JP2021036841A - Improved method for producing hyaluronic acid - Google Patents

Abstract

To provide a more efficient method for producing hyaluronic acid using a microorganism of the genus Streptococcus thermophilus.SOLUTION: A method for producing hyaluronic acid comprises the steps of: (a) pre-culturing a Streptococcus thermophilus microorganism in a medium containing at least one selected from glucose and lactose as saccharide; and (b) inoculating the microorganism pre-cultured in the above step into a medium containing a sugar different from the sugar used in the pre-culture as the saccharide and culturing the mixture.SELECTED DRAWING: None

Description

The present invention relates to an improved method for producing hyaluronic acid by lactic acid bacteria.

Hyaluronic acid is an expensive glucosaminoglucan together with chondroitin sulfate and heparin, and is known to be widely distributed in the connective tissue of mammals in a trace amount and also to produce microorganisms. High-purity hyaluronic acid extracted and purified from these has properties such as high moisturizing property, high viscosity, and wound healing property. Therefore, paying attention to its function, it is used as a cosmetic compound material and during eye surgery. Widely used as a protective agent, a therapeutic agent for arthritis, etc.
In recent years, consumers have been paying more attention to added values such as health effects and functionality in the food field, and hyaluronic acid can be expected to have various effects depending on its functionality. It is also attracting attention as one.

Conventionally, hyaluronic acid has been produced by an industrial method by extracting it from chicken crown, cow joint or whale cartilage, but hyaluronic acid is present in a very small amount in the living body and in the tissue. Since it forms a complex with proteins and other mucopolysaccharides, it is difficult to produce in large quantities because it requires complicated steps to separate and purify the proteins and mucopolysaccharides. The method for producing hyaluronic acid according to the above is generally practiced.

Examples of hyaluronic acid-producing microorganisms include Streptococcus pyogenes, Streptococcus zooepidemicus, Streptococcus equi, Streptococcus equi, Streptococcus equicisim Streptococcus spp., Etc. are known. In addition, the applicant has found that Streptococcus thermophilus microorganisms such as Streptococcus thermophilus YIT2084 strain (FERM BP-10879) have excellent hyaluronic acid producing ability (Patent Document 1).

Japanese Patent No. 4920552

The present invention relates to providing a more efficient method for producing hyaluronic acid using a microorganism of the genus Streptococcus thermophilus.

When producing hyaluronic acid using a microorganism of the genus Streptococcus thermophilus, the present inventors preculture the microorganism in a medium containing one or more selected from glucose and lactose as saccharides, and in the main culture, the microorganism is precultured. , It was found that the production amount of hyaluronic acid is improved when the culture medium contains a sugar different from the sugar used in the preculture.

That is, the present invention relates to the following (1) to (4).
(1) The following process;
a) A step of pre-culturing a Streptococcus thermophilus microorganism in a medium containing one or more selected from glucose and lactose as sugars.
b) A step of inoculating the microorganism pre-cultured in the above step into a medium containing a sugar different from the sugar used in the pre-culture as a sugar and culturing the mixture.
A method for producing hyaluronic acid, including.
(2) The method of (1), wherein the medium in step a contains lactose as a saccharide, and the medium in step b contains glucose as a saccharide.
(3) The method of (1), wherein the medium in step a contains glucose as a saccharide, and the medium in step b contains lactose as a saccharide.
(4) The method according to any one of (1) to (3), wherein the Streptococcus thermophilus microorganism is Streptococcus thermophilus YIT2084 strain (FERM BP-10879).

According to the method of the present invention, hyaluronic acid that can be blended in pharmaceuticals, cosmetics, foods and drinks, etc. can be efficiently, inexpensively and easily produced.

The method for producing hyaluronic acid of the present invention includes the following steps a and b.
a) A step of pre-culturing a Streptococcus thermophilus microorganism in a medium containing one or more selected from glucose and lactose as sugars.
b) A step of inoculating the microorganism pre-cultured in the above step into a medium containing a sugar different from the sugar used in the pre-culture as a sugar and culturing.

