JPS5856692A - Preparation of hyaluronic acid - Google Patents

Abstract

PURPOSE:To prepare the titled substance, by culturing hyaluronic acid-producing bacteria belonging to Streptococcus genus in a nutrient medium containing more than specific amount of a saccharide component as a main carbon source, and separating the objective substance produced and accumulated in the medium. CONSTITUTION:Hyaluronic acid-producing bacteria belonging to Streptococcus genus, e.g. Streptococcus pyogenes, Streptococcus equi, Streptococcus equisimilis, etc. are cultured in a medium containing >=3% saccharide component such as glucose, sucrose, galactose, etc. as a main carbon source, and a nitrogen source, inorganic salts, minor organic nutrients, etc. under aeration and agitation, and the accumulated hyaluronic acid is separated from the medium. The separation can be carried out by conventional method for the separation of polysaccharides.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing hyaluronic acid using microorganisms. Conventionally, hyaluronic acid has been isolated from cow eye glass fluid, chicken crest, umbilical cord, synovial fluid, etc. It binds with protein and water to maintain a jelly-like state, acting as a lubricant, protecting against bacterial invasion, and retaining moisture.

However, the problem was that it was extremely expensive.

On the other hand, regarding the production of hyaluronan using bacteria of the genus Streptococcus and Kas, Strebutov and Kas pyogenes (5
treptococcus pyogenea),
Strebutco, Kas Eki (5treptococcu)
sequi), strebtococcus, cas.
equiaimilis) Strebutco, Kas disgalactiie (5tr8ptococcus dy
sgalactiae) and streptococcus.

Cas zooepidemicus (5treptococcus)
It is already known that hyaluronic acid is produced by bacteria such as B. zooepidemicus. The report was published by Holmström (E, Ho1m5trδm Appl.
, Microbial 1967), J.P.
Woolcock, Ri (, T, B, Woolcock 85.3
72 He375 J, Gen.

Microbial 1974), Lee Kiem (
E, Kjem Acta Pathol.

Microbial, 5cand 1976) et al. However, none of them are intended for mass production, and are carried out under conditions such as glucose 1%, culture time M hours, pH 70 to 76, and temperature 30 to 376 C, and the yield is low at 0.6 g// or less. Ta.

The present inventors conducted extensive research with the intention of stably producing hyaluronic acid in good yields using a simple and inexpensive medium.
Cultivating the above-mentioned bacteria under specific conditions That is, the present invention involves culturing with aeration agitation of a microorganism of the genus Streptococcus that has the ability to produce hyaluronic acid in a nutrient medium containing 3% or more of sugar components as the main carbon source. Hyaluronic acid is produced in the liquid and collected.

(Left below) 1! I-opening R58-50G92 (2) Bacteria having the ability to produce hyaluronic acid of the present invention include Streptococcus pyogenes, Streptococcus equi, Streptococcus excimilis, Streptococcus dysgalactiae, Streptococcus zooepidemicus, and the like.

In the present invention, production of hyaluronic acid is carried out by culturing the above-mentioned hyaluronic acid-producing bacteria in a specific nutrient medium. As a medium, a medium containing a carbon source, a t-source, an inorganic salt, and other organic micronutrients if necessary is preferable. There are various carbon sources such as organic acids and aliphatic alcohols, but in the present invention, starch hydrolysates, glucose, etc.
Common sources such as essential sugars such as sucrose, galactose, and 7-lactose, mixtures of various amino acids, and yeast extract are used.

In addition, sodium chloride, phosphate sulfates or carbon salts of magnesium, potassium, iron, calcium, etc., and trace amounts of vitamins are added as necessary.

Particularly good results can be obtained when glucose is used as a carbon source to increase the yield of hyaluronic acid. When adding sugar, it is better to divide it into small amounts and add it at appropriate times than to add a large amount at once. When the concentration of sugar is 3% or more, the amount of hyaluronic acid produced increases significantly compared to when the sugar concentration is 1%. It is sufficient to add 6 to 8% of the thick hemp, and adding more will not increase the yield. If the amount is less than 3%, the effect of the present invention cannot be achieved.Table 2 shows the results of culturing with varying amounts of addition.

It can be seen that when 3% or more glucose is added, the yield increases considerably compared to 1%.

That is, Table 1: Comparison of medium composition PH55-9, OS humidity blade ~44°02 ventilation i 1-2
l/mix culture time (1 to 4 days) The yield of hyaluronic acid when 8% glucose was added was 40 l/l as a result of purification according to a conventional method.

Aeration and stirring are essential during culture. Methods for aeration and agitation include shaking culture and normal aeration and agitation culture using air blowing, either of which may be used. In this case, better results can be obtained by using a weak stirring force and increasing the amount of aeration. Table H shows the presence or absence of ventilation. It can be seen that the yield clearly increases by aeration.

Furthermore, during culturing, adjust the pH to 55-90 using an alkaline water bath.
When adjusted to , hyaluronic acid can be stably produced, which is more preferable. As the alkaline aqueous solution, a single or mixed solution of commonly used alkalis such as sodium hydroxide, potassium hydroxide, basic amino acids, lower amines, etc. can be used.

When separating and collecting hyaluronic acid accumulated in the culture medium after culturing, the medium is made acidic to improve its purity and make it easier to process.

