CN111172222B

CN111172222B - Method for producing hyaluronic acid by fermentation and application thereof

Info

Publication number: CN111172222B
Application number: CN201911379590.2A
Authority: CN
Inventors: 李斌; 徐飞; 祝俊; 余允东; 马晶晶
Original assignee: Jiangsu Chengxin Pharmaceutical Co ltd
Current assignee: Jiangsu Chengxin Pharmaceutical Co ltd
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2024-03-26
Anticipated expiration: 2039-12-27
Also published as: CN111172222A

Abstract

The invention provides a method for producing hyaluronic acid by fermentation and application thereof. The method comprises the following steps: and (3) after the streptococcus zooepidemicus is subjected to activation and propagation, fermenting, adding a carbon source for one time in the fermentation process to obtain a hyaluronic acid fermentation liquid, and purifying to obtain hyaluronic acid. According to the fermentation method provided by the invention, the primary carbon source is added in the fermentation process, so that the glucose concentration in the fermentation liquid can be maintained, the fermentation period is shortened, the risk of bacterial contamination caused by adding the carbon source for many times is avoided, the normal growth and metabolism of thalli are facilitated, the problem of unbalance between the metabolism of streptococcus zooepidemicus and the expression of products is solved, and the obtained hyaluronic acid has high yield and large molecular weight.

Description

Method for producing hyaluronic acid by fermentation and application thereof

Technical Field

The invention belongs to the technical field of fermentation, and particularly relates to a method for producing hyaluronic acid by fermentation, in particular to a method for producing high-molecular medical-grade hyaluronic acid by fermentation and application thereof.

Background

Hyaluronic acid (HA for short) is a macromolecular mucopolysaccharide, and is a chain high polymer formed by repeatedly and alternately connecting glucuronic acid and N-acetamido glucosamine through beta-1, 3 and beta-1, 4 glycosidic bonds. Based on high viscosity, lubricity, water retention, good biocompatibility and special physiological effects of hyaluronic acid, the hyaluronic acid has wide application in the fields of medicine, beauty, health care food and the like. HA for different applications HAs different requirements on molecular weight, HA with medium molecular weight (molecular weight range 1,000-1,800 kda) is mainly used in cosmetics, while HA with high molecular weight (molecular weight range 1,800-2,200 kda) can be used in ophthalmic adhesive surgery, for treating arthrosis, soft tissue repair and as a drug carrier, etc., and HAs high application value in preventing and reducing postoperative tissue adhesion in particular.

The hyaluronic acid production method includes animal organ extraction method and bacterial fermentation method. When the extraction method is adopted to extract HA from animal viscera, the raw materials are limited, the production cost is high, the commodity price is high, and the wide application of the HA in medicines and cosmetics is limited. Compared with animal tissue extraction, the bacterial fermentation method HAs the advantages that the production scale is not limited by animal raw materials, HA in fermentation liquor exists in a free form, separation and purification are easy, cost is low, large-scale industrial production is easy to form, the risk of animal-derived pathogenic virus pollution is avoided, and the like.

CN101935678A discloses a method for producing hyaluronic acid fermentation liquor, which adds microelement liquor in a culture medium, wherein the microelement liquor consists of calcium chloride, zinc chloride, manganese sulfate and copper sulfate, and uses water for injection to prepare a seed culture medium and a fermentation culture medium, so that the problems of reduced hyaluronic acid yield and quality caused by lack of nutrient substances or improper preparation among various nutrient substances in the process of producing hyaluronic acid by bacterial fermentation are solved, however, the yield of hyaluronic acid is 6-7g/L, and the yield is still lower.

CN109536550a discloses a method for preparing sodium hyaluronate, which improves the yield of sodium hyaluronate, controls the molecular weight of sodium hyaluronate and expands the application field of sodium hyaluronate through reasonable planning of fermentation process and optimized preparation of fermentation medium. The method adopts a batch feeding method to supplement carbon sources during fermentation, and respectively supplements the carbon sources for five times at different times, so that the yield of the obtained hyaluronic acid is higher, but the method has the defects that the carbon sources are supplemented for many times, the fermentation period is longer, the fermentation liquid is easily polluted by miscellaneous bacteria, the quality of the hyaluronic acid is influenced, and the molecular weight of the obtained hyaluronic acid is lower.

