CN1102435A - Method for producing sodium hyaluronate by microbial fermentation - Google Patents
Method for producing sodium hyaluronate by microbial fermentation Download PDFInfo
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- CN1102435A CN1102435A CN 94114885 CN94114885A CN1102435A CN 1102435 A CN1102435 A CN 1102435A CN 94114885 CN94114885 CN 94114885 CN 94114885 A CN94114885 A CN 94114885A CN 1102435 A CN1102435 A CN 1102435A
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- fermentation
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- hyaluronate sodium
- cctcc94034
- sodium hyaluronate
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- 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 30
- 238000000855 fermentation Methods 0.000 title claims abstract description 23
- 230000004151 fermentation Effects 0.000 title claims abstract description 23
- 230000000813 microbial effect Effects 0.000 title claims abstract description 7
- 229920002385 Sodium hyaluronate Polymers 0.000 title abstract 5
- 229940010747 sodium hyaluronate Drugs 0.000 title abstract 5
- 238000004519 manufacturing process Methods 0.000 title description 6
- 238000000034 method Methods 0.000 claims abstract description 46
- 241000194048 Streptococcus equi Species 0.000 claims abstract description 15
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 6
- 230000001580 bacterial effect Effects 0.000 claims description 28
- 244000005700 microbiome Species 0.000 claims description 17
- 239000001963 growth medium Substances 0.000 claims description 14
- 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 description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 5
- 239000001888 Peptone Substances 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 4
- 235000019319 peptone Nutrition 0.000 claims description 4
- 239000001632 sodium acetate Substances 0.000 claims description 4
- 235000017281 sodium acetate Nutrition 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 3
- 238000001179 sorption measurement Methods 0.000 claims description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- 239000012716 precipitator Substances 0.000 claims description 2
- 239000007788 liquid Substances 0.000 abstract description 10
- 241000894006 Bacteria Species 0.000 abstract description 6
- 239000002537 cosmetic Substances 0.000 abstract description 4
- 239000002994 raw material Substances 0.000 abstract description 4
- 238000012869 ethanol precipitation Methods 0.000 abstract description 3
- 239000005909 Kieselgur Substances 0.000 abstract 1
- 239000003610 charcoal Substances 0.000 abstract 1
- 238000001035 drying Methods 0.000 abstract 1
- 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 24
- 229920002674 hyaluronan Polymers 0.000 description 24
- 229960003160 hyaluronic acid Drugs 0.000 description 24
- 239000000047 product Substances 0.000 description 8
- 238000001556 precipitation Methods 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000009395 breeding Methods 0.000 description 4
- 230000001488 breeding effect Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
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- 108090000623 proteins and genes Proteins 0.000 description 3
- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000001263 FEMA 3042 Substances 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 description 2
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- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 229920002258 tannic acid Polymers 0.000 description 2
- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 description 2
- 229940033123 tannic acid Drugs 0.000 description 2
- 235000015523 tannic acid Nutrition 0.000 description 2
- MEIRRNXMZYDVDW-MQQKCMAXSA-N (2E,4E)-2,4-hexadien-1-ol Chemical compound C\C=C\C=C\CO MEIRRNXMZYDVDW-MQQKCMAXSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000561734 Celosia cristata Species 0.000 description 1
- 241000732800 Cymbidium Species 0.000 description 1
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 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
- 241000726221 Gemma Species 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 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 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
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Sodium hyaluronate is produced by fermentation of Streptococcus equi CCTCC94034 in a liquid medium, with diatomaceous earth and/or viable bacteria after fermentation has been terminatedRemoving microbial cells in the culture solution by using charcoal for suction, separating and collecting sodium hyaluronate in the culture solution by using an ethanol precipitation method, and drying the sodium hyaluronate in vacuum to obtain a finished product. The yield of the sodium hyaluronate is more than 4g/l, and the molecular weight is about 4.5 multiplied by 105Daltons, protein content below 0.03% by weight. The product is suitable for use as raw material for high-grade cosmetics.
Description
The present invention relates to microorganism and the intermediate carrier that is used for daily cosmetics, particularly relate to a kind of method that is used for the hyaluronate sodium of makeup with microbial fermentation engineering production.
Hyaluronic acid (Hyaluronic acid) is a kind of linear polymer that is alternately connected to form by glucuronic acid and N-acetylglucosamine, and the molecular weight majority is between 200,000-3,500,000 dalton or higher.Product is a white powder, exists with the form of hyaluroni.