Conventional methods for separating and collecting polysaccharides can be used to separate hyaluronic acid. For example, bacterial cells and other insoluble components in the culture solution are separated and removed by filtration or centrifugation. Proteins mixed in the solution can be removed with a mixture of trichloroacetic acid, chloroform, and isoamyl alcohol, or with an adsorbent such as activated clay or activated carbon, or with a proteolytic enzyme such as pepsin, papain, or pronase.

In addition, mixed low-molecular substances can be separated and removed by ultrafiltration, dialysis, or organic solvent reprecipitation.The hyaluronic acid obtained in this way is soluble in water, methanol, ethanol, acetone, and chloroform. It is a white, fibrous, tasteless, odorless dry product that is insoluble in organic solvents such as ether.

Bell.

Color reaction Anthrone-sulfuric acid reaction: Green carbazole-sulfuric acid reaction: arE color infrared absorption spectrum The infrared absorption spectrum measured by the KBr tablet method is shown in FIG.

Specific optical rotation of 01% aqueous solution: [α] - 675° Analysis by electrophoresis A chart of isotachophoresis is shown in FIG.

The electrophoresis conditions were as follows.
Tobaria (potential Unit Val
ue ) was 023, which confirmed that the mucilage was hyaluronic acid.

Also, in filter paper electrophoresis using a cellulose acetate membrane, this polysaccharide was found to be identical to hyaluronic acid using a 0.1 M zinc acetate electrode solution. At that time, fungal hyaluronidase (stre
ptomyces hyalurolyticus)
The samples analyzed by electrophoresis after being treated with
It was confirmed that it was hyaluronic acid because it was degraded by this enzyme and showed no oxidation.

Example 1 Glucose 8%, yeast extract O5%, peptone 15%,
A medium with a composition of 02% potassium monohydrogen phosphate, 611% sodium thiosulfate, and 002% sodium sulfite was placed in a jar fermenter (MA type 50, manufactured by Iwashiya).
After heat sterilizing at 120°C for 115 minutes and inoculating with pre-cultured Streptococcus zooepidemicus, the mixture was aerated and stirred at 33°C for 4 days (aeration volume 2t, 'min, rotation speed). 200rpm)
Cultured.

After completion of the culture, the bacterial cells and other impurities were removed from the culture solution by centrifugation, and when twice the amount of ethanol was added to the supernatant with stirring, fibrous substances were precipitated. After taking this from house to house, thoroughly washing it with ether and then ethanol, dissolving it again in water, adding cetylpyridinium chloride, and taking the resulting precipitate from house to house, thoroughly washing it with water and O, 1M Na0j aqueous solution, and dipping it into 0.5M NaC7. After extracting hyaluronic acid and desalting the solution by dialysis, the solution was freeze-dried to obtain 40 g of hyaluronic acid from culture solution 11. This product was electrophoresed in the same position as the standard hyaluronic acid by p-paper electrophoresis, and the yaw, tobar, and - values of isokinetic electrophoresis were 0.23, which matched that of the standard. Furthermore, the infrared absorption spectrum matches the spectrum of Hinoki products.

On the other hand, when Streptococcus zooepidemicus was similarly cultured in a medium to which 1% glucose was added instead of 8% glucose, only 5 q/l of hyaluronic acid was obtained from culture solution 11.

Example 2 The amount of glucose in the medium used in Example 1 was reduced to 4%.
Streptococcus and Zooepidemicus were cultured in the same manner as in Example 1 using the replaced medium. The yield of hyaluronic acid was 2.19/l◇However, the yield when aeration and stirring under these conditions was not performed was as low as 0.005 l/l.

Example 3 The amount of glucose in the medium used in Example 1 was reduced to 3%.
Streptococcus zooepidemica was cultured and treated in the same manner as in Example 1 using the replaced medium.

The yield of hyaluronic acid was zO/l. Example 4 Streptococcus equi (A
TOO9527) was cultured, hyaluronic acid was isolated and purified from the culture solution, and 389 hyaluronic acid was obtained from culture solution 11.

[Brief explanation of the drawing]

FIG. 1 shows an infrared absorption spectrum of hyaluronic acid obtained by main culture. FIG. 2 is a chart of isotachophoresis of hyaluronic acid obtained by main culture. Patent applicant: Shiseido Co., Ltd. 1st figure Woe ground seal (, 1f-z figure procedural amendment (voluntary) November q, 1980 Commissioner of the Patent Office Shima 1) Tono Haruki 1, Indication of the case 1975 Patent Application No. 152355 3 Person making the amendment Cm Episode number 40 Tokyo (A721+XXZ 14211
] 4. "Detailed Description of the Invention" column 5 of the specification to be amended Contents of the amendment (1) Page 4, line 17 to line 189 of the specification 11
Correct "raw materials" to "raw materials". (2) "Carbonate" in the first line of page 5 of the specification will be corrected to "carbonate." (3) "Water bath liquid" on page 7, line 10 of the specification is corrected to "aqueous solution." (4) [[α] 薯--67 on page 9, line 10 of the specification
, 5°” is corrected to “[α]□−, j57.5°”. (5) l' on page 11, line 5 to line 6 of the specification
-17 Jarfur Mentor (MA-type 5 made by Iwashiya)
00m1! Mini jar)" to "Iwashiya MA type 500"
m1 mini jar” and capture it 2

Claims (1)

[Claims] (1) Streptococcus microorganisms capable of producing hyaluronic acid are cultured with aeration in a nutrient medium containing 3% or more sugar content as the main carbon source, and hyaluronic acid is produced and accumulated in the culture solution. A method for producing hyaluronic acid using a microorganism, the method comprising collecting hyaluronic acid.