Accordingly, there is a need to provide a fermentation process for preparing hyaluronic acid with a higher yield and a higher molecular weight of hyaluronic acid to meet the market demand.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention aims to provide a method for producing hyaluronic acid by fermentation and application thereof. The method is simple to operate, can shorten the fermentation period, increases the yield of the hyaluronic acid, and has higher molecular weight. To achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a method for fermentatively producing hyaluronic acid, comprising the steps of: and (3) after the streptococcus zooepidemicus is subjected to activation and propagation, fermenting, adding a carbon source for one time in the fermentation process to obtain a hyaluronic acid fermentation liquid, and purifying to obtain hyaluronic acid.

In the fermentation process, the initial glucose concentration is too high, which can inhibit the growth of strains, so that the fermentation period is prolonged, and the too low glucose concentration can influence the yield of hyaluronic acid.

Preferably, the content of hyaluronic acid in the hyaluronic acid fermentation broth is 10-15.5g/L, and can be 10g/L, 10.2g/L, 10.5g/L, 11g/L, 11.5g/L, 12g/L, 13g/L, 14g/L, 14.5g/L, 15g/L, 15.5g/L or the like.

Preferably, the molecular weight of the hyaluronic acid is 2200-4500kDa, for example 2200kDa, 2500kDa, 2800kDa, 3000kDa, 3500kDa, 4000kDa or 4500kDa, etc.

The hyaluronic acid obtained by the invention has higher yield which can basically reach 10-15.5g/L, and the molecular weight of the product is higher, can be used as a medicine-level high-molecular-weight raw material, and can be further developed into orthopaedics injection, eye drops, ophthalmic operation viscoelastic agent, injection beauty filling product, operation anti-adhesion product, and has wider application range and higher added value.

As a preferable technical scheme of the invention, the carbon source is glucose.

Preferably, the mass of the carbon source added to each liter of fermentation broth is 10-20g, and for example, 10g, 11g, 12g, 13g, 14g, 15g, 16g, 17g, 18g, 19g or 20g, etc. can be used.

Preferably, the time for supplementing the carbon source is 4 to 18 hours after transferring the strain to the fermentation medium after the expansion culture, and may be, for example, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours or 18 hours, preferably 16 hours.

Preferably, the method comprises the steps of, the fermentation medium used in the fermentation includes 50-100g/L (e.g., 50g/L, 55g/L, 60g/L, 65g/L, 70g/L, 75g/L, 80g/L, 85g/L, 90g/L, 95g/L, 100g/L, etc.), 5-10g/L (e.g., 5g/L, 5.5g/L, 6g/L, 6.5g/L, 7g/L, 7.5g/L, 8g/L, 8.5g/L, 9g/L, 9.5g/L, 10g/L, etc.), yeast powder 10-20g/L (e.g., 10g/L, 12g/L, 14g/L, 15g/L, 16g/L, 18g/L, etc.), yeast powder 19g/L or 20g/L, etc.), peptone 0.5-2g/L (e.g., may be 0.5g/L, 0.8g/L, 1g/L, 1.2g/L, 1.5g/L, 1.8g/L, or 2g/L, etc.), magnesium sulfate 0.5-2g/L (e.g., may be 0.5g/L, 0.8g/L, 1g/L, 1.2g/L, 1.5g/L, 1.8g/L, or 2g/L, etc.), monopotassium phosphate 10-20g/L (e.g., may be 10g/L, 12g/L, 14g/L, 15g/L, 16g/L, 18g/L, 19g/L, or 20g/L, etc.) sodium glutamate and 1-5g/L (e.g., may be 1g/L, 1.5g/L, 2g/L, etc.), 2.5g/L, 3g/L, 3.5g/L, 4g/L, 4.5g/L, 5g/L, etc.) arginine hydrochloride.

