KR20120077726A - A method for purifying hyaluronic acid or salt there of - Google Patents

Abstract

PURPOSE: A method for purifying hyaluronic acid and salt thereof is provided to shorten purification time and to effectively suppress microbial contamination. CONSTITUTION: A method for purifying hyaluronic acid and salt thereof comprises: a step of adding acid to a medium of a hyaluronic acid-producing strain for adjusting pH concentration; a step of performing thermal treatment of the medium for adjusting viscosity; and a step of adding alcohol to the medium and stirring. The method additionally comprises a step of adjusting viscosity of the medium for culturing the strain. The method further comprises a step of adding diatomite or activated charcoal and filtering.

Description

Purification method of hyaluronic acid and its salt {A METHOD FOR PURIFYING HYALURONIC ACID OR SALT THERE OF}

The present invention relates to a method for purifying hyaluronic acid and salts thereof, and more particularly, to a method for efficiently purifying hyaluronic acid and salts thereof produced by microbial fermentation.

Hyaluronic acid (HA) is a polymer having a molecular weight of 1,000 to 13,000,000 Da, usually 50,000 to 10,000,000 Da, and is a kind of glycosaminoglycan existing in vivo, and is a water-soluble, colorless, high viscosity straight chain. Of polysaccharides. The structure of hyaluronic acid is the same repeating molecular structure of glucuronic acid (D-glucuronic acid) and N-acetylglucosamine (N-acetylglucosamine), that is, glucuronic acid and N-acetylglucosamine β-1 , 3 bonds and β-1,4 bonds are alternately connected.

Hyaluronic acid has various effects and excellent physical properties such as moisturizing effect, lubricating effect against physical friction, and protection against invasion of bacteria, etc., and therefore, cosmetic additives, arthritis treatment agent, surgical aid for ophthalmic surgery, and adhesion inhibitor after surgery Widely used in cosmetics, medicines and quasi-drug materials and foods.

More specifically, the hyaluronic acid has a lubricating effect to facilitate the movement of the body, and the role of mediators and extracellular matrix to induce the differentiation and growth of cells by the specific action with the cells to control the movement of physiologically active substances Collagen, elastin, chondroitin sulfate and other proteins that support tissues and glycoproteins have an effect related to the support role, it is known to be excellent in water content, excellent viscosity and elasticity.

Therefore, due to these characteristics, hyaluronic acid is used as a therapeutic agent for the treatment of degenerative arthritis and pain relief according to aging, and as a main component of surgical aid and dry eye medication during ophthalmic surgery, and also supplements wrinkles and depressed skin tissue. It is also used as a molding aid, and is also used as an additive for cosmetics and an additive for food.

Since hyaluronic acid was first discovered in cow's eye, that is, the eye's vitreous juice, it is contained in cow's eye, chicken crust, animal buffer tissue, muscle and skin, umbilical cord, placenta, and so on almost all parts of the body. It is known to exist.

Therefore, the hyaluronic acid is produced by extracting from an example of chicken tissues, such as chicken tissue, it is also produced by the method of fermenting microorganisms and recovering the product.

The method of extracting from the animal tissue may have an excellent point in terms of efficacy, but since the hyaluronic acid extract contains a large amount of impurities such as chondroitin sulfate and glycosamino glycan sulfate, a complex purification process for removing them is performed. There is a problem that it is difficult to mass-produce at a high cost accordingly required.

Therefore, when a special purpose is not required, a method of producing hyaluronic acid by culturing microorganisms such as Streptococcus sp.

The method of producing hyaluronic acid by culturing the microorganism is relatively low in production cost compared to the method of producing hyaluronic acid by extracting it into animal tissue, so it is not a special condition such as high specific demand or high molecular weight. In this case, the method for producing hyaluronic acid by culturing the microorganism is more economical and efficient.

In the case of producing hyaluronic acid by culturing the microorganisms, a process of separating and purifying hyaluronic acid from the microbial culture solution is required.

Regarding the method of purifying hyaluronic acid, a method of purifying hyaluronic acid through a precipitation method using an organic solvent is usually mainly used after removing a strain from a microbial culture.

In the case of the above method, there is a problem that the purification time is long and the purification efficiency is low due to the high viscosity due to the high molecular weight of hyaluronic acid, and a special facility is required due to the high possibility of contamination by microorganisms during the long-term purification process. However, when special equipment is not provided, there is a disadvantage that additional work is required to prevent or improve pollution.

In order to improve the problems of the prior art as described above, an object of the present invention is to provide a more efficient and economical method capable of purifying hyaluronic acid with high purity.

