JP2009011315A - Production method of hyaluronic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently producing hyaluronic acid by accumulating the hyaluronic acid to be high concentration in a culture solution. <P>SOLUTION: The production method of hyaluronic acid includes cultivating a microorganism having hyaluronic acid-production ability in the presence of living yeast. The microorganism having hyaluronic acid-production ability is a microorganism belonging to the genus Streptococcus, especially Streptococcus zooepidemicus or its variant, and the living yeast is preferably Saccharomyces cerevisiae. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an efficient method for producing hyaluronic acid.

  In recent years, hyaluronic acid has also been produced by fermentation. This is because the hyaluronic acid produced by the fermentation method is produced under certain conditions, so that the quality of the product is kept constant, and thus the industrial utility value of the obtained hyaluronic acid is great.

  Also in the fermentation method, a method for efficiently producing hyaluronic acid has been studied.

  For example, (1) a method of culturing by controlling the pH of the culture medium to neutral, (2) a method of culturing under an aerobic condition, and (3) a method of culturing by adding glutamine or glutamic acid as a fermentation raw material (non-patented) Reference 1), (4) Method of culturing by adding arginine and / or glutamic acid as fermentation raw material (see Patent Document 1), (5) Method of adding uridine as fermentation raw material and culturing (Patent Document 2), etc. Is mentioned.

However, in the above method, as a result of accumulating a large amount of lactic acid during fermentation, assimilation of glucose, which is the main carbon source of the microorganism used in the fermentation method, is suppressed. As a result, since the synthesis of hyaluronic acid is suppressed, there is a problem that hyaluronic acid cannot be accumulated at a high concentration in the culture solution.
J.Soc.Cosmet.Chem.Japan Vol.22, No1 1988 JP-A 62-289198 Japanese Patent No. 2547965

  An object of the present invention is to efficiently obtain hyaluronic acid by accumulating hyaluronic acid in a culture solution at a high concentration.

  As a result of intensive studies to achieve the above object, the present inventor has found that the above object can be achieved by culturing a microorganism having hyaluronic acid producing ability in the presence of live yeast, and has completed the present invention. .

  That is, the present invention is a method for producing hyaluronic acid, comprising culturing a microorganism having hyaluronic acid-producing ability in the presence of live yeast.

  According to the present invention, a high concentration of hyaluronic acid can be accumulated in the culture medium, so that hyaluronic acid can be produced more efficiently.

  Hereafter, the manufacturing method of this invention hyaluronic acid is demonstrated in detail.

<Microorganisms capable of producing hyaluronic acid>
The microorganism having the ability to produce hyaluronic acid used in the present invention is a microorganism capable of producing hyaluronic acid by ordinary culture, and the kind is not limited. For example, microorganisms belonging to the genus Streptococcus can be preferably used. Even microorganisms that do not belong to the genus Streptococcus can be used microorganisms that have obtained the ability to produce hyaluronic acid using ordinary genetic engineering techniques.

  It is known that microorganisms belonging to the genus Streptococcus having the ability to produce hyaluronic acid are generally present in the bovine nasal mucosa and the bovine eyeball. In the present invention, microorganisms isolated therefrom can also be used.

  Examples of microorganisms belonging to the genus Streptococcus include Streptococcus zooepidemicus, Streptococcus equi, and Streptococcus pyogenes. Streptococcus zooepidemicus can be preferably used.

  Furthermore, by treating a microorganism having the ability to produce hyaluronic acid such as a microorganism belonging to the genus Streptococcus with ultraviolet light, NTG (N-methyl-N′-nitro-N-nitrosoguanidine), methylmethanesulfonic acid, etc., hyaluronidase-free It is more preferable to improve to production bacteria or non-hemolytic bacteria. This is because the possibility of adverse effects on the human body or animals is reduced. Examples of strains deficient in the hyaluronidase activity and hemolysis include Streptococcus zooepidemicus NH-131 (FERM P-7580), Streptococcus zooepidemicus HA-116 (ATCC39920), Streptococcus zooepidemicus MK5 (FERM P-21487), and Streptococcus cerevisiae. Zooepidemicas YIT2030 (FERM BP-1305) is preferable, and Streptococcus zooepidemicus MK5 (FERM P-21487) and Streptococcus zooepidemicus YIT2030 (FERM BP-1305) are particularly preferable.

  Of these, the FERM strain is available from the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary. The ATCC strain can be obtained from the American Type Culture Collection.

<Culture>
The culture conditions are not limited as long as the microorganism can produce hyaluronic acid, and normal culture conditions according to the type of microorganism can be used. For example, the medium includes monosaccharides such as glucose and fructose as a carbon source, disaccharides such as lactose, sucrose and maltose as a carbon source, organic nitrogen sources such as polypeptone and yeast extract as a nitrogen source, and free amino acids such as arginine, glutamic acid and glutamine. Those using a hyaluronidase inhibitor having a phenolic hydroxyl group such as tannin, vitamins, inorganic salts, and the like can be used. The culture conditions are usually preferably aerobically controlled at a pH of 7 to 7.5 and a temperature of 30 to 37 ° C.

