JPH046356B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a method for producing hyaluronic acid. More specifically, bacteria belonging to the genus Streptococcus, which have the ability to produce hyaluronic acid but are non-hyaluronidase-producing and non-hemolytic, are cultured in a medium to efficiently produce high-molecular-weight hyaluronic acid that does not contain hemolysin. Concerning how to manufacture well. Conventional technology Today, hyaluronic acid is recognized to exist in all parts of the connective tissue of animals, and is industrially obtained from living tissues such as chicken crests and velvets by extraction methods. Its functions are to retain water between cells, to form a jelly-like matrix within tissues to retain cells, to control the movement of substances between cells, and to prevent external physical shocks and infections such as bacteria. This is mentioned. Utilizing these functions, hyaluronic acid is used in pharmaceuticals (arthritis treatments, eye drops, wound healing agents, etc.), cosmetics, and the like. However, the production of hyaluronic acid by extraction from living tissue is difficult and extremely expensive to mass produce due to the complexity of separation and purification. This has closed off the path to developing new uses for hyaluronic acid. Regarding the production of hyaluronic acid by microorganisms, bacteria of the genus Streptococcus, type A, C, and D of the Lancefield serogroup, such as Streptococcus pyogenes, Streptococcus zooepidemicus ( Streptococcus
zooepidemicus), Streptococcus equi, Streptococcus equisimilis, Streptococcus dysgalactiae and Streptococcus faecalis var. zymogenes) and Pasteurella multocida (Pasteurella
multocida) etc., are already known to produce hyaluronic acid; for example, Kendall et al. (FE
Kendall et al., J.Biol.Chem., 118, 61,
1937), WAPierce et al., J.
Bact., 63, 301, 1952), Pine Crenan (AP
MacLennan, J.Gen.Microbiol., 14, 134−142,
1956; J.Gen.Microbiol., 15, 485-491, 1956),
B. Holmström et al.
Appl.Microbiol., 15, 1409−1413, 1967), JBWoolcock, J.Gen.Microbiol.
85, 372-375, 1974), E. Kjems et al.
al., Acta Path.Microbiol.Scand.Sect.B, 84,
162-164, 1976), T. Bergan et al.
al., Acta Path.Microbiol.Scand., 75, 97−
103, 1969) and JACifonelli,
Carbohyd. Res., 14, 272-276, 1970). These reports were not aimed at mass production of hyaluronic acid, but were cultured using 1-1.5% glucose as a carbon source, and the amount of hyaluronic acid produced was 0.5-0.6g/
The yield based on sugar was 6% or less. In the above report, Matsukrennan reported that culturing a type of Lansfield serogroup C bacterium of the genus Streptococcus under aerobic conditions may promote the production of hyaluronic acid. Among the above-mentioned microorganisms that produce hyaluronic acid, Lancefield serogroup A bacteria of the genus Streptococcus and Pasteurella are known as pathogenic bacteria to humans, and are actually unsuitable for mass culture. JP-A-58-56692 discloses a method for industrially culturing hyaluronic acid-producing bacteria of the genus Streptococcus and extracting and purifying hyaluronic acid from the culture solution. This method is to obtain a large amount of hyaluronic acid by culturing Lancefield serogroup A and C bacteria of the genus Streptococcus.
g/g of hyaluronic acid is obtained. In this case, the yield of hyaluronic acid based on sugar was 5%, and remained unchanged even when the amount of glucose added was changed from 1% to 8%. Therefore, this sugar yield (5%) is almost the same as the sugar yield of hyaluronic acid producing bacteria in previous reports. In addition, there is a method for obtaining hyaluronic acid using Streptococcus bacteria.