Preferably, the transfer amount of the strain transferred to the fermentation medium after the expansion culture is 3-5%, for example, 3%, 3.2%, 3.5%, 3.8%, 4%, 4.2%, 4.5%, 4.8% or 5%, etc.

Preferably, the stirring speed at the time of fermentation is 100-500rpm, and for example, 100rpm, 150rpm, 200rpm, 250rpm, 300rpm, 350rpm, 400rpm, 450rpm, 500rpm, or the like may be used.

Preferably, the fermentation temperature is 33-37 ℃, for example, 33 ℃, 33.5 ℃, 34 ℃, 34.5 ℃, 35 ℃, 35.5 ℃, 36 ℃, 36.5 ℃, 37 ℃ or the like can be used.

Preferably, the fermentation time is 20-24h, and may be, for example, 20h, 20.5h, 21h, 21.5h, 22h, 22.5h, 23h, 23.5h, 24h, or the like.

Preferably, the aeration rate in the fermentation is 1 to 3 vm, for example, 1 vm, 1.2 vm, 1.5 vm, 2 vm, 2.2 vm, 2.5 vm, 2.6 vm, 2.8 vm, 3 vm, or the like.

As a preferable technical scheme of the invention, the fermentation process further comprises the step of adjusting the pH of the fermentation liquor by using a pH regulator.

Preferably, the pH adjuster is sodium hydroxide.

Preferably, the pH of the fermentation broth is maintained between 7.0 and 8.0, and may be, for example, 7.0, 7.2, 7.4, 7.6, 7.8, 7.9, 8.0, etc.

As a preferable embodiment of the present invention, the temperature at the time of activation in the method may be 33℃to 37℃such as 33.5℃to 34℃to 34.5℃to 35℃to 35.5℃to 36℃to 36.5℃or 37℃; the time is 10-14h, and can be, for example, 10h, 10.5h, 11h, 11.5h, 12h, 12.5h, 13h or 13.5h, etc.

Preferably, the medium used in the activation is shake flask seed medium.

Preferably, the method comprises the steps of, the shake flask seed medium comprises 1-10g/L (e.g., 1g/L, 2g/L, 4g/L, 5g/L, 6g/L, 8g/L, 9g/L, or 10g/L, etc.) glucose, 5-10g/L (e.g., 5g/L, 5.5g/L, 6g/L, 6.5g/L, 7g/L, 7.5g/L, 8g/L, 8.5g/L, 9g/L, 9.5g/L, or 10g/L, etc.), yeast powder, 10-20g/L (e.g., 10g/L, 12g/L, 14g/L, 15g/L, 16g/L, 18g/L, 19g/L, or 20g/L, etc.) peptone, 0.5-2g/L (e.g., 0.5g/L, 0.8g/L, 1.2g, 1.5g/L, 1.2g, 2g, 2.1g, 2g, 2.5g, 2g, 2.1g, 2g, etc.), potassium sulfate, etc.

As a preferable embodiment of the present invention, the temperature at the time of the expansion culture in the method is 33 to 37℃and may be, for example, 33℃33.5℃34℃34.5℃35℃35.5℃36℃36.5℃37℃or the like; the time is 8-12 hours, and can be 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours or 12 hours, for example.

Between the zooepidemicus seed culture and the fermentation culture stage, a seed tank expanding culture step is added, so that the strain is quickly adapted to the nutrition environment provided by the fermentation culture medium, enters the fermentation stage, can quickly utilize the nutrition in the fermentation culture for growth, and shortens the fermentation period.

Preferably, the culture medium used in the expansion culture is a seed tank seed culture medium.

Preferably, the method comprises the steps of, the seed tank seed medium comprises 1-10g/L (e.g., 1g/L, 2g/L, 4g/L, 5g/L, 6g/L, 8g/L, 9g/L, or 10g/L, etc.) glucose, 5-10g/L (e.g., 5g/L, 5.5g/L, 6g/L, 6.5g/L, 7g/L, 7.5g/L, 8g/L, 8.5g/L, 9g/L, 9.5g/L, or 10g/L, etc.), yeast powder, 10-20g/L (e.g., 10g/L, 12g/L, 14g/L, 15g/L, 16g/L, 18g/L, etc.), yeast powder, and yeast powder 19g/L or 20g/L, etc.), 0.5-2g/L (e.g., 0.5g/L, 0.8g/L, 1g/L, 1.2g/L, 1.5g/L, 1.8g/L, or 2g/L, etc.), and 10-20g/L (e.g., 10g/L, 12g/L, 14g/L, 15g/L, 16g/L, 18g/L, 19g/L, or 20g/L, etc.) of monosodium glutamate.