Hyaluronic acid has fabulous water-retentivity and assists the characteristic of infiltration, is applied to clinical medicine as binding agent, and cosmetic industry is the senior water conservation factor of glycerine as an alternative, and its demand increases day by day, and Application Areas is progressively expanded, and is paid attention to by countries in the world.
The traditional extracting method of hyaluronic acid is that employing animal tissues is a raw material, extracts hyaluronic acid from the vitreum of people's umbilical cord, cockscomb, cattle and sheep pig eye, and these animal tissuess must be fresh collections, and through freezing treatment.Said animal tissues source difficulty cost an arm and a leg, and hyaluronic content is very low in these animal tissuess, and product yield is low.In leaching process, must use a large amount of organic solvent and enzyme, complex process, operating unit is many, and hyaluronic cost is heightened.Use the hyaluronic acid foreign matter content height of the method production of from animal tissues, extracting in addition, refining difficulty.
The method of producing hyaluronate sodium with microbial fermentation engineering that the object of the present invention is to provide that a kind of technology is simple, cost is low.
Technical solution scheme of the present invention is such: adopt plurality of raw materials to produce hyaluronic acid with microbe fermentation method, the source of required substratum is wide, and is unrestricted, and price is more cheap, has reduced hyaluronic cost, helps suitability for industrialized production simultaneously.Because hyaluronic acid is the exocellular polysaccharide of microorganism secretion to cells in vitro, in leaching process, does not need cytoclasis, protein content in the fermented liquid is few, and this makes extraction process become simply, does not need a large amount of organic solvents, more do not need proteolytic enzyme, hyaluronic cost is reduced greatly.And the hyaluronic yield of fermentative Production is than higher.Hyaluronic acid with fermentative Production also helps further making with extra care, and makes than pure preparation, is used for ophthalmologic operation and other medical field.
Adopt microbe fermentation method to produce hyaluronic acid, the measure of most critical is to screen and to cultivate a kind of can the manufacturing hyaluronic bacterial strain and filter out a kind of substratum that is most appropriate to cultivate this bacterial strain in good quality and high output ground.
The inventor obtains a kind of hyaluronic bacterial classification that is suitable for producing in nineteen ninety from professor Ji Yanchang of Institute for Medical Research of Tokyo Univ Japan, its formal name used at school is Streptococcus equi, Chinese streptococcus equi by name.Yet, when making hyaluronate sodium with this bacterial strain, obtain protein content higher (3.68 weight %) in the product, when the hyaluronate sodium that will contain higher protein is used for makeup, cause allergicly easily, so this original strain is not ideal enough.
Inventor's decision is carried out mutagenesis and screening with above-mentioned streptococcus equi as original strain, cultivates a kind of better bacterial strain.The mutafacient system that is adopted is to shine original strain 60-90 second with the 15W ultraviolet lamp in about 30cm distance, then by measuring the method for hyaluronic acid enzyme activity, select single bacterium colony that the hyaluronic acid enzyme activity is weak or do not have, cultivate through 8 months repeated screenings, it is strong to have obtained a kind of adaptability, vitality is vigorous, and hyaluronic acid (hereinafter referred HA) output is high, the new bacterial strain of mutant (strain number N90006) of safety.
The inventor identifies with following method new bacterial strain.At first by microbiological ordinary method at the mutant of determining aspect form and the Physiology and biochemistry two that N90006 number new bacterial strain is a kind of streptococcus equi.Identify on the flat board with Japanese strain at Unidasa then to compare, prove that this new bacterial strain is the very weak mutation type of a kind of hyaluronic acid enzyme activity as original strain.When identifying, used evaluation flat board is the culture medium flat plate that contains 1% bovine serum albumin, 0.2% hyaluronic acid, 1% agarose, pH6.8 ± 1.Identify by the inoculation of method of scoring or photolithography dull and stereotypedly, be cultured to bacterium colony in 37 ℃ and form (usually 40 little more than), covered 10 minutes with 2N acetate then that the size of transparent circle of observing periphery of bacterial colonies is to compare.Japanese strain is identifying that the transparent circle that forms on the flat board is very big, and the transparent circle that new bacterial strain N90006 forms is then very little or form transparent circle hardly.The hyaluronic acid enzyme activity of this explanation Japanese strain is stronger, and the very little or essentially no vigor of the hyaluronic acid enzyme activity of new bacterial strain of the present invention.The mutant that provable thus N90006 number new bacterial strain is a kind of streptococcus equi.