The present invention relates to a method for purifying hyaluronic acid and salts thereof, and more particularly, to a method for efficiently purifying hyaluronic acid and salts thereof produced by microbial fermentation.

The inventors of the present invention during the purification process of the hyaluronic acid produced by microbial fermentation, during the study to solve the problems caused by the characteristics of hyaluronic acid having high viscosity by high molecular weight, the culture solution of the hyaluronic acid producing strain After adding acid, heat treatment to adjust the viscosity, and adding a certain amount of ethanol and stirring, the purification time is shortened and the purification efficiency can be improved and microbial contamination can be effectively suppressed. The present invention has been completed.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a method for purifying hyaluronic acid and salts thereof, and more particularly, to a method for efficiently purifying hyaluronic acid and salts thereof produced by microbial fermentation.

Specifically, the present invention comprises the steps of adjusting the pH by adding an acid to the culture of the hyaluronic acid producing strain; Adjusting the viscosity by heat-treating the culture solution of the hyaluronic acid producing strain adjusting the pH; And it relates to a method of purifying hyaluronic acid and its salt comprising the step of adding alcohol and stirring the culture medium of the hyaluronic acid production strain with the viscosity adjusted.

The adjusting the pH may be characterized by adjusting the pH to pH 4 to pH 5, wherein the acid may be a weak acid and the weak acid may be lactic acid.

In addition, adjusting the pH may further include adjusting the viscosity of the culture solution of the hyaluronic acid producing strain.

In addition, the process of adjusting the viscosity may be to adjust the viscosity of the culture solution of the hyaluronic acid producing strain to 6,000 cps to 9,000 cps. Adjusting the viscosity may be carried out at 40 ° C to 80 ° C, and may be performed for 1 to 300 minutes at the temperature conditions in more detail.

In addition, the addition amount of the alcohol in the step of adding and stirring the alcohol is 1% (v / v) to 40% (v / v), preferably based on the volume of the culture medium of the hyaluronic acid producing strain in which the viscosity is adjusted May be 5% (v / v) to 30% (v / v), more preferably 10% (v / v) to 20% (v / v). The alcohol may be ethanol or an ethanol aqueous solution, and the ethanol aqueous solution may be 50% to 99% ethanol aqueous solution.

In addition, the method of purifying the hyaluronic acid and salts thereof may further include adding and filtering one or more selected from the group consisting of diatomaceous earth and activated carbon to the culture solution of the hyaluronic acid producing strain to which the alcohol is added and stirred.

The filtering may include adding at least one selected from the group consisting of diatomaceous earth and activated carbon to the culture solution of the hyaluronic acid producing strain, and performing primary filtration until the OD value is 0.4 to 0.6 and diatomaceous earth to the primary filtered culture medium. And adding at least one selected from the group consisting of activated carbon and performing secondary filtration until the OD value becomes 0.001 to 0.007.

In addition, the filtration step may further include the step of precipitating hyaluronic acid by adding an organic solvent to the filtrate obtained by the filtration step.

The step of adjusting the pH may be performed by adjusting the pH of the culture solution of the hyaluronic acid producing strain by adding an acid.

The hyaluronic acid producing strain refers to a strain capable of producing hyaluronic acid, that is, a strain capable of producing hyaluronic acid as a common metabolite, and is not limited to a specific strain. The strain may be, for example, a microorganism of the genus Streptococcus sp. It may be, but is not limited to, Streptococcus equimilis.

The culture solution of the hyaluronic acid producing strain may be prepared by culturing the hyaluronic acid producing strain in a conventional culture method.

The culture solution of the hyaluronic acid producing strain may be a viscosity of 4,000 cps to 10,000 cps or 6,000 cps to 9,000 cps or 7,000 cps to 8,000 cps, for example, the process of adjusting the viscosity to the viscosity of the range when out of the range of the viscosity Can be performed further.

In addition, in the present invention, since ethanol is added before the ethanol is added for precipitation in the purification process, that is, the viscosity is adjusted, it is possible to control the contamination of microorganisms, so that the process of removing the cells from the culture solution is essential. Not.

However, if necessary, a process of removing the cells from the culture medium may be further performed, and the process of removing the cells may be performed before adjusting the pH. Removing the cells may be carried out by centrifugation or filtration.

The step of adjusting the pH may be adjusted to pH 4 to pH 5, preferably pH 4.25 to pH 4.75. The acid added may be a strong acid or a weak acid. The strong acid refers to an acid having an pH of 3 or less, and may be, for example, hydrogen chloride, sulfuric acid, nitric acid, perchloric acid, formic acid, oxalic acid, or picric acid, but is not limited thereto. The weak acid refers to an acid having a pH of 3 or more, and may be, for example, lactic acid, acetic acid, phosphoric acid, or hydrogen cyanide, but is not limited thereto.