JP-A-60-500597, JP-A-60-133894, JP-A-61
-15698, but there are problems such as the hyaluronic acid obtained has a low molecular weight and the yield is low. Furthermore, it is known that the hyaluronic acid-producing bacterial strains in both cases produce streptolysin (soluble hemolysin) and exhibit β-hemolytic properties. When attempting to produce hyaluronic acid by culturing a large amount of such bacteria, there is a risk that the hemolysin may be mixed into the hyaluronic acid product, and it is not preferable to incorporate such hyaluronic acid into cosmetics or pharmaceuticals. In order to improve this drawback, a method for obtaining hyaluronic acid by culturing a hyaluronic acid-producing strain that lacks the ability to produce streptolysin through mutation treatment with a chemical mutating agent was proposed in JP-A-60-251898.
has been disclosed. This contains 6 glucose
It is stated that 3.6 g/g of hyaluronic acid was obtained by adding 3.6% of hyaluronic acid, and the yield of hyaluronic acid to sugar was 6%. It is low. Problems to be Solved by the Invention In view of the above problems, as a result of intensive research with the aim of efficiently producing large amounts of high molecular weight hyaluronic acid that does not contain hemolysin (streptolysin), we have isolated it from nature. By mutating a strain that no longer exhibits hemolytic properties obtained through mutation treatment from a bacterium belonging to the genus Streptococcus that has the ability to produce hyaluronic acid, the strain loses the ability to produce hyaluronidase and produces high-molecular-weight hyaluronic acid. We obtained a bacterial strain with extremely high production ability, and found that this strain satisfies the above objectives. The present invention has been completed based on this knowledge, and therefore, the present invention uses bacteria belonging to the genus Streptococcus that have hyaluronic acid-producing ability, non-hyaluronidase production, and non-hemolytic properties in a culture medium. The present invention provides a method for efficiently producing hemolysin-free, high-molecular-weight hyaluronic acid by culturing. Means for Solving the Problems (1) Obtaining a Mutant Strain In order to achieve the object of the present invention, the present inventors obtained a new mutant strain by the following method. First, Streptococcus zooepidemicus belongs to Lancefield serogroup C, which has a strong ability to produce hyaluronidase (hyaluronic acid degrading enzyme) and produces hyaluronic acid, from the bovine nasal mucosa (the identification of this bacterium was carried out by the Verges Manual). of Determinative Bacteriology, 8th edition, 1974). This strain is known as MacLennan (MacLennan, J.Gen.
Microbiol., 14, 134-142, 1956) pointed out that when hyaluronic acid is produced well under aerobic conditions and glucose is used as the carbon source,
By adding 4% glucose, 2 g/hyaluronic acid was produced (yield of hyaluronic acid based on sugar was 5%). The molecular weight of the hyaluronic acid obtained at this time was 300,000 to 600,000. This strain was treated with ultraviolet rays and chemical agents (N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethylmethane sulfur, The treated bacterial cells were plated on a blood agar medium, a colony that did not show hemolysis was collected, and this strain was then subjected to mutation treatment again to produce cells containing hyaluronic acid. Spread on nutrient agar medium,
A mutant strain of Streptococcus zooepidemicus was obtained by collecting a colony that did not degrade hyaluronic acid (see Reference Example 1 below). (2) Mycological properties Mutant strain of Streptococcus zooepidemicus obtained in Reference Example 1 below (hereinafter referred to as the main strain)
Todd Huyt Broth
It forms transparent colonies with extremely strong viscosity on agar medium (Hewitt broth), and is non-hemolytic (β-
Hemolytic: negative), non-production of hyaluronidase,
This bacterium is a streptococcus belonging to Lancefield serogroup C, and its bacteriological properties are as follows. (a) Gram staining: positive (b) 10℃ growth: negative (c) 45℃ growth: negative (d) 0.1% methylene blue resistance: negative (e) 6.5% salt resistance: negative (f) 40 %Bile resistance: Negative (g) Bacitracin resistance: Positive (h) PH9.6 resistance: Negative (i) 60℃, 30 minutes resistance: Negative (j) Gelatin decomposition: Negative (k) Starch decomposition :Positive (l) Sodium hippuric acid degradability: Negative (m) Aesculin degradability: Weakly positive (n) Arginine degradability: Positive (o) Sugar fermentability: Glucose, galactose,
Positive for sucrose, lactose, maltose, sorbitol and salicin, negative for glycerin, mannitol, trehalose and arabinose. Based on the mycological properties of this bacterium, the present inventors identified this bacterium as Streptococcus suoepidemicus.
