FR2617849A1 - New process for obtaining hyaluronic acid of bacterial origin - Google Patents

Abstract

Process for obtaining hyaluronic acid of high molecular weight by fermentation of a Streptococcus which produces hyaluronic acid and does not produce enzymes which degrade hyaluronic acid. Application of hyaluronic acid thus obtained to cosmetological compositions.

Description

 The present invention relates to a new process for obtaining hyaluronic acid of high molecular weight by fermentation.

 It relates more particularly to the production of hyaluronic acid of high molecular weight by culturing a microorganism belonging to the genus Streptococcus.

Hyaluronic acid is a natural high-molecular polymer of 3-0-ss-D glucoronopyranosyl-N-acetyl D-glucosamine dioxide.

 Hyaluronic acid is found in many animal connective tissues, such as skin and cartilage.

 Some organs are particularly rich in hyaluronic acid like the umbilical cord, the vitreous humor of the bull's eye, the rooster's crest. The synovial fluid is also very rich in hyaluronic acid.

 The main biological properties of hyaluronic acid are related to its ability to fix water and form gels giving it moisturizing properties for tissues and lubricating the joints.

 The processes for obtaining hyaluronic acid have long used extractions from the anatomical sources mentioned above. But the use of these limited sources in the implementation of low yield led to very high cost products.

The high demand for hyaluronic acid at acceptable prices has led to the search for other modes of production. This is how publications
B. Hömstrom - Appl. Microbiol. (1967) p. 1400-1413
JB Woolcock - J. Gen. Microbiol. (1974) 85, 372-375
G and W Kohler-Zbl. Bakt., I.Abt. Orig. (1970) 215, 187195, and K. sugahara-J. Biol. Chem. (1979) 254, 14, 625.

6261 describe fermentation processes of hyaluronic acid-producing bacteria in the culture medium
The most widely used bacterium is s treptococcus.

 WO 84/03303 and WO 86/04355 describe processes for producing hyaluronic acid by fer tation of Streptococcus zooepidemicus and Streptococcus pyogenes. However, despite fairly long fermentations (24 to 72 hours on average), the yields remain low and the purification delicate. In addition, during fermentation the production of hyaluronisases, enzymes partially degrading hyaluronic acid into low molecular weight fragments devoid of biological interest, is deplored.

 FR-A-2,564,859 describes a process for the fermentation of Streptococcus zooepidemicu, a better yield of hyaluronic acid. However, this process does not solve the hyaluronic acid degradation problems, hyaluronidases.

 The object of the invention is a novel process for producing hyaluronic acid of bacterial origin.

 The aim of the invention is a process for obtaining hyaluronic acid without producing degrading enzymes.

 The process comprises three steps: fermentation, then isolation and purification of hyaluronic acid.

The Streptococcus that can be used during the implementation of the method according to the invention is a mutant belonging to the genus Streptococcus pyogenes of group A, registered under No. I 672 in the National Collection of
Cultures of Microorganisms of the Pasteur Institute, July 2, 1987, 28 rue du Docteur I>u> cux - 75724 PARIS CEDEX 15.

 Maintenance conditions consisting in periodically inoculating the strain in an animal and then isolating it from the blood of the latter, several times in succession, have not only made it possible to exacerbate the virulence of this strain, but also its capacity to produce hyaluronic acid in large quantities, without leading to the production of hyaluronidases.

 Due to the exacerbation of this production capacity, it is advantageous to obtain high concentrations of hyaluronic acid in the culture medium with much shorter fermentation times (24 hours maximum) than those described in the prior art.

 According to a preferred embodiment of this method, the strain is inoculated 9 times successively peritoneally to the mouse, then 24 hours later, isolated from the blood of the animal.

 At the end of this series of inoculations, the strain is ready to be used in a fermentor in the context of the implementation of the process according to the invention.

 According to the invention, the Streptococcus mutant can be cultured on a culture medium containing sources of nitrogen, carbon and mineral salts.