The acid to be added may preferably be a weak acid, more preferably lactic acid.

Adjusting the viscosity may be carried out at 40 ℃ to 80 ℃, preferably 50 ℃ to 70 ℃ 1 minute to 300 minutes, preferably 5 minutes to 150 minutes, more preferably 10 minutes to 90 minutes have.

In the step of adding and stirring the alcohol, the amount of the alcohol is 1% (v / v) to 40% (v / v), preferably 5, based on the volume of the culture medium of the hyaluronic acid producing strain having the viscosity adjusted. % (v / v) to 30% (v / v), more preferably 10% (v / v) to 20% (v / v). The alcohol may be ethanol or an ethanol aqueous solution, and the ethanol aqueous solution may be 50% to 99% ethanol aqueous solution.

Adding and filtering the at least one selected from the group consisting of diatomaceous earth and activated charcoal to the culture solution of the hyaluronic acid producing strain to which the alcohol is added and stirred is at least one selected from the population consisting of diatomaceous earth and activated carbon to the culture solution of the hyaluronic acid producing strain. It can be carried out by adding the above, stirring and then removing the activated carbon. In adding at least one selected from the group consisting of diatomaceous earth and activated carbon, salt (NaCl) may be further added to the culture solution of the hyaluronic acid producing strain.

The amount of one or more selected from the group consisting of diatomaceous earth and activated carbon may be adjusted within the range of ordinary knowledge in the art to which the present invention pertains, for example, one type selected from the group consisting of diatomaceous earth and activated carbon for the total culture volume The amount may be 0.1 wt% to 10 wt%, but is not limited thereto.

The filtering may be performed by dividing into two stages. More specifically, at least one selected from the group consisting of diatomaceous earth and activated carbon is added to the culture solution of the hyaluronic acid producing strain, and until the OD value is 0.4 to 0.6. It may be carried out by a method comprising the step of filtering the primary and the primary filtration culture medium, at least one selected from the group consisting of diatomaceous earth and activated carbon, and secondary filtration until the OD value is 0.001 to 0.007. .

In addition, the filtration step may further include the step of precipitating hyaluronic acid by adding an organic solvent to the filtrate obtained by the filtration step.

Precipitating the hyaluronic acid may be carried out by adding an organic solvent to the filtrate obtained by the filtration step to precipitate hyaluronic acid, and further by drying the hyaluronic acid obtained through the precipitation. can do. The organic solvent may be acetone, methanol, ethanol, propanol, isopropanol or acetonitrile as a water-soluble organic solvent, preferably ethanol.

In addition, the precipitation of the hyaluronic acid may be performed by adding salt (NaCl) to the organic solvent, and the concentration of the salt may be added to the aqueous hyaluronic acid solution at a concentration of 0.5 to 3 M, but is not limited thereto. It doesn't happen.

Purification method of the present invention is a method of adding an acid to the culture solution of the hyaluronic acid producing strain, heat treatment to adjust the viscosity, and then adding a certain amount of ethanol, and performing a filtration and purification process, according to the present invention the purification time is It is economical and efficient compared to the conventional purification method because it is shortened and the purification efficiency is improved and economical as well as it can effectively suppress the contamination of microorganisms. It can be widely used for the production of hyaluronic acid for the purpose.

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to specific examples and comparative examples in order to help understanding of the present invention. However, the following examples are only intended to more clearly understand the present invention, and are not limited to the following examples of the present invention.

Example 1

After adjusting the pH to pH 4.5 by adding lactic acid 1% to 2% to the hyaluronic acid culture having a viscosity value of about 7,000 cps to 8,000 cps, and after heat treatment at 50 ℃ to 70 ℃ to adjust the viscosity to 100 cps to 2,000 cps , 10% to 20% of ethanol was added and stirred well. Diatomaceous earth and activated carbon were added by a predetermined amount based on 20 L of the culture solution, followed by filtration stepwise using a Filterpress. In the case of primary filtration, 1% of activated carbon was added, followed by filtration until the OD value was about 0.5, and in the case of secondary filtration, 4% of activated carbon was added, followed by filtration until the OD value was 0.005 or less. Was carried out to complete purification. After purification was completed, excess ethanol was added to precipitate hyaluronic acid, and the obtained hyaluronic acid was washed and dried to obtain a white fine powder.