Preferably, the method used for purifying the hyaluronic acid fermentation broth is an ethanol precipitation method.

The ethanol precipitation method is a common method for separating various polysaccharides, and can be used for effectively dehydrating and decoloring hyaluronic acid, so that the product quality of the hyaluronic acid is improved, in order to completely precipitate the hyaluronic acid, about 1% of NaCl or NaAc is often added to achieve proper ion concentration, and the addition amount of ethanol is generally 2 times of the volume of fermentation liquor.

As a preferred technical scheme of the invention, the method comprises the following steps:

(1) Inoculating streptococcus zooepidemicus into shake flask seed culture medium, culturing at 33-37deg.C for 10-14 hr, transferring to seed tank seed culture medium, and culturing at 33-37deg.C for 8-12 hr;

(2) Inoculating Streptococcus zooepidemicus in seed culture medium of seed tank into fermentation culture medium at 3-5% inoculum size, fermenting at 33-37deg.C under stirring at 100-500rpm, introducing air volume of 1-3vvm, and maintaining pH of fermentation liquid at 7.0-8.0;

(3) Transferring the strain to a fermentation medium, adding a carbon source for 4-16 hours, wherein the fermentation time is 20-24 hours, obtaining hyaluronic acid fermentation liquor after fermentation, and purifying by an ethanol precipitation method to obtain hyaluronic acid.

In a second aspect, the present invention provides the use of a process as described in the first aspect for the preparation of pharmaceutical grade hyaluronic acid and/or hyaluronate.

The numerical ranges recited herein include not only the above-listed point values, but also any point values between the above-listed numerical ranges that are not listed, and are limited in space and for the sake of brevity, the present invention is not intended to be exhaustive of the specific point values that the stated ranges include.

Compared with the prior art, the invention has the beneficial effects that:

(1) The method for producing the hyaluronic acid by fermentation can maintain the concentration of glucose in fermentation liquor, is beneficial to the normal growth and metabolism of thalli, shortens the fermentation period, avoids the risk of bacterial contamination caused by adding carbon sources for many times, and solves the problems of small hyaluronic acid yield and molecular weight caused by unbalance between the metabolism of streptococcus zooepidemicus and the expression of products;

(2) The hyaluronic acid prepared by the method has high yield, the hyaluronic acid content in the hyaluronic acid fermentation liquor can reach 15.5g/L, the molecular weight is large, the application value of the hyaluronic acid with high molecular weight is high, and meanwhile, the method is simple to operate, short in fermentation period and high in production efficiency, and is beneficial to industrial production.

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.

In the following examples, the seed solid medium used was formulated as follows: glucose 10g/L, yeast powder 5g/L, peptone 10g/L, magnesium sulfate 1g/L, potassium dihydrogen phosphate 1g/L, and agar 20g; the sterilization temperature is 115 ℃ and the sterilization time is 20 minutes after the culture medium is prepared.

In the following examples, the shake flask seed medium was used with the following formulation: glucose 10g/L, yeast powder 5g/L, peptone 10g/L, magnesium sulfate 1g/L, and potassium dihydrogen phosphate 1g/L; the sterilization temperature is 115 ℃ and the sterilization time is 20 minutes after the culture medium is prepared.

In the following examples, the formulation of the seed tank seed medium used was: glucose 10g/L, yeast powder 5g/L, peptone 10g/L, magnesium sulfate 1g/L, potassium dihydrogen phosphate 1g/L, and sodium glutamate 20g/L; the sterilization temperature is 115 ℃ and the sterilization time is 20 minutes after the culture medium is prepared.