It was named YIT2030 and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microtechnology Research Article No. 1305. (3) Production of hyaluronic acid using this bacterium The medium for producing hyaluronic acid by culturing this bacterium preferably contains a carbon source, an organic/inorganic nitrogen source, an inorganic salt, and other organic micronutrients as necessary. . As the carbon source, those containing sugar such as glucose, galactose, sucrose, lactose, fructose, maltose, sorbitol, and starch hydrolyzate are preferred, and organic acids, aliphatic alcohols, and the like may also be used. As a nitrogen source, common organic and inorganic materials may be used, such as various meat extracts, amino acid mixtures,
Peptone, yeast extract, etc. are preferred. Furthermore, chlorides, sulfates, phosphates, nitrates of sodium, potassium, calcium, magnesium, iron, etc.
Carbonates, vitamins, etc. may be added as necessary. Aerobic conditions are essential for culturing, and it is better to increase the stirring speed as the viscosity of the culture solution increases, but excessive stirring is not preferred. The culture temperature is generally 25-38°C, which is the temperature at which bacteria grow. Furthermore, during culturing, this bacterium produces lactic acid, which inhibits the growth of the bacterium and the production of hyaluronic acid. Therefore, an alkaline aqueous solution is added to neutralize the lactic acid, and the pH is within the range of 6-8. It is necessary to adjust within the range. The alkaline aqueous solution used at this time may be an aqueous solution of sodium hydroxide, potassium hydroxide, or aqueous ammonia. This bacterium contains high molecular weight hyaluronic acid (molecular weight 200
-3 million) at an extremely high yield and production rate, and particularly good results are obtained when glucose is used as the carbon source. Added amount of sugar 3
% or less, the sugar yield was 14-15%, and if the amount added was more than that, the sugar yield tended to decrease slightly. When the sugar addition was 6% (see Example 1), the viscosity of the culture solution became 8000 centipoise (cP) at 36°C, and the culture solution became almost non-fluid, reaching the limit of culture without any effect even if the stirring speed was increased. Summer. Table 1 shows the amount of hyaluronic acid produced when culturing was performed with varying amounts of glucose added in the medium. That is, as a result of purifying the produced hyaluronic acid using a conventional method, the sugar yield was 15 when 1% glucose was added.
% is 1.5g/, and when 6% is added, the sugar yield is 11%.
6.7 g/hyaluronic acid was obtained.

[Table] Next, a medium with a composition of 2% glucose was used,
When continuous culture was carried out at a dilution rate of 0.3 (hr ^-1 ), hyaluronic acid could be produced continuously and extremely stably at a high yield and production rate with a low viscosity that was easy to operate. The yield of hyaluronic acid based on sugar is 15%, and its productivity is 0.9g/
/hr, and 21.6 g/hr of hyaluronic acid could be produced per day (see Example 2). Since this bacterium is a polymeric substance and does not accumulate substances other than hyaluronic acid in the culture solution, it is easy to separate and purify the hyaluronic acid accumulated in the culture solution after culturing, and it is already known to separate and purify polysaccharides. Just use the law. An example of a method for separating and purifying hyaluronic acid is shown. Dilute the culture solution with water to an appropriate viscosity (preferably 100 centipoise or less), and adjust the pH to 4 or less with trichloroacetic acid. Next, the bacterial cells are separated and removed by centrifugation or membrane filtration (pore size 0.2 μm or less). Next, the low molecular weight substances dissolved in the solution are removed by ultrafiltration, dialysis, organic solvent precipitation, or adsorption using an ion exchange resin, followed