The inventor should be sent to Chinese typical culture collection center preservation by new bacterial strain, and preserving number is CCTCC NO, M94034(C TCC94034), preservation day is on June 20th, 1994.
Feature description to the CCTCC94034 bacterial strain is as follows below:
A, thalline: diameter is spheroidal or the ovoid shape of 0.6-1 μ m, often is arranged in chain, and the length of bacterium chain is relevant with growing environment, easily is the long-chain shape in the liquid medium within, easily is the short chain shape on solid medium.
B, bacterium colony: transparent, flash of light, little protuberance reveals and drips shape.
C, the cymbidium Albert'stain Albert positive are cultivated and are negative no gemma long afterwards.
D, amphimicrobian.
E, haemolysis: periphery of bacterial colonies forms opaque green circle on the blood agar.
F, Lan Shi (Lance field): the D that classifies
G, security: the animal beyond people and the horse is not had infectivity.
H, fermentation character:
Sorbyl alcohol+sodium hippurate-
Lactose-Vitamin C2-
Synanthrin-arginine-
The inventor as Object of Development, filters out the substratum of this strain growth of a kind of optimum and breeding with the CCTCC94034 bacterial strain then.This substratum is a kind of substratum of water solution system.Every liter of liquid medium contains following material:
Peptone 5-15g KH
2PO
45-15g
Sodium acetate 3-15g NaHCO
32-5g
MgSO
4·7H
2O 0.1-1.0g FeSO
4·7H
2O 0.05-0.2g
MnCl
2·4H
2O 0.05-0.2g CaCl
20.02-0.2g
Yeast powder 3-10g glucose 20-60g
Weibull 0.01-0.3g
Should illustrate, above-mentioned culture medium prescription is the substratum of CCTCC94034 bacterial strain optimum breeding of the present invention, because this bacterial strain is the bacterial strain that a kind of adaptability is very strong and vitality is vigorous, so also can be used to cultivate the CCTCC94034 bacterial strain with akin other substratum of above-mentioned prescription, yet this also should be considered to belong to scope of the present invention.Therefore, the method that every use CCTCC94034 bacterial strain is produced hyaluronate sodium all should be considered to belong to the application's protection domain.
Because the CCTCC94034 bacterial strain is constantly made hyaluronic acid (HA) and it is excreted in the substratum in the process of its growth and breeding, the sodium hydroxide effect in HA and the substratum, thus generated hyaluronate sodium.When bacterial strain stopped or slowing down growth and breeding, regeneration HA so the pH value of substratum no longer descends, according to this point, can not stop fermentation.In order to obtain the hyaluronate sodium product, remove as long as microorganism separated, and then hyaluronate sodium is precipitated out from culture medium solution and collected get final product.These steps all are known technologies, therefore, no matter what method of employing is separated remove microorganism and how to precipitate hyaluronate sodium, as long as use the CCTCC94034 bacterial strain, promptly should think scope of the present invention.
When separate microorganism, can earlier it be killed, can not kill, as long as can be removed yet.Isolating means have varied, for example diatomite adsorption method, centrifuging etc.The method of precipitation hyaluronate sodium also has varied, for example the precipitator method of ethanol precipitation and other precipitation agents etc.
That is to say, the invention provides a kind of new bacterial strain that is suitable for producing by fermentation hyaluronate sodium, it is characterized in that it is a kind of mutant of streptococcus equi (Streptococcus equi), its preserving number is CCTCC94034.
In addition, the invention provides a kind of method of producing hyaluronate sodium by microbial fermentation, it is characterized in that, streptococcus equi CCTCC94034 is fermented in substratum, pH value with substratum maintains between the 6.5-7.5 during the fermentation, after the termination fermentation, microorganism cells is separated with culture medium solution, and then separate and collect the hyaluronate sodium of generation the culture medium solution after removing microorganism cells.
The present invention provides a kind of method of further qualification based on the above method, it is characterized in that, wherein, every liter of said culture medium solution contains following material:
Peptone 5-15g KH
2PO
45-15g
Sodium acetate 3-10g NaHCO
32-5g
MgSO
4·7H
2O 0.1-1.0g FeSO
4·7H
2O 0.05-0.2g
MnCl
2·4H
2O 0.05-0.2g CaCl
20.02-0.2g
Yeast powder 3-10g glucose 20-60g
Weibull 0.01-0.3g
Other further being characterized as of limiting of the inventive method: the pH value of fermenting process is controlled between the 7.2-7.5; Temperature is 32-38 ℃, is preferably 37 ± 1 ℃; Stir during fermentation, stirring velocity is preferably 50-150rpm; After fermentation stops, be diatomite and/or active carbon adsorption with microorganism cells and the isolating method of culture medium solution; The method of separating hyaluronate sodium the culture medium solution after removing microorganism cells is an ethanol precipitation.