 As a nitrogen source, yeast extract, soy peptone will be used; the mineral elements will be provided by sodium chloride. The carbohydrates will be amnes by the glucose.

These different constituents are incorporated in the culture medium in the following proportions
Yeast extract ..................... 10 to 40 g / l,
Soya peptone ..................... 5 to 20 g / l, NaCl .................. ... 5 gll,
Glucose ................. 50 to 100 g / l.

According to a preferred embodiment of the invention, the above compounds incorporated into the culture medium are in the following proportions
Yeast extract ..................... 30 g / l
Soya peptone ....................... 10 g / l.

Glucose ............................... 75 g / l.

 Only a portion of the glucose is incorporated into the culture medium before fermentation.

 About two-thirds of the glucose is incorporated into the culture medium during fermentation, in small quantities and at regular intervals. According to a preferred embodiment of the process according to the invention, 12.5 g / l of glucose are added to the 11th, 14th, 17th and 20th hours, ie 50 g / l of glucose in total.

 According to the method, the cultivation of Streptococcus pyogenes is preferably aerobic, the concentrations of dissolved oxygen ranging from 20% to 80% saturation.

 During the entire fermentation, the pH is kept constant, equal to a value between 6.5 and 7.5, preferably equal to 7.

 The fermentation temperature is generally between 350C and 400C, preferably equal to 370C.

 The fermentation is preferably under constant stirring.

 When the concentration of hyaluric acid in the culture medium reaches 4 g / l, the fermentation can be stopped by heat.

 According to a preferred implementation of the process according to the invention, 24 hours after the beginning of the fermentation under the conditions described above, the temperature is rapidly raised above 400C, preferably up to 450C.

 Immediately after the end of fermentation, hyaluronic acid is isolated by continuous centrifugation of the biomass.

 The clarified supernatant containing hyaluronic acid is collected and then cooled.

The purification phase may be carried out in different ways, but preferably it comprises the following steps: a) Preparation of the hyaluronic acid from the on
clarified swimming, b) enzymatic treatment of the precipitate obtained in a), c) ultrafiltration after dilution in water, the composition
obtained, d) successive intakes of the solution according to
c), e) spinning and drying the precipitate obtained in d).

 Many organic solvents can be used to precipitate hyaluronic acid.

 Quaternary ammoniums such as cetyl trimethyl ammonium bromide (CTAB) are commonly used in the prior art. The use of acetone in the context of the process according to the invention makes it possible, for a small added volume, to obtain, by simple decantation, a precipitate of highly soluble hyaluronic acid.

 The enzymatic treatment is intended to specifically eliminate nucleic acids and contaminating proteins, thanks to the action of RNases, DNases, proteinases, in particular.

According to a preferred embodiment of the method, the enzymatic treatment can be done by the action of
RNase type I, pancreatic DNase and proteinase K.

Ultrafiltration of the composition obtained after
enzymatic digestion is carried out on a membrane with a threshold i
cut suitably chosen, and allows elimination
simultaneous products of enzymatic digestion and
salts.

The purification is then completed by a series of
alcoholic cipitations. According to a preferred mode of bet el
process, precipitation takes place in the presence of
95% ethyl alcohol.

Finally, the hyaluronic acid composition is
collected, drained and dried.

The hyaluronic acid composition thus obtained
has the following characteristics as a percentage of dry weight
Uronic acids 30% to 46%
Hexosamines 25% to 35%
N-Acetylhexosamines 35% to 49%
Nucleic acids <0.1% to 5%
Total nitrogen 2% to 6%
Amino acids <0.1% to 5%
Ash 5% to 10%
Water content 5% to 15%
Molecular weight 000 000-1 500 000 D
High-molecular-weight hyaluronic acid compositions obtained by carrying out the process according to the invention are advantageously used in cosmetology.

 Other features and advantages of the present invention will become apparent from the following examples.

EXAMPLE 1
Production of a high molecular weight hyaluronic acid composition by fermentation of Streptococcus pyogenes
I 672
Streptococcus pyogenes I 672 is fermented in 400 L of the following medium
Yeast extract 30 g / l
Soy peptone 10 g / l
Glucose 25 g / l
NaCl 5 g / l.