[Example 2]

Purification was carried out in the same manner as in Example 1 except adding 0.2% to 0.5% hydrochloric acid to the hyaluronic acid culture. After purification was completed, excess ethanol was added to precipitate hyaluronic acid, and the obtained hyaluronic acid was washed and dried to obtain a white fine powder.

Comparative Example 1

After adjusting the viscosity through acid treatment and heat treatment to the hyaluronic acid culture solution, purification was performed in the same manner as in Example 1, except that 5% of ethanol was added and sufficiently stirred. After purification was completed, excess ethanol was added to precipitate hyaluronic acid, and the obtained hyaluronic acid was washed and dried to obtain a white fine powder.

Comparative Example 2

After adjusting the viscosity through the acid treatment and heat treatment to the hyaluronic acid culture solution, purification was carried out in the same manner as in Example 1 except that 30% of ethanol was added and sufficiently stirred. After purification was completed, excess ethanol was added to precipitate hyaluronic acid, and the obtained hyaluronic acid was washed and dried to obtain a white fine powder.

[Experimental Example 1]

(1) productivity measurement

The amount of hyaluronic acid in the hyaluronic acid culture medium in which the microorganism culture was performed was measured by the following method.

(2) OD value measurement

After adjusting the viscosity through the acid treatment and heat treatment using a UV spectrometer (Varian 210), the transparency of the hyaluronic acid culture solution added with ethanol was measured. The measurement of the OD value measured the OD value of the hyaluronic acid culture medium and the culture solution for each purification step performed the microbial culture at 600 nm.

[Experimental Example 2]

(1) protein / HA content / color / microbial / tablet yield measurement

The protein content and hyaluronic acid content of the hyaluronic acid obtained in Examples 1, 2 and Comparative Example 1 and Comparative Example 2 were lowered (European pharmacopoeia, 6.0, 2905) and Cabazole (European pharmacopoeia, 6.0, 2906). Measured by

In addition, the color was observed through the naked eye, the microorganisms were carried out by measuring the total number of bacteria.

More specifically, the total bacterial count refers to the number of mesophilic bacteria that can be developed in standard agar medium among the bacteria present in the sample. Briefly, the sample and the standard agar medium were mixed and coagulated in a Petri dish to calculate the number of live bacteria in the sample from the colony numbers of the bacteria generated after the culture.

The total bacterial count was measured as follows. First, as a reagent and a reagent for measuring the total bacterial count, plate count agar was added to sterile physiological distilled water in 5.0 g of tryptone, 2.5 g of yeast extract, 1.0 g of dextrose, and 15.0 of agar. After making, the medium was adjusted to pH 7.0 ± 0.2. The pH-adjusted medium was prepared by sterilization at 121 ° C. for 15 minutes. The sterile physiological saline solution was used by sterilizing for 15 minutes at 121 ℃ in 0.9% by weight aqueous sodium chloride solution.

The total bacterial count was measured by dissolving 0.5 g of each hyaluronic acid purified by the method of Example 1, Example 2, Comparative Example 1 and Comparative Example 2 in 50 mL of the sterile saline solution. When the sterilized standard agar medium was cooled to 43 ° C. to 45 ° C., for each 250 mL of the standard agar medium, 50 mL of a solution in which the sample was dissolved in physiological saline was added and shaken to prepare a mixed solution. Before solidification, the mixed solution was poured into a Petri dish and cooled to coagulate. The Petri dish, to which the mixed solution was coagulated, was inverted and incubated at 37 ± 1 ° C. for 24 to 48 hours. After the incubation, the number of colonies produced (colonies) was measured and divided by 0.5 g to determine the total number of bacteria (cfu / g).

Purification yield measurement was based on the hyaluronic acid in the culture solution confirmed through the productivity measurement, the purification yield was calculated by the following equation.

[formula]

Purification yield = (final dry amount / amount contained in initial culture) X 100 (%)

The protein content, hyaluronic acid content (HA), color, microbial total bacterial count results, and purification yield measurement results of the hyaluronic acid powders of Examples 1, 2, and Comparative Examples 1 and 2 obtained by purification under the above conditions Table 1 shows.