  The culture method in the present invention is not particularly limited, and any of a batch culture method, a continuous culture method, and a flow-down culture method is possible.

  In addition, the culture is usually performed after pre-culture. Pre-culture conditions include carbon sources such as glucose and fructose, nitrogen sources such as polypeptone, yeast extract, and malt extract, vitamins and inorganic salts, pH 4 to 8, temperature 30 to 37 ° C. It is preferable to control the aerobic culture.

  In the present invention, it is important to add (inoculate) live yeast when culturing (main culture) a microorganism having the ability to produce hyaluronic acid. The timing of addition (inoculation) of live yeast to the main culture is not particularly limited, and may be at the start of culture or may be during culture or before completion of culture.

  Degradation or consumption of the lactic acid accumulated in the medium by the live yeast can prevent a decrease in the ability of microorganisms to produce hyaluronic acid due to lactic acid. Therefore, a higher concentration of hyaluronic acid can be accumulated in the medium, and hyaluronic acid can be obtained efficiently.

  The live yeast used in the present invention is not limited as long as it can decompose or consume lactic acid, but from the viewpoint of ease of use or availability, Saccharomyces cerevisiae is preferable, and Saccharomyces cerevisiae. NBRC0216 is particularly preferred. This strain can be obtained from the Biological Resource Department of the National Institute for Product Evaluation.

  Live yeast can also be pre-cultured under conditions such as glucose, fructose, lactic acid and other carbon sources, polypeptone, yeast extract, malt extract and other nitrogen sources, vitamins, inorganic salts, etc. Is preferably aerobically cultured at a temperature of 4 to 8 and a temperature of 30 to 37 ° C.

  The hyaluronic acid obtained by the present invention can be used for various uses such as pharmaceuticals, cosmetics, and food additives.

<Example 1>
Lactose 6%, Polypeptone (Wako Pure Chemical Industries, Ltd.) 1.5%, Yeast extract (Oriental Yeast Industry) 0.5%, Dibasic potassium phosphate 0.2%, Magnesium sulfate heptahydrate 0.1%, Calcium chloride 0.005%, Glutamic acid Streptococcus zooepidemicus MK5 (FERM P-21487) containing 2 L of a medium (pH 7.0) composed of 0.1% sodium and Adecanol L61 (antifoaming agent, manufactured by Asahi Denka Kogyo) in a 3 L jar fermenter, sterilized and pre-cultured And 1% each of Saccharomyces cerevisiae NBRC0216, and cultured at 37 ° C. for 23 hours while controlling the culture pH to 7.4 with a 12% aqueous sodium hydroxide solution. Lactose was sterilized separately and added at once at the start of culture.

  Pre-culture conditions are as follows: For both bacteria, 100 ml of a medium (pH 7.0) composed of 0.2% glucose, 2.0% polypeptone, 0.5% yeast extract and 0.05% magnesium sulfate heptahydrate is placed in a 500 ml Erlenmeyer flask with cotton plug. After sterilization, one platinum thread was inoculated for each colony formed on an agar plate containing the same composition, and Streptococcus zooepidemicus MK5 was cultured at 30 ° C. for 18 hours and Saccharomyces cerevisiae NBRC0216 for 42 hours.

  The cultured broth (hyaluronic acid-containing liquid) is diluted 10-fold with ion-exchanged water, 5 g of activated carbon (Shirakaba RW50-T manufactured by Takeda Pharmaceutical Co., Ltd.) and pearlite (Mitsui Mining & Mining Co., Ltd.) 30 g of Roca Help # 409) from the company was added, treated for 1 hour, and filtered using Nutsche. This operation was repeated twice to remove organic components in the medium.

  3 g of sodium chloride is dissolved in 1 L of hyaluronic acid-containing liquid from which organic components have been removed, adjusted to pH 7, precipitated with 2-propanol 6 L, and vacuum dried at 40 ° C. to obtain 0.7 g or more of sodium hyaluronate. It was.

<Comparative Example 1>
The same operation as in Example 1 was performed except that 1% of Saccharomyces cerevisiae NBRC0216 precultured in the Example was not inoculated. Only 0.6 g or less of sodium hyaluronate was obtained.

Claims (6)

  A method for producing hyaluronic acid, comprising culturing a microorganism capable of producing hyaluronic acid in the presence of live yeast.   The method according to claim 1, wherein the microorganism having the ability to produce hyaluronic acid is a microorganism belonging to the genus Streptococcus.   The method according to claim 2, wherein the microorganism belonging to the genus Streptococcus is Streptococcus zooepidemicus or a variant thereof.   The method according to claim 3, wherein the Streptococcus zooepidemicus is Streptococcus zooepidemicus MK5 (FERM P-21487).   The method according to any one of claims 1 to 4, wherein the live yeast is Saccharomyces cerevisiae.   The method according to claim 5, wherein the Saccharomyces cerevisiae is Saccharomyces cerevisiae NBRC0216.