The Streptococcus thermophilus microorganism used in the method of the present invention may be any microorganism having a hyaluronic acid-producing ability, and can be applied without particular limitation. Specifically, Streptococcus thermophilus. YIT2084 strain (FERM BP-10879) can be mentioned. The microbial strain belonging to Streptococcus thermophilus has been deposited at the Patent Microorganisms Depositary, Incorporated Administrative Agency.

1. 1. Step a
In the present invention, in order to culture Streptococcus thermophilus microorganisms and obtain hyaluronic acid, the preserved cells are first inoculated into a small amount of medium and subcultured, and then sequentially inoculated into a large volume medium. Scale-up is done while doing it.
In the present invention, step a means culturing (preculture) in the process of scaling up while sequentially subculturing microbial cells, and step b is culturing in the final step (preculture) performed to recover hyaluronic acid. (Culture) means.

In the present invention, the preculture is carried out in a medium containing at least one selected from glucose and lactose as a saccharide which is one of the carbon sources, but it is preferable to use glucose or lactose alone. The concentration of the saccharide in the medium is preferably 0.1 g / L to 50 g / L, more preferably 1 g / L to 30 g / L, and even more preferably 10 g / L to 20 g / L.

The pre-culture of the present invention can be carried out in a medium containing a medium component generally used for the pre-culture of lactic acid bacteria, except that the above-mentioned sugar is used as the sugar, but the cells to be inoculated into the culture in step b can be as much as possible. From the viewpoint of increasing, it is preferable to culture in a nutrient medium having high proliferative ability.
For example, as the nitrogen source, organic / inorganic nitrogen compounds such as meat extract, casein, peptone, yeast extract, dried yeast, germ, soybean flour, urea, amino acid, and ammonium salt can be used, and sodium salt, potassium salt, and the like can be used. Inorganic salts such as calcium salt, magnesium salt, phosphate, iron salt, copper salt, zinc salt and cobalt salt can be appropriately added as needed. Further, in addition to growth-promoting substances such as biotin, vitamin B1, cystine, methyl oleate, and lard oil, defoaming agents such as silicone oil and surfactant can be added.
The carbon source may contain an organic carbon compound such as oils and fats other than the above-mentioned saccharides, but it is preferable to use only the above-mentioned saccharides as a carbon source.
Specifically, for example, an ILS medium containing lactose or glucose as a carbon source shown in Table 1 described later can be mentioned.

Generally, after inoculating the bacterium into the medium, the first-stage preculture is performed in which the culture is started to grow the bacterium, and then this culture solution is used for the next-stage preculture (second stage). It can be inoculated into a medium and cultured in the same manner. That is, the culture in the pre-culture is appropriately repeated depending on the liquid volume of the main culture, and is usually carried out in two or more steps, preferably in two or three steps. Moreover, it is preferable to increase the amount of liquid in the preculture with the number of stages. In the present invention, the culture performed in the pre-culture (when the pre-culture is performed in a plurality of stages, at least one stage of the culture is arbitrary) is referred to as step a. When the pre-culture is performed in a plurality of stages, the step a may be continuously or intermittently performed a plurality of times, and it is preferable that the pre-culture one step before the main culture is the step a, and the pre-culture is performed a plurality of times. It is more preferable that all of the above steps are repeated in step a.

The culture temperature is 25 to 42 ° C., preferably 30 to 40 ° C., and the culture time is 12 to 72 hours, preferably 24 to 48 hours. The culture form may be any of static culture, shaking culture, stirring culture and the like, but stirring culture without aeration is preferable.
The pH of the medium is preferably adjusted to about 6 to 8, and more preferably in the neutral region.

