acquisition
Support base；By Sodium Hyaluronate streptococcus vaccination into culture medium shaking table culture, condition of culture are as follows: 36.5 DEG C, shaking speed
Seed liquor reaches defined 1.5 or more absorbance after cultivating 17h under 200rpm.
Embodiment 3
A kind of streptococcic culture medium of Sodium Hyaluronate, every 1L culture medium are composed of the following components: glucose 70g, yeast
Powder 32.5g, magnesium sulfate 3g, sodium dihydrogen phosphate 2.5g, CuSO4·5H2O 5.12×10-5G, MnSO4 4.785×10-4G, ZnCl2
1.235×10-4G, CaCl2 3.8×10-5g。
Cultural method: glucose, yeast powder are added to the water, and the stirring at 25 DEG C is until obtain uniform mixed liquor；
Magnesium sulfate, sodium dihydrogen phosphate and microelement are added into mixed liquor, stirring obtains the training until the uniform mixed liquor of acquisition
Support base；By Sodium Hyaluronate streptococcus vaccination into culture medium shaking table culture, condition of culture are as follows: 37 DEG C, shaking speed 200rpm
Seed liquor reaches defined 1.5 or more absorbance after lower culture 16h.
Embodiment 4
Embodiment 4 and the difference of embodiment 3 are, do not include CuSO in embodiment 44·5H2O、CaCl2, remaining component with
Cultural method is same as Example 3.
Embodiment 5
Embodiment 5 and the difference of embodiment 3 are, do not include MnSO in embodiment 54、ZnCl2, remaining component and culture side
Method is same as Example 3.
Comparative example 1
The difference of this comparative example and embodiment 3 is, does not include CuSO in this comparative example4·5H2O、MnSO4、ZnCl2、
CaCl2, remaining component and cultural method are same as Example 3.
Test example
It will be diluted after the sampling of the culture medium seed liquor of embodiment 3- embodiment 5 and comparative example with sterile saline, take 1ml
Dilution counts the bacterium colony on plate to be coated with on plate.Statistical result is as shown in table 1:
Table 1
By the comparison of embodiment 3- embodiment 5 and comparative example it is found that under identical cultivation cycle, use is provided by the invention
Culture medium and cultural method culture Sodium Hyaluronate hammer bacteria concentration are higher than the seed liquor concentration for being not added with metal ion；Four kinds of gold
Concentration when belonging to ion while adding, when seed liquor concentration is also above only two kinds of Mn, Zn or Cu, Ca ions of addition.
In conclusion the streptococcic culture medium of Sodium Hyaluronate provided by the invention and cultural method, with prior art phase
Than, the cell concentration under identical cultivation cycle can be promoted, incubation time needed for streptococcus seed liquor reaches demand concentration is shortened,
Effectively evade the generation of adverse reaction, so that big degree reduces production cost and shortens the production cycle of Sodium Hyaluronate, is suitable for
Large-scale industrialization promotion.
Although the present invention is not limited thereto to the present invention have been described in detail by way of preferred embodiment.In
Under the premise of not departing from spirit and substance of the present invention, those of ordinary skill in the art can carry out the embodiment of the present invention each
Kind equivalent modifications or substitutions, and these modifications or substitutions all should in covering scope of the invention/any be familiar with this technology neck
The technical staff in domain in the technical scope disclosed by the present invention, can easily think of the change or the replacement, and should all cover of the invention
Within protection scope.Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (9)
1. a kind of streptococcic culture medium of Sodium Hyaluronate, which is characterized in that culture medium includes following components: glucose, yeast
Powder, magnesium sulfate, sodium dihydrogen phosphate, water and microelement.
2. a kind of streptococcic culture medium of Sodium Hyaluronate as described in claim 1, which is characterized in that the culture medium includes
Glucose 50-100g/L, yeast powder 20-40g/L, magnesium sulfate 1-5g/L, sodium dihydrogen phosphate 1-4g/L, microelement 4 × 10-3-5
×10-3g/L。
3. a kind of streptococcic culture medium of Sodium Hyaluronate as claimed in claim 2, which is characterized in that the microelement packet
Include the combination of any one of Cu, Mn, Zn, Ca or at least two.
4. a kind of streptococcic culture medium of Sodium Hyaluronate as claimed in claim 3, which is characterized in that the microelement with
Ionic species exists.
5. a kind of streptococcic culture medium of Sodium Hyaluronate as claimed in claim 3, which is characterized in that the Cu is by CuSO4·
5H2O is provided, and Mn is by MnSO4It provides, Zn is by ZnCl2It provides, Ca is by CaCl2It provides.
6. a kind of streptococcic culture medium of Sodium Hyaluronate as claimed in claim 5, which is characterized in that the culture medium includes
Glucose 70g/L, yeast powder 32.5g/L, magnesium sulfate 3g/L, sodium dihydrogen phosphate 2.5g/L, CuSO4·5H2O 5.12×10-5g/
L, MnSO4 4.785×10-4G/L, ZnCl2 1.235×10-4G/L, CaCl23.8×10-3g/L。
7. a kind of streptococcic culture medium of Sodium Hyaluronate as described in claim 1, which is characterized in that the pH value of culture medium is
7.0-7.5。
8. a kind of Sodium Hyaluronate streptococcus cultural method using the culture medium as described in claim any one of 1-7, feature
It is, method includes:
(1) culture medium is prepared；
(2) by Sodium Hyaluronate streptococcus vaccination into culture medium shaking table culture, condition of culture are as follows: 36-38 DEG C, shaking speed
16-18h is cultivated under 200rpm.
9. Sodium Hyaluronate streptococcus cultural method as claimed in claim 8, which is characterized in that the preparation of culture medium include with
Lower step:
(1) glucose, yeast powder are added to the water, the stirring at 25 DEG C is until obtain uniform mixed liquor；
(2) magnesium sulfate, sodium dihydrogen phosphate and microelement are added into mixed liquor, stirring obtains until the uniform mixed liquor of acquisition
To the culture medium.
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