by organic solvent precipitation, freeze drying, or spray drying. Hyaluronic acid can be obtained using the following methods (see Example 1). As a result of performing various studies on the hyaluronic acid obtained by extraction and purification from the above culture solution and comparing it with a hyaluronic acid standard (manufactured by Sigma), it was confirmed that this product is hyaluronic acid. . Its properties are shown below. (1) Shows the same mobility as the standard sample in electrophoresis using cellulose acetate membrane. (2) When it is degraded by actinomycete hyaluronidase (manufactured by Amano Pharmaceutical) and the degraded product is subjected to silica gel thin layer chromatography, two spots appear with the same mobility as the standard degraded product after treatment. (3) Analysis of the chemical composition reveals that N-acetyl-D-
Glucosamine and D-glucuronic acid in a molar ratio of 1:
1 exists. (4) The specific luminosity is [α] ²⁰ _D = -69°. (5) The infrared absorption spectrum measured by the thin film method is shown in Figure 3 and is the same as the standard product. (6) The ¹³ C-NMR spectrum measured after dissolving in heavy water is shown in Figure 4 and is the same as the standard. (7) Molecular weight is determined by viscosity measurement method (TCLaurent et al.,
Biochim. Biophys. Acta, 42, 476−485, 1960)
The result was 2-3 million. The present invention will be explained in further detail with reference to Reference Examples and Examples below. Reference Example 1 Streptococcus zooepidemicus, which exhibits β-hemolytic properties, produces hyaluronidase, and produces hyaluronic acid, collected from bovine nasal mucosa, was incubated at 37°C for 10 hours in Todd Huyt Broth Medium (manufactured by Difco). After culturing, the cells in the logarithmic growth phase were collected by centrifugation, and washed twice aseptically with 0.05M Tris-maleate buffer (PH6.0) while repeating centrifugation at low temperature. ×
Suspend in the same buffer solution to a bacterial concentration of 10 ⁸ /ml,
Add NTG to this at 200μg/ml and hold at 37°C.
The mixture was shaken for 30 minutes. Next, the bacterial cells at low temperature were
2 with 0.05M Tris-maleate buffer (PH6.0)
After washing twice, the cells were inoculated into Todo Huyt's broth medium and cultured at 37°C for 18 hours. This culture solution was diluted with sterile physiological saline to a concentration of 1 x 10 ³ /ml, and 0.1 ml of this was poured onto blood (rabbit defibrinated blood) agar (20 ml) and cultured, after which it showed hemolytic properties. We sampled villages where there were no. The acquisition frequency of this mutant strain is approximately 4
It was ×10 ^-6 . This non-hemolytic strain was then added to Todd Huyt's Broth medium as above.
Culture at 37℃, collect cells in logarithmic growth phase, and add 0.05M
After washing with Tris-maleate buffer (PH6.0),
The mixture was shaken at 37°C for 20 minutes in the same buffer containing 200 μg/ml of NTG. Next, after washing the microbial cells at low temperature with the same buffer solution, the ultrafiltration medium (Totdo Huyt Broth medium to ultrafiltration membrane YM made by Amicon) was used.
-10 to remove the high molecular fraction).
Cultured at 37°C for 18 hours. This culture solution was diluted with sterile physiological saline to a concentration of 1-5 x 10 ² /ml, and 0.1 ml of the solution was applied onto the ultrafiltrated medium agar (high purity agar) containing 0.1% sodium hyaluronate. do
Culture in a moisture chamber at 37℃ for 20-40 hours, collect the grown bacteria using the replica method, and spray a 10% cetylpyridinium chloride aqueous solution onto the agar. We obtained a non-hyaluronidase-producing mutant strain Streptococcus zooepidemicus YIT2030, which forms colonies with cloudy surroundings, from among ten thousand bacterial strains. The above method for identifying hyaluronidase-producing and non-producing bacteria is based on the method of Erika Balke et al.