2. Step b
In this step, the medium used for culturing the Streptococcus thermophilus microorganism contains a carbon source in which the sugar used as the saccharide is different from the sugar used in the step a. Here, examples of the sugar include monosaccharides such as glucose and fructose, disaccharides such as lactose, sucrose and maltose, oligosaccharides and the like. However, when lactose is used as the saccharide in the preculture, it is used as the medium in this step. For sugars other than lactose, for example, glucose, sucrose and the like are used, and glucose is preferably used. When glucose is used as the sugar in the preculture, sugars other than glucose, such as lactose and sucrose, are used as the medium in this step, and lactose is preferably used.
The concentration of the saccharide in the medium is preferably 1 g / L to 200 g / L, more preferably 5 g / L to 100 g / L, and even more preferably 10 g / L to 50 g / L.

As a component other than the carbon source, a component used for culturing ordinary lactic acid bacteria can be used. Such components include inorganic salts such as monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium sulfite, sodium thiosulfate, ammonium phosphate, and organic nutrient sources such as polypeptone, yeast extract, and corn steep liquor. Various amino acids and the like can be mentioned as needed. In addition, soybean peptide can be added from the viewpoint of increasing the amount of hyaluronic acid produced.

The amount of the pre-cultured Streptococcus thermophilus microorganisms inoculated into the medium in this step is not particularly limited, but from the viewpoint of hyaluronic acid productivity, 0.1 to 5% of the main culture solution, It is preferably 0.5 to 3%, more preferably 1 to 2%.
The culture in this step may be appropriately selected from known methods such as shaking culture and aeration stirring culture.
The culture temperature is 25 to 42 ° C., preferably 30 to 40 ° C., and the culture time is 12 to 72 hours, preferably 24 to 48 hours. In addition, the pH of the culture solution decreases with the growth of the lactic acid bacterium and may affect the amount of hyaluronic acid produced. Therefore, about 6 to 8 is preferable using various pH adjusters such as caustic soda, caustic potash, and ammonia. Is preferably adjusted to 6.5-7.5.

After completion of the culture, hyaluronic acid may be separated / collected from the culture solution according to a usual method for separating / collecting polysaccharides. For example, insoluble matter such as bacterial cells in the culture solution can be separated by filtration or centrifugation, and then purified hyaluronic acid can be separated from this solution using a solvent precipitant such as ethanol.

The hyaluronic acid thus obtained by the method of the present invention has an average molecular weight of about 300,000 to 1,500,000, and further about 300,000 to 1,000,000. The molecular weight distribution is in the range of about 2 million to 20,000, and the maximum peak is around 400,000 to 500,000.

The hyaluronic acid obtained by the method of the present invention can be used in the form of pharmaceuticals, cosmetics, etc., as in the conventional case, and is produced by Streptococcus thermophilus microorganisms that can be used in the production of yogurt, etc. , Can also be administered in the form of food. For example, when the hyaluronic acid obtained by the method of the present invention is used in the form of a drug or dietary supplement that appeals for effects such as beauty and anti-aging, it is a solid preparation such as a capsule, a granule, a tablet, or a powder. Alternatively, it can be orally administered as a liquid preparation such as a syrup.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples and the like.

Examples 1 to 4, Comparative Examples 1 to 5
(1) Strain Streptococcus thermophilus YIT2084 strain (FERM BP-10879)

(2) Pre-culture The lactose (L) -ILS medium shown in Table 1 and the G-ILS and S-ILS media in which the carbon source was changed to glucose (G) or sucrose (S) were used. After mixing the components excluding the carbon source and autoclaving, a carbon source sterilized by filtration was added to prepare the mixture.

20 μL of Streptococcus thermophilus YIT 2084 glycerol stock was inoculated into 2 mL of L-ILS, G-ILS or S-ILS medium, and statically cultured at 40 ° C. for 24 hours. And said. 100 μL of the first-stage preculture solution was inoculated into 10 mL of L-ILS, G-ILS or S-ILS medium, and the mixture was cultured under the same conditions for 24 hours to prepare the second-stage preculture solution.