Example 1 Example 2 Comparative Example 1 Comparative Example 2 Purification time Tier 1 (standard OD 0.5) 5 ~ 6 5 ~ 6 8-9 10-12 Tier 2 (Base OD 0,005) 12-14 12-14 9-10 16-18 Protein content 0.07-0.10% 0.09-0.15% 0.05-0.57% 0.07-0.09% HA content 97-103% 85-95% 80-105% 95-98 Microbial total bacteria 3 ~ 30cfu / g 3 ~ 30cfu / g 30 ~ 500cfu / g 3 ~ 20cfu / g Purification yield 92-95% 92-95% 90-95% 75-85% pH (based on 1% solution) 5.5 5.0 5.5 5.5

As shown in Table 1, in Example 1, the total purification time for managing up to the reference OD value is about 17 hours to 20 hours, it can be seen that both the protein and the content and microorganisms are obtained above the reference value. In addition, the purification yield was also very good results of more than 90%. In the case of Example 2, the purification time is similar to Example 1, but the content was found to be 85% to 90% lower than the reference value. It is estimated that the content is low because strong acid remains in the culture even after the purification process. This can be confirmed by measuring the pH of the powder obtained.

In the case of Comparative Example 1 in which the ethanol addition amount is 5%, the total purification time may be slightly longer than that of Example 1. In addition, it can be seen that the range of change of protein and content is very large. This means that when only 5% of ethanol is used, problems such as microbial contamination may occur due to external factors during the purification process, thereby increasing the protein and decreasing the content of hyaluronic acid. When 20% or more of ethanol was used, the purification time increased more than two times, but the protein and content were found to be very stable. In particular, the microorganisms were found to be very safe. However, 75% to 85% of the purification yield was obtained, presumably because the excessive amount of ethanol increased some of the insoluble portion and the purification yield was lowered.

Claims (16)

Adjusting pH by adding acid to a culture solution of hyaluronic acid producing strain;
Adjusting the viscosity by heat-treating the culture solution of the hyaluronic acid producing strain adjusting the pH; And
Purifying the hyaluronic acid and its salt, characterized in that it comprises the step of adding and stirring alcohol to the culture medium of the hyaluronic acid production strain with the viscosity adjusted. The method of claim 1,
The step of adjusting the pH is a method of purifying hyaluronic acid and its salt, characterized in that the pH is adjusted to pH 4 to pH 5. The method of claim 1,
The method of purifying hyaluronic acid and its salts, characterized in that the acid in the step of adjusting the pH is a weak acid. The method of claim 3,
The weak acid is lactic acid (lactic acid), characterized in that the hyaluronic acid and its salt purification method. The method of claim 1,
The step of adjusting the pH further comprises the step of adjusting the viscosity of the culture solution of the hyaluronic acid production strain hyaluronic acid and its salt purification method. The method of claim 5,
The step of adjusting the viscosity is hyaluronic acid and the salt purification method of adjusting the viscosity of the culture solution of the hyaluronic acid producing strain to 6,000 cps to 9,000 cps. The method of claim 1,
The step of adjusting the viscosity is hyaluronic acid and a method for purifying the salt, characterized in that carried out at 40 ℃ to 80 ℃. The method of claim 7, wherein
Adjusting the viscosity is a method for purifying hyaluronic acid and its salt, characterized in that performed for 1 to 300 minutes. The method of claim 1,
The step of adding and stirring the alcohol is the addition amount of alcohol is 1% (v / v) to 40% (v / v) of the hyaluronic acid and its salt based on the volume of the culture medium of the hyaluronic acid producing strain adjusted the viscosity Purification method. 10. The method of claim 9,
The addition amount of the alcohol is 5% (v / v) to 30% (v / v) hyaluronic acid and a method for purifying the salt thereof. The method of claim 10,
The addition amount of the alcohol is 10% (v / v) to 20% (v / v) of the hyaluronic acid and its purification method. 10. The method of claim 9,
The alcohol is a method of purifying hyaluronic acid and its salt that is ethanol or ethanol aqueous solution. The method of claim 12,
The aqueous ethanol solution is a 50% to 99% aqueous ethanol solution of the hyaluronic acid and its salt purification method. The method of claim 1,
The method of purifying hyaluronic acid and its salt further comprising the step of adding and filtering at least one selected from the group consisting of diatomaceous earth and activated carbon to the culture solution of the hyaluronic acid producing strain to which the alcohol is added and stirred. The method of claim 14,
The filtering may include adding at least one selected from the group consisting of diatomaceous earth and activated carbon to the culture solution of the hyaluronic acid producing strain, and performing primary filtration until the OD value is 0.4 to 0.6 and diatomaceous earth to the primary filtered culture medium. And adding at least one selected from the group consisting of activated charcoal and performing secondary filtration until the OD value is 0.001 to 0.007. The method of claim 14,
The method of purifying hyaluronic acid and its salt further comprising the step of precipitating hyaluronic acid by adding an organic solvent to the filtrate obtained by the filtering step in addition to the filtering step.