 The pH is maintained at 7, the temperature at 370C.

 The dissolved oxygen level is maintained at 20% saturation.

 Glucose is added as a sterile concentrated solution at a rate of 12.5 g / l at the 11th, 14th, 17th, and 20th hour.

 The culture medium is constantly stirred for the duration of the fermentation.

 After 24 hours of culture under the conditions above, the temperature inside the fermenter is rapidly raised to 450C and the biomass is immediately removed by continuous centrifugation. The clarified supernatant containing the free hyaluronic acid is collected and then cooled.

 The hyaluronic acid is then precipitated by addition to the clarified medium of 1 volume of acetone with rapid stirring. The stirring is then stopped and the precipitate left in decantation for 2 hours in the reactor, then withdrawn and drained.

 The enzymatic treatment proceeds under the following conditions.

 The precipitate obtained above is taken up in water in 100 l of water in a thermostatically controlled reactor. The pH is adjusted to 7.0 with concentrated NaOH.

10 mg / l of RNase type I and 10 mg / l of
Pancreatic DNase. The temperature is raised to 25 ° C and the hydrolysis is allowed to continue overnight at this temperature.

 50 mgol of proteinase K are then added and the enzymatic degradation is continued for 5 hours at 370 ° C. at pH 7.0.

 The concentration of hyaluronic acid in the solution is then adjusted to 10 g / l by dilution with 1 ', followed by diafiltration on 100,000 dalton cut-off membrane at constant volume until the resistivity becomes constant. It is then concentrated to 1/4 of the initial volume.

 The alcoholic precipitation proceeds as follows.

 To the above concentrated solution, sodium acetate is added to obtain a molarity of 0.1 M. The purified hyaluronic acid is then precipitated from this solutic by gradual addition, with stirring, of 2 volumes of ethyl alcohol. 95%.

 The precipitate is collected by decantation and then resuspended in 1 volume of 95% alcohol. The washed precipitate is then collected by decantation and then spin.

 It is finally dried in a vacuum oven for 24 hours at 500C.

Analytical characteristics of the hyaluronic acid thus prepared
The results are expressed as a percentage of the dry weight.

Uronic acids 40.9%
Hexosamines 3-7.2%
N-acetyl hexosamines 46.1%
Nucleic acids 0.6%
Total nitrogen 3.2%
Amino acids <0.1%
7.5% sulfuric ash
Moisture content 10.0%
Molecular weight> 800,000 D
EXAMPLE 2
ANALYTICAL COMPARISON BETWEEN ACID COMPOSITIONS
KNOWN HYALURONICS AND THAT OBTAINED BY THE PROCESS
ACCORDING TO THE INVENTION
The comparison is made from commercially available hyaluronic acid-containing compositions 1) Umbilical cord (Sigma), 2) Rooster crest (Cerva), 3) Hyaluronic acid of bacterial origin (Shiseido)
The principle of the comparison is as follows: The enzymatic hydrolysis of hyaluronic acid by a Streptomyces hyalurolite 5 eliminase releases characteristic unsaturated oligosaccharides, detectable in UV

 . tetrasaccharides (HA.DA),. Hexasaccharides (HA.D6),. Octosaccharides (HA.D8), Decasaccharides (HA.D10),. Dodecasaccharides (H.D12).

 The oligosaccharides thus obtained are separated by HPLC chromatography on anion exchange columns with 8 mM trifluoroacetic acid and detected in UV at 240 nm.

 Isolated fractions I, II, III, IV are identified by negative FAB mass spectrometer (tastAtom Bombardment) as tetra, octa, deca and dodecasaccharide, respectively.

 Figure 1 shows the HPLC chromatogram of hyaluronic acid from the umbilical cord and that from the rooster crest.

 Figure 2 shows the chromatograms of hyaluronic acid shiseido and that of hyaluron acid; obtained by the process according to the invention.