(3) In the main culture (2), the second-stage preculture solution cultured in L-ILS, G-ILS or S-ILS medium, respectively, was prepared with a 50 g / L carbon source and 20 g / L. The cells were cultured by inoculating 1% of the medium containing yeast extract in 120 mL of medium. For culturing, a 200 mL 8-series mini jar (Bio Jr. 8, Able) was used, and the culture was performed under the conditions of 34 ° C., 200 rpm, pH 6.8, and no aeration. Various measurements were performed using the culture solution 24 hours after the start of the culture. The test was repeated at least twice and the average value was shown.

(4) Extraction / Purification of Hyaluronic Acid 100% (w / v) trichloroacetic acid was added to the culture solution so that the final concentration was 10% (w / v), and the mixture was allowed to stand at 4 ° C. for 2 hours. The mixture was centrifuged at 16,900 × g at 4 ° C. for 30 minutes, the supernatant was filtered through a 0.45 μm filter, an equal amount of 99.5% cold ethanol was added, and the mixture was allowed to stand at 4 ° C. overnight. After centrifugation at 16,900 × g at 4 ° C. for 30 minutes, RO water is added to the precipitate to dissolve it, and dialyzed using a dialysis membrane (Spectrum Laboratory) having a molecular weight cut off of 12,000 to obtain a polymer fraction. It was. This was further fractionated with an ultrafiltration membrane (Centricon Plus 70, Merckmillipore) having a molecular weight cut off of 100,000, and the polymer fraction was freeze-dried.

(5) Results Table 2 shows the amount of hyaluronic acid produced. In addition, the amount of hyaluronic acid produced when the sugar used in the preculture was changed to sucrose and the main culture was performed in the same manner is also shown as a comparative example.

When glucose or lactose was used as the carbon source for the preculture and the carbon source of the main culture was changed (Examples 1 to 4), the amount of hyaluronic acid produced was improved. In particular, when pre-cultured with lactose and glucose was used in the main culture (Example 1), the amount of hyaluronic acid produced was very high. On the other hand, when sucrose was used in the preculture (Comparative Examples 3 to 5), the amount of hyaluronic acid produced was low, and when the sugar was changed in the main culture, the amount of production decreased significantly.

_{(6) Cell concentration and productivity In Table 2, the turbidity (OD 660} ) (cell concentration) and the amount of hyaluronic acid produced per cell were found in Examples 1 and 3 in which the amount of hyaluronic acid produced was particularly high. The results of measuring (productivity) are shown in Tables 3 and 4. OD ₆₆₀ was measured using Cary 50 (Agilent) and used as an index of mycelium concentration.
As shown in Tables 3 and 4, when glucose or lactose was used as the carbon source for the preculture and the carbon source of the main culture was changed (Examples 1 and 3), the turbidity (OD ₆₆₀ ) (cell concentration) and bacteria The amount of hyaluronic acid produced per body (productivity) was improved. On the other hand, when sucrose was used for the preculture (Comparative Examples 3 and 5), the turbidity (OD ₆₆₀ ) (cell concentration) and the amount of hyaluronic acid produced per cell (productivity) were low, and sugar was used in the main culture. When changed, the turbidity (OD ₆₆₀ ) (cell concentration) and the amount of hyaluronic acid produced per cell (productivity) were significantly reduced.

Claims (4)

The following process;
a) A step of pre-culturing a Streptococcus thermophilus microorganism in a medium containing one or more selected from glucose and lactose as sugars.
b) A step of inoculating the microorganism pre-cultured in the above step into a medium containing a sugar different from the sugar used in the pre-culture as a sugar and culturing the mixture.
A method for producing hyaluronic acid, including. The method according to claim 1, wherein the medium in step a contains lactose as a saccharide, and the medium in step b contains glucose as a saccharide. The method according to claim 1, wherein the medium in step a contains glucose as a saccharide, and the medium in step b contains lactose as a saccharide. The method according to any one of claims 1 to 3, wherein the Streptococcus thermophilus microorganism is Streptococcus thermophilus YIT2084 strain (FERM BP-10879).