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US4780414A
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Method of producing high molecular weight sodium hyallronate by fermentation of streptococcus
Assignee: Bio Technology General Israel Ltd
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Filing: 1986-01-09
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Granted: 1988-10-25
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Expires: 2006-01-09
Abstract
Abstract A novel mutant microorganism Streptococcus zooepidemicus HA-116 ATCC 39920, has been produced. The microorganism produces large amounts of high molecular weight hyaluronic acid. The invention provides a method of obtaining such microorganisms. The invention also concerns a method of obtaining sodium hyaluronate which comprises growing with vigorous agitation a microorganism of the genus Streptococcus under appropriate conditions in a suitable nutrient medium containing a sugar component as a carbon source. The sugar component is present in a substantially constant concentration between 0.2 and 10 grams per liter. The medium has a substantially constant pH between about 6.0 and 7.5 and includes a substantially constant magnesium ion concentration above 0.05 grams per liter. The sodium hyaluronate excreted into the medium by the organism is purified using methods involving precipation, redissolving and reprecipating the hyaluronate. Composition of sodium hyaluronate which are characterized by an absence of pyrogenicity and skin irritation are obtained.
Inventors
- 👤 Abraham Nimrod
- 👤 Benjamin Greenman
- 👤 Dov Kanner
- 👤 Moshe Landsberg
- 👤 Yaffa Beck
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 10:54
CPC Classifications
A; A61; A61K; A61K31/00; A61K31/70; A61K31/715; A61K8/00; A61K8/18; A61K8/72; A61K8/73; A61K8/735; A61P; A61P27/00; A61P27/02; A61P29/00; A61Q; A61Q19/00; C; C08; C08B; C08B37/00; C08B37/006; C08B37/0063; C08B37/0072; C12; C12N; C12N1/00; C12N1/20; C12N1/205; C12P; C12P19/00; C12P19/26; A61K2800/00; A61K2800/74; A61K2800/75; C12R; C12R2001/00; C12R2001/01; C12R2001/46; Y; Y10; Y10S; Y10S435/00; Y10S435/803; Y10S435/818; Y10S435/8215; Y10S435/822; Y10S435/885
Claims Analysis
Total Claims: 40
Claim 1
Independent Claim
12 words
The microorganism Streptococcus zooepidemicus HA-116 ATTC No. 39920 and mutants derived therefrom.
Claim 2
Dependent Claim
12 words
The microorganism Streptococcus zooepidemicus HA-116 ATTC No. 39920 and mutants derived therefrom.
Claim 3
Independent Claim
109 words
A method of obtaining sodium hyaluronate which comprises (a) growing a microorganism of the genus Streptococcus under appropriate conditions in a suitable nutrient medium, the conditions comprising vigorous agitation and aeration of the medium and the medium including a sugar component as the carbon source in a substantially constant concentration between about 0.2 and 10 grams per liter, having a substantially constant pH between about 6.5 and 7.5 and including a substantially constant magnesium ion concentration above about 0.05 grams per liter, such that the microorganism produces sodium hyaluronate and excretes the sodium hyaluronate so produced into the medium, and (b) then recovering the sodium hyaluronate from the medium.
Claim 4
Independent Claim
109 words
A method of obtaining sodium hyaluronate which comprises (a) growing a microorganism of the genus Streptococcus under appropriate conditions in a suitable nutrient medium, the conditions comprising vigorous agitation and aeration of the medium and the medium including a sugar component as the carbon source in a substantially constant concentration between about 0.2 and 10 grams per liter, having a substantially constant pH between about 6.5 and 7.5 and including a substantially constant magnesium ion concentration above about 0.05 grams per liter, such that the microorganism produces sodium hyaluronate and excretes the sodium hyaluronate so produced into the medium, and (b) then recovering the sodium hyaluronate from the medium.
Claim 5
Dependent Claim
14 words
A method of claim 2, wherein the microorganism is of the species Streptococcus zooepidemicus.
Claim 6
Dependent Claim
14 words
A method of claim 3, wherein the microorganism is Streptococcus zooepidemicus, HA-116, ATCC 39920.