Compare with the microorganism of prior art, the adaptability of streptococcus equi CCTCC94034 of the present invention is strong, and vitality is vigorous, and the vigor of its Unidasa is very weak, makes the low percentages of protein in the strong and hyaluronic acid made of hyaluronic ability.
Compare with the method that prior art is produced hyaluronate sodium with microbial fermentation, the present invention uses streptococcus equi CCTCC94034, technological process is simple, processing ease, cost is lower, and the yield of hyaluronate sodium is higher (generally to reach the 4.0g/l fermented liquid, reach the 8g/l fermented liquid when the highest), low percentages of protein in the hyaluronate sodium product (be generally 0.03 weight %, reach 0.008 weight % when minimum) therefore is more suitable for the raw material as superior cosmetics.
Enumerate embodiment below and further explain the present invention.
Embodiment 1
Use streptococcus equi CCTCC94034 as producing hyaluronic microorganism, use liquid nutrient medium to cultivate, after stopping fermentation, separate the microorganism of removing in the substratum, precipitation reclaims hyaluronate sodium from this substratum then.Whole technological process comprises the steps:
1, take by weighing various compositions respectively by following prescription:
Peptone 10g KH
2PO
410g
Sodium acetate 5g NaHCO
33g
MgSO
4·7H
2O 0.1g FeSO
4·7H
2O 0.1g
MnCl
2·4H
2O 0.1g CaCl
20.05g
Yeast powder 5g glucose 40g
Weibull 0.2g
2, the preparation of culture medium solution and processing
(1) with glucose 40g dissolved in distilled water, be diluted to 150ml, sterilized 15 minutes separately with 121 ℃, standby.
(2) with Weibull 0.2g dissolved in distilled water, dilute main 40ml, sterilized 15 minutes separately with 121 ℃, standby.
(3) other compositions in the above-mentioned prescription are mixed, use dissolved in distilled water, be diluted to 760ml, its pH value is adjusted to 7.2, with 121 ℃ of sterilizations 25 minutes, then it is transferred in three mouthfuls of vials that are installed in the water bath with thermostatic control, temperature is controlled at 37 ± 1 ℃, and start stirrer, with the stirring velocity stirring of 100rpm.
3, inoculation fermentation
The pre-nutrient solution of 50ml streptococcus equi CCTCC94034 is inoculated in the 760ml nutrient solution in the vial, feeds nitrogen simultaneously to guarantee anaerobic condition.Then the side mouth by vial drips above-mentioned glucose solution 150ml and tannic acid solution 40ml.At this moment because the carrying out and the CCTCC94034 bacterial strain of fermenting process constantly produce hyaluronic acid, make pH value begin to descend.NaOH solution with 40 weight % is controlled at pH between the 7.2-7.5.Above-mentioned glucose solution and tannic acid solution dripped in 10 hours, and at this moment the cumulative volume of complete soln is 1 liter.Continue fermentation 20 hours, at this moment the pH value of nutrient solution has stopped descending, so stop fermentation.
4, the extraction of hyaluronate sodium
(1) the NaCl solution with 1.5M is diluted to 1.5 liters with fermented liquid.
(2) add 45g diatomite and 2g gac, stir 2 hours with impurity and the color that removes solution such as absorption microorganism cellss;
(3) filter cleaner;
(4) filtrate is carried out centrifugation, with further Ex-all impurity;
(5) add 1.5 times of volume of ethanol in the clear liquid after centrifugal so that the hyaluronate sodium precipitation is filtered the collecting precipitation thing;
(6) with of the NaCl solution 1100ml dissolving of obtain precipitation, toward wherein adding 15g diatomite and 1g gac, stirred 1 hour, then filter cleaner then with 0.15M;
(7) filtrate is carried out a centrifugation again;
(8) with the clear liquid after centrifugal with precise and tiny filter filtration sterilization;
(9) will the go out filtrate of bacterium is slowly injected 2 liters of ethanol, leaves standstill 8 hours, stirs once, and then leaves standstill 6 hours;
(10) collecting precipitation thing, with carrying out vacuum-drying behind the absolute ethanol washing, the result obtains the hyaluronate sodium 4.6g of white, and this is finished product.Because the initial incubation based sols is 1l, so the yield of hyaluronate sodium is the 4.6g/l nutrient solution.