In the following examples, the fermentation medium used was formulated as follows: the concentration of glucose is 90g/L, the concentration of yeast powder is 10g/L, the concentration of peptone is 20g/L, the concentration of magnesium sulfate is 0.5g/L, the concentration of potassium dihydrogen phosphate is 2g/L, the concentration of sodium glutamate is 20g/L, and the concentration of arginine hydrochloride is 5g/L; the sterilization temperature is 121 ℃ and the sterilization time is 30min after the culture medium is prepared.

Example 1

The embodiment provides a method for producing hyaluronic acid by fermentation, which comprises the following steps:

1. streptococcus zooepidemicus is used as a fermentation strain, inoculated onto a seed solid culture medium and cultured for 16 hours at 33 ℃;

2. inoculating streptococcus zooepidemicus into a shake flask seed culture medium for culture, and performing shake culture at 33 ℃ for 12 hours to obtain a streptococcus zooepidemicus shake flask seed culture solution;

3. inoculating the streptococcus zooepidemicus shake flask seed culture solution into a seed tank culture medium for culture, adjusting the pH of the culture solution by using 30% sodium hydroxide at the culture temperature of 33 ℃ to maintain the pH at 7.5, and culturing for 12 hours to obtain the streptococcus zooepidemicus seed tank seed culture solution;

4. inoculating seed culture solution of streptococcus zooepidemicus seed tank into a 50L fermentation tank, wherein the fermentation tank contains 30L fermentation medium, the inoculation amount is 5%, and the stirring speed is 250rpm after inoculation; culturing for 4 hours at 33 ℃ and pH value of 7.5 and ventilation of 1vvm, supplementing 10g glucose to each liter of fermentation liquor, continuing culturing, regulating the pH value of the fermentation liquor by 30% sodium hydroxide in the fermentation process to keep the pH value at 7.5+/-0.5, and fermenting for 24 hours to obtain 30L of hyaluronic acid fermentation liquor;

5. 200g of NaCl is added into the obtained fermentation liquor, 58L of ethanol is added to separate out sodium hyaluronate to obtain sodium hyaluronate, the amount of hyaluronic acid is converted according to the yield of sodium hyaluronate, meanwhile, the intrinsic viscosity is measured by adopting a Ping Shi viscometer, the relative molecular weight is converted, the yield of the obtained hyaluronic acid is 10.2g/L, and the molecular weight of the hyaluronic acid is 2200kDa.

Example 2

The same as in example 1, except that 10g of glucose was fed per liter of fermentation broth at 8 hours after transfer to the fermentation medium; the yield of the obtained hyaluronic acid was 10.6g/L, and the molecular weight of the hyaluronic acid was 2400kDa.

Example 3

The same as in example 1, except that 10g of glucose was fed per liter of fermentation broth at 12 hours after transfer to the fermentation medium; the yield of the obtained hyaluronic acid was 10.7g/L, and the molecular weight of the hyaluronic acid was 3000kDa.

Example 4

The same as in example 1, except that 10g of glucose was fed per liter of fermentation broth at 16 hours after transfer to the fermentation medium; the yield of the obtained hyaluronic acid was 12.4g/L, and the molecular weight of the hyaluronic acid was 3600kDa.

Example 5

The same as in example 1, except that 10g of glucose was fed per liter of fermentation broth at 18 hours after transfer to the fermentation medium; the yield of the obtained hyaluronic acid was 11.2g/L, and the molecular weight of the hyaluronic acid was 3400kDa.

Example 6

The same as in example 1, except that a 500L fermenter was used in this example, the volume of the fermentation medium was 300L, and 10g of glucose was fed per liter of fermentation broth 16 hours after switching to the fermentation medium; the yield of the obtained hyaluronic acid was 12.8g/L, and the molecular weight of the hyaluronic acid was 3800kDa.

Example 7

The same as in example 1, except that 5g of glucose per liter of fermentation broth was fed in 16 hours after switching to the fermentation medium; the yield of the obtained hyaluronic acid was 9.3g/L, and the molecular weight of the hyaluronic acid was 3200kDa.