al., Zbl.Bakt.Hyg.A, 259, 194−200, 1985)
I modified it. Example 1 Batch culture Glucose 6%, Polypeptone (Daigo Nutrient Chemical) 1.5%, Baker's yeast extract (Oriental Yeast Co., Ltd.) 0.5%, Potassium phosphate 0.2%, Magnesium sulfate heptahydrate 0.1%, Calcium chloride 0.005 %, ADEKA NOL LG-109 (antifoaming agent manufactured by Asahi Denka Kogyo)
Streptococcus zooepidemicus YIT2030 was pre-cultured after sterilization by placing 0.001% composition medium (PH7.0) in 10 jar fermenters.
was inoculated at 1%, and cultured with aeration and stirring at 37° C. for 42 hours while continuously controlling the culture pH to 7 with a 6N-sodium hydroxide aqueous solution. Glucose was sterilized separately and added all at once at the start of culture. The progress of the culture at this time is shown in FIG. Culture in which hyaluronic acid accumulated over the course of culture29
After 42 hours of incubation, the viscosity of the culture solution reached nearly 8000 centipoise and became almost non-liquid.
The culture was terminated when the glucose in the culture solution reached zero. Since the harvested culture solution had no fluidity, it was diluted with water to a viscosity of 100 centipoise or less. Next, PH this solution with trichloroacetic acid.
4 or less, pass through a hollow fiber microfilter module (PW-103 manufactured by Asahi Kasei) to remove bacterial cells and insoluble components, and then pass the filtrated liquid through a hollow fiber ultrafiltration membrane (H1P30-43 manufactured by Amicon). The low molecular weight substances in the solution were removed by adding water to the solution.
This solution was then dried by freeze-drying to obtain 6.7 g of hyaluronic acid per culture solution. Example 2 Continuous culture Example 1 except that the glucose concentration was 2.5%
Pour 5 media of the same composition into 10 jar fermenters, and after sterilization (glucose is sterilized separately),
Inoculate 1% of the pre-cultured Streptococcus zooepidemicus YIT2030 and grow at 37°C while adjusting the culture pH to 7 with a 6N-sodium hydroxide aqueous solution.
Culture was carried out with aeration and stirring for 15 hours. Thereafter, a medium having the same composition as in Example 1 except that the glucose concentration was changed to 2% was continuously added at a dilution rate of 0.3 (hr ^-1 ).
Continuous culture with aeration and stirring was carried out for one week at 37°C and pH 7. The progress of the culture at this time is shown in FIG. The culture solution flowing out of the culture tank was collected at regular intervals, and the culture solution was collected in Example 1.
Hyaluronic acid was extracted and purified in the same manner. As a result, the yield of hyaluronic acid based on sugar was 15%, the productivity was 0.9 g/hr, and 21.6 g/hr of hyaluronic acid could be obtained. Effects of the Invention By culturing the Streptococcus zooepidemicus mutant strain of the present invention, the yield of Streptococcus zooepidemicus far exceeds that of the previously reported method for producing hyaluronic acid using Streptococcus bacteria. High molecular weight hyaluronic acid completely free of lysine contamination can be obtained at high yield and production rate at low cost. Hyaluronic acid produced in this manner is optimal as a raw material for cosmetics and pharmaceuticals.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the culture progress of hyaluronic acid production in Example 1 of the present invention, FIG. 2 is a diagram showing the culture progress in Example 2, and FIGS. 3 and 4 are diagrams showing the culture progress in Example 2. 1 is a diagram showing an infrared absorption spectrum and an NMR spectrum of hyaluronic acid obtained in Example 1.

Claims (1)

[Scope of Claims] 1. A method comprising culturing in a medium bacteria belonging to the genus Streptococcus, which has the ability to produce hyaluronic acid, does not produce hyaluronidase, and is non-hemolytic, and collects hyaluronic acid from the culture. A method for producing hyaluronic acid. 2. The method for producing hyaluronic acid according to claim 1, wherein the bacterium belonging to the genus Streptococcus is Streptococcus zooepidemicus. 3. The method for producing hyaluronic acid according to claim 1 or 2, wherein the bacterium belonging to the genus Streptococcus is Streptococcus zooepidemicus YIT2030 (Feikoken Jokyo No. 1305).