To the result of this product analysis, record its molecular weight and be about 4.6 * 10
5Dalton, the protein content in the product is 0.028 weight %.
Claims (9)
1, a kind of new bacterial strain is characterized in that, it is a kind of mutant of streptococcus equi (Streptococcu-s equi), and its preserving number is CCTCC94034.
2, a kind of method of producing hyaluronate sodium by microbial fermentation, it is characterized in that, streptococcus equi CCTCC94034 is fermented in substratum, pH value with substratum maintains between the 6.5-7.5 during the fermentation, after the termination fermentation, microorganism cells is separated with culture medium solution, and then separate and collect the hyaluronate sodium of generation the culture medium solution after removing microorganism cells.
3, method as claimed in claim 2 is characterized in that, contains following material in every liter of said culture medium solution:
Peptone 5-15g KH
2PO
45-15g
Sodium acetate 3-10g NaHCO
32-5g
MgSO
4·7H
2O 0.1-1.0g FeSO
4·7H
2O 0.05-0.2g
MnCl
2·4H
2O 0.05-0.2g CaCl
20.02-0.2g
Yeast powder 3-10g glucose 20-60g
Weibull 0.01-0.3g
As the method for claim 2 or 3, it is characterized in that 4, the pH value of fermenting process is controlled between the 7.2-7.5.
As the method for claim 2 or 3, it is characterized in that 5, the temperature of fermenting process is controlled between 32-38 ℃.
6, method as claimed in claim 5 is characterized in that, the temperature of fermenting process is controlled at 37 ± 1 ℃.
As the method for claim 2 or 3, it is characterized in that 7, the stirring velocity of fermenting process is 50-150rpm.
8, as the method for claim 2 or 3, it is characterized in that, is diatomite and/or active carbon adsorption with microorganism cells and the isolating method of culture medium solution.
As the method for claim 2 or 3, it is characterized in that 9, the method for separating hyaluronate sodium the culture medium solution after removing microorganism cells is the acetate precipitator method.
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|---|---|---|---|
| CN 94114885 CN1102435A (en) | 1994-08-17 | 1994-08-17 | Method for producing sodium hyaluronate by microbial fermentation |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 94114885 CN1102435A (en) | 1994-08-17 | 1994-08-17 | Method for producing sodium hyaluronate by microbial fermentation |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100478360C (en) * | 2006-10-31 | 2009-04-15 | 山东福瑞达生物化工有限公司 | Process for producing hyaluronic acid or its salt by concentration |
| CN101935362B (en) * | 2009-06-30 | 2012-07-25 | 上海佰加壹医药有限公司 | Method for purifying hyaluronic acid by pre-laying filter aid |
| CN105194096A (en) * | 2015-09-21 | 2015-12-30 | 成都圣雪贝佳化妆品有限公司 | Preparation fast relieving skin red, swelling, heat, pain and itch and preparing method thereof |
| CN106434444A (en) * | 2016-09-23 | 2017-02-22 | 东辰控股集团有限公司 | Streptococcus equi subsp and production technology for preparing hyaluronic acid through streptococcus equi subsp |
| CN106434443A (en) * | 2016-09-23 | 2017-02-22 | 东辰控股集团有限公司 | Production process of sodium hyaluronate |
-
1994
- 1994-08-17 CN CN 94114885 patent/CN1102435A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100478360C (en) * | 2006-10-31 | 2009-04-15 | 山东福瑞达生物化工有限公司 | Process for producing hyaluronic acid or its salt by concentration |
| CN101935362B (en) * | 2009-06-30 | 2012-07-25 | 上海佰加壹医药有限公司 | Method for purifying hyaluronic acid by pre-laying filter aid |
| CN105194096A (en) * | 2015-09-21 | 2015-12-30 | 成都圣雪贝佳化妆品有限公司 | Preparation fast relieving skin red, swelling, heat, pain and itch and preparing method thereof |
| CN106434444A (en) * | 2016-09-23 | 2017-02-22 | 东辰控股集团有限公司 | Streptococcus equi subsp and production technology for preparing hyaluronic acid through streptococcus equi subsp |
| CN106434443A (en) * | 2016-09-23 | 2017-02-22 | 东辰控股集团有限公司 | Production process of sodium hyaluronate |
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