Example 8

The same as in example 1, except that 8g of glucose was fed per liter of fermentation broth at 16 hours after transfer to the fermentation medium; the yield of the obtained hyaluronic acid was 10.6g/L, and the molecular weight of the hyaluronic acid was 3400kDa.

Example 9

The same as in example 1, except that 15g of glucose per liter of fermentation broth was fed in 16 hours after switching to the fermentation medium; the yield of the obtained hyaluronic acid was 14.2g/L, and the molecular weight of the hyaluronic acid was 4000kDa.

Example 10

The same as in example 1, except that 20g of glucose was fed per liter of fermentation broth at 16 hours after transfer to the fermentation medium; the yield of hyaluronic acid obtained was 15.5g/L, the molecular weight of which was 4200kDa.

Example 11

The same as in example 1, except that 25g of glucose per liter of fermentation broth was fed in 16 hours after switching to the fermentation medium; the yield of the obtained hyaluronic acid was 15.0g/L, and the molecular weight of the hyaluronic acid was 4200kDa.

Example 12

The same as in example 1, except that a 500L fermenter was used in this example, the volume of the fermentation medium was 300L, and 15g of glucose was fed per liter of fermentation broth 16 hours after switching to the fermentation medium; the yield of the obtained hyaluronic acid was 14.4g/L, and the molecular weight of the hyaluronic acid was 4500kDa.

Comparative example 1

The method for producing the hyaluronic acid fermentation liquor by taking streptococcus zooepidemicus as a fermentation strain comprises the following steps:

1. inoculating streptococcus zooepidemicus to seed solid culture for culturing, and culturing at 33 ℃ for 16 hours;

2. preparing a shake flask seed culture medium: glucose 10g/L, yeast powder 5g/L, peptone 10g/L, magnesium sulfate 1g/L, and potassium dihydrogen phosphate 1g/L; the sterilization temperature is 115 ℃ and the sterilization time is 20 minutes after the culture medium is prepared.

Inoculating streptococcus zooepidemicus into a seed culture medium for culture, and carrying out shaking culture at 33 ℃ for 12 hours to obtain streptococcus zooepidemicus seed culture solution.

3. Preparing a seed tank seed culture medium: glucose 10g/L, yeast powder 5g/L, peptone 10g/L, magnesium sulfate 1g/L, potassium dihydrogen phosphate 1g/L, sodium glutamate 20g/L, and sterilizing temperature 115 deg.C for 20 min.

Inoculating streptococcus zooepidemicus into a seed tank culture medium for culture, wherein the seed culture condition is that the temperature is 33 ℃, the pH is regulated to 6.0-8.0 by 30% sodium hydroxide, and the culture is carried out for 8-12 hours to obtain the streptococcus zooepidemicus seed culture solution.

4. Preparing a fermentation tank culture medium

The concentration of glucose is 100g/L, the concentration of yeast powder is 10g/L, the concentration of peptone is 20g/L, the concentration of magnesium sulfate is 0.5g/L, the concentration of potassium dihydrogen phosphate is 2g/L, and the concentration of sodium glutamate is 20g/L; arginine hydrochloride 5g/L, sterilizing temperature 121 deg.C, sterilizing time 30min.

Inoculating streptococcus zooepidemicus seed culture solution to fermentation culture solution, regulating the pH value of the fermentation solution by 30% sodium hydroxide in the fermentation process under the conditions of stirring rotation speed of 200rpm, temperature of 33 ℃, pH value of 7.5 and ventilation of 1vvm, so that the pH value is kept at 7.5+/-0.5, and culturing for 24 hours to obtain hyaluronic acid fermentation solution;

5. 200g of NaCl was added to the obtained fermentation broth, and 58L of ethanol was added to precipitate sodium hyaluronate to obtain sodium hyaluronate, whereby the yield of the obtained hyaluronic acid was 8.1g/L, and the molecular weight of the hyaluronic acid was 1800kDa.

Comparative example 2

Inoculating streptococcus zooepidemicus into a seed tank culture medium for culture, wherein the seed culture condition is that the temperature is 33 ℃, the pH is regulated to 8.0 by 30% sodium hydroxide, and the culture is carried out for 8-12 hours to obtain the streptococcus zooepidemicus seed culture solution.