Claim 7
Dependent Claim
28 words
The method of claim 2, wherein the aeration of the medium is at a rate greater than about 0.5 volume of air per volume of medium per minute.
Claim 8
Dependent Claim
52 words
The method of claim 2, wherein the suitable nutrient medium comprises in the concentration of grams per liter of the medium the following components: ______________________________________
component concentration
______________________________________
Casein hydrolysate about 10-30
Yeast extract about 5-15
NaCl about 2
MgSO.sub.4.7H.sub.2 O
above about 0.5
K.sub.2 HPO.sub.4 about 2.5
Glucose about 2-15
______________________________________
Claim 9
Dependent Claim
42 words
The method of claim 2, wherein recovering the sodium hyaluronate comprises treating the medium containing the microorganisms so as to remove the microorganism and other materials insoluble in the medium, precipitating the sodium hyaluronate from the medium and then recovering the precipitate.
Claim 10
Dependent Claim
13 words
The method of claim 7 further comprising grinding and then drying the precipitate.
Claim 11
Dependent Claim
48 words
The method of claim 7, further comprising adjusting the pH of the medium containing the microorganism to about 5.0 and then heating the medium for a suitable period of time at a temperature between about 80° and 95° C. prior to treating the medium to remove the microorganism.
Claim 12
Dependent Claim
18 words
The method of claim 9, wherein the medium is heated for about 20 minutes at about 90° C.
Claim 13
Dependent Claim
18 words
The method of claim 9, wherein the medium is heated for about 40 minutes at about 80° C.
Claim 14
Dependent Claim
10 words
The method of claim 7, wherein the treating comprises filtration.
Claim 15
Dependent Claim
14 words
The method of claim 12, wherein the filtration comprises filtration on a diatomaceous earth.
Claim 16
Dependent Claim
72 words
The method of claim 7, wherein the precipitation comprises adding a first organic solvent to the medium to produce a precipitate, redissolving the precipitate in 3% aqueous sodium acetate, adding a second organic solvent to produce a precipitate, redissolving the precipitate in 3% aqueous sodium acetate, adding activated charcoal to form a suspension, filtering the suspension and adding a third organic solvent to the filtrate to produce a precipitate of sodium hyaluronate.
Claim 17
Dependent Claim
20 words
The method of claim 14, wherein each of the first, second and third organic solvents are isopropanol, ethanol or acetone.
Claim 18
Dependent Claim
15 words
The method of claim 15, wherein the first, second and third organic solvents are isopropanol.
Claim 19
Dependent Claim
50 words
The method of claim 7, further comprising adjusting the pH of the medium containing the microorganism to about 7.0, cooling the medium to a temperature between about 4° and 15° C. and then diluting the medium with 3% aqueous sodium acetate prior to treating the medium to remove the microorganism.
Claim 20
Dependent Claim
50 words
The method of claim 7, further comprising adjusting the pH of the medium containing the microorganism to about 7.0, cooling the medium to a temperature between about 4° and 20° C. and then diluting the medium with 3% aqueous sodium acetate prior to treating the medium to remove the microorganism.
Claim 21
Dependent Claim
50 words
The method of claim 14, further comprising adjusting the pH of the medium containing the microorganism to about 7.0, cooling the medium to a temperature between about 4° and 15° C. and then diluting the medium with 3% aqueous sodium acetate prior to treating the medium to remove the microorganism.
Claim 22
Dependent Claim
46 words
The method of claim 19, further comprising redissolving the precipitate in 0.15M squeous NaCl, adding cetyl-pyridinium chloride to form the cetyl-pyridinium salt of hyaluronic acid, dissolving the cetyl-pyridinium salt in aqueous NaCl (at least about 1M) and ethanol, adding organic solvent and recovering the sodium hyaluronate.
Claim 23
Dependent Claim
88 words
The method of claim 20, further comprising redissolving the recovered sodium hyaluronate in 0.15M aqueous NaCl, adding cetyl-pyridinium chloride to again form the cetyl-pyridinium salt of hycluronic acid, dissolving the cetyl-pyridinium salt in aqueous NaCl (at least about 1M) and 10% ethanol, precipitating the sodium hyaluronate with an organic solvent, dissolving the sodium salt in NaCl solution, contacting the resulting solution with magnesium silicate absorbent to remove impurities and residual cetyl-pyridinium ions, sterilizing the solution and adding sterile organic solvent to precipitate the sodium hyaluronate from the solution.