Figure 3 shows the mass spectrogram of HAD6)
Figure 4 shows the mass spectrogram of (HAD8).

 Figure 5 shows the mass spectrogram of (HAD10).

 Figure 6 shows the mass spectrogram of (HAD12).

Compared to reference hyaluronic acid
Sigma isolated from the umbilical cord, it can be concluded that the hyaluronic acid isolated from the crest of rooster and that obtained by the method of the invention, have characteristics that are strictly identical as regards their relative oligosaccharide compositions after enzymatic hydrolysis.

 In contrast, hyaluronic acid.Shiseido take a very different chromatographic profile both by its com in oligosaccharides than by the presence of shoulders and additional peaks likely corresponding to impurities.

Claims (19)

 1. Process for obtaining high molecular weight hyaluronic acid by fermentation of a hyaluronic acid producing and non-hyaluronic acid-degrading enzyme Streptococcus.  2. Method according to claim 1, characterized in that, prior to fermentation, the strain is repeatedly inoculated in the mouse and then isolated from the blood of the latter.  3. Process according to claim 1, characterized in that hyaluronic acid of high molecular weight is obtained by fermentation of a Group A Streptococcus pyogenes registered under No. 672 in the National Collection of Microorganism Cultures at the same time. Pastor Institute.  4. Method according to claims 1 to 3, characterized in that the Streptococcus is cultured in a medium containing yeast extract, soy peptone, glucose and sodium chloride in the following concentrations Yeast extract 10 to 40 g / l, Soy peptone 5 to 20 g / l, NaCl 5 g / l, Glucose 50 to 100 g / l.  5. Method according to claim 4, characterized in that the concentrations of yeast extract, soya peptone, glucose and sodium chloride are as follows Yeast extract 30 g / l Soy peptone 10 g / l Glucose 25 g / l  6. Process according to claims 1 to 3,  characterized in that the culture of the microorganism is  aerobically.  7. Method according to claim 6, characterize  in that the concentration of dissolved oxygen is between 2 <  and 80 8 of saturation.  8. Process according to claims 1 to 7, carac  in that during the cultivation of the microorganism, the  pH is maintained between 6.5 and 7.5, preferably 7,  9. Process according to claim 1 to 8, characterized in that 2/3 of the total glucose is added to the millet  of culture during fermentation, in small quantities  and at regular intervals of time.  10. Process according to claim 9, characterized  in that the glucose is added to the culture medium at  12.5 g / l every 3 hours from the 11th hour  11. Process according to claim 10, characterized  in that 50 g / l of glucose is added to the culture medium  during fermentation.  12. Process according to claims 1 to 9, cara  in that the fermentation is stopped when the con  Hyaluronic acid concentration in the culture medium  reaches 4 g / l.  13. The method of claim 12, characterized  in that the fermentation is stopped by a rise of  the temperature above 400C, preferably up to 4  14. Process according to claims 1 to 9  a fermentation step, an isolation step of  hyaluronic acid from the culture medium, and a  purification step of the supernatant containing hya acid  ronic, said supernatant being obtained by centrifugation  continuous biomass.  15. The method of claim 14, characterized  in that said purification comprises the following steps a) precipitation of hyaluronic acid from the on  clarified swimming, b) enzymatic treatment of the precipitate obtained in a), c) ultrafiltration, after dilution in water, of the compound  sition thus obtained, d) successive alcoholic precipitations of the solution  obtained according to c), e) spinning and drying of the precipitate obtained according to d).  16. The method of claim 15, characterized in that said enzymatic treatment comprises the use of RNase, DNase, and proteinase.  17. The method of claim 16 comprising the use of RNase type I, pancreatic DNase, proteinase K.  18. Hyaluronic acid of high molecular weight obtained by the method according to one of claims 1 to 17, characterized in that the molecular weight of said hyaluronic acid is between 500,000 D and 1,500,000 D.  19. Application of the hyaluronic acid composition obtained according to the method according to one of claims 1 to 18 to cosmetological compositions.