4. Preparing a fermentation tank culture medium

The concentration of glucose is 90g/L, the concentration of yeast powder is 10g/L, the concentration of peptone is 20g/L, the concentration of magnesium sulfate is 0.5g/L, the concentration of potassium dihydrogen phosphate is 2g/L, and the concentration of sodium glutamate is 20g/L; arginine hydrochloride 5g/L, sterilizing temperature 121 deg.C, sterilizing time 30min.

Inoculating streptococcus zooepidemicus seed culture solution to fermentation culture solution, culturing for 4 hours under the conditions that the stirring rotation speed is 200rpm, the temperature is 33 ℃, the pH value is 7.5 and the ventilation rate is 1vvm, supplementing 2g glucose per liter of fermentation solution in 8 hours and supplementing 6g glucose per liter of fermentation solution in 16 hours; regulating the pH value of the fermentation liquor by 30% sodium hydroxide in the fermentation process to ensure that the pH value is maintained at 7.5+/-0.5, and culturing for 24 hours to obtain hyaluronic acid fermentation liquor;

5. 286g NaCl was added to the obtained fermentation broth, and 72L ethanol was further added to precipitate sodium hyaluronate to obtain sodium hyaluronate, whereby the yield of the obtained hyaluronic acid was found to be 12.1g/L, and the molecular weight of the hyaluronic acid was found to be 3500kDa.

The glucose addition times, addition amounts, and resultant hyaluronic acid yields and hyaluronic acid molecular weights in examples 1 to 12 and comparative examples 1 to 2 above are shown in table 1.

TABLE 1

From the data in Table 1, it is clear from comparison between example 4 and comparative examples 1 to 2 that the hyaluronic acid obtained in example 4 has a higher yield and a higher molecular weight, compared with the case of directly increasing the carbon source content in the fermentation medium without adding the carbon source in comparative example 1, in which the carbon source is added in an equal amount for a plurality of times in comparative example 2; as can be seen from comparison of example 4 and example 6 and comparison of example 9 and example 12, the 500L tank and the 50L tank are different in stirring form, the 500L tank is more fully stirred, the aeration and dissolved oxygen state is better, the bacterial growth is facilitated, and the yield and molecular weight of hyaluronic acid can be improved; however, when the added amount of glucose is further increased during the amplification experiment, the yields of hyaluronic acid obtained from the 500L tank and the 50L tank are more and more similar to the molecular weight; as is clear from a comparison of example 4 and example 5, the optimum time point for adding the carbon source was 16 hours, 18 hours, and the yield was reduced, and the molecular weight was no longer increased; as is clear from comparison of examples 4 and examples 7 to 11, the yield of hyaluronic acid increases with the amount of carbon source supplementation, and the higher the supplementation amount, the higher the yield of hyaluronic acid and the higher the molecular weight, but when the carbon source supplementation amount exceeds 20g/L, for example, the addition amount in example 11 is 25g/L, the yield of hyaluronic acid is not increased any more, indicating that the yield of hyaluronic acid is limited when the carbon source concentration exceeds a certain range.

In summary, the fermentation method provided by the invention can maintain the glucose concentration in the fermentation liquid, shortens the fermentation period, avoids the risk of bacterial contamination caused by adding carbon sources for many times, is beneficial to normal growth and metabolism of thalli, and solves the problem of unbalance between metabolism of streptococcus zooepidemicus and expression of products, and the obtained hyaluronic acid has high yield and large molecular weight.

The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.

Claims

1. A method for producing hyaluronic acid by fermentation, comprising the steps of:

activating and expanding culture streptococcus zooepidemicus, fermenting, adding a carbon source for one time in the fermentation process to obtain hyaluronic acid fermentation liquor, and purifying to obtain hyaluronic acid;

the fermentation culture medium used in the fermentation comprises 90-100g/L glucose, 5-10g/L yeast powder, 10-20g/L peptone, 0.5-2g/L magnesium sulfate, 0.5-2