Claim 24
Dependent Claim
11 words
The method of claim 21, wherein the organic solvent is isopropanol.
Claim 25
Dependent Claim
15 words
The method of claim 21, further comprising airdrying the sodium hyaluronate precipitate under sterile conditions.
Claim 26
Dependent Claim
57 words
The method of claim 2, wherein recovering the sodium hyaluronate comprises adding a first organic solvent to the medium to produce a precipitate, washing the precipitate with more organic solvent, redissolving the precipitate in a suitable aqueous solution, adding activated charcoal to form a suspension and filtering the suspension to remove residual microorganisms and other insoluble materials.
Claim 27
Dependent Claim
45 words
The method of claim 24, further comprising adjusting the pH of the medium containing the microorganism to about 5.0 and heating the medium for a suitable period of time at a temperature between about 80° and 95° C. prior to adding the first organic solvent.
Claim 28
Dependent Claim
20 words
The method of claim 25, wherein the suitable period of time is 40 minutes and the temperature is 80° C.
Claim 29
Dependent Claim
14 words
The method of claim 24, wherein the filtration comprises filtration on a diatomaceous earth.
Claim 30
Dependent Claim
13 words
The method of claim 24, wherein the suitable aqueous solution 3% sodium acetate.
Claim 31
Dependent Claim
46 words
The method of claim 24, further comprising adding a second organic solvent to the filtrate to produce a precipitate, redissolving the precipitate in 3% aqueous sodium actate, filtering the solution and adding a third organic solvent to the filtrate to produce a precipitate of sodium hyaluronate.
Claim 32
Dependent Claim
20 words
The method of claim 29, wherein each of the first, second and third organic solvents are isopropanol, ethanol or acetone.
Claim 33
Dependent Claim
15 words
The method of claim 29, wherein the first, second and third organic solvents are ethanol.
Claim 34
Dependent Claim
18 words
The method of claim 29, further comprising grinding and then drying the precipitate of the third organic solvent.
Claim 35
Dependent Claim
42 words
The method of claim 24, 25 or 29, further comprising adjusting the pH of the medium containing the microorganism to about 7.0 and cooling the medium to a temperature between about 4° and 20° C. prior to adding the first organic solvent.
Claim 36
Dependent Claim
21 words
The method of claim 24, wherein the suitable aqueous solution is 0.15M aqueous NaCl solution containing 0.1% parahydrozybenzoic acid methyl ester.
Claim 37
Dependent Claim
48 words
The method of claim 34, further comprising adding cetyl-pyridinium chloride in 0.15M NaCl to the hyaluronate solution to form the cetyl-pyridinium salt to hyaluronic acid, redissolving the cetyl-pyridinium salt in aqueous NaCl (at least about 1M) containing 10% ethanol and adding organic solvent to recover the sodium hyaluronate.
Claim 38
Dependent Claim
89 words
The method of claim 35, further comprising redissolving the recovered sodium hyaluronate in 0.15M aqueous NaCl, adding cetyl-pyridinium chloride to again form the cetyl-pyridinium salt of hyaluronic acid, redissolving the cetyl-pyridinium salt in aqueous NaCl (at least about 1M) containing 10% ethanol, adding organic solvent to precipitate the sodium hyaluronate, redissolving the precipitate in sterile 1M aqueous NaCl, contacting the resulting solution with magnesium silicate absorbent to remove impurities and residual cetyl-pyridinium ions, sterilizing the solution, and adding sterile organic solvent to precipitate the sodium hyaluronate from the solution.
Claim 39
Dependent Claim
11 words
The method of claim 36, wherein the organic solvent is ethanol.
Claim 40
Dependent Claim
16 words
The method of claim 36, further comprising drying the sodium hyaluronate by nitrogen under sterile conditions.
References Cited (Backward Citations)
JPS5837001A
Cited
US4517295A
Cited
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 7.5 Years (Year 8) | 1995-10-25 | 1996-04-25 | 1996-10-25 | $3760.00 | Window Closed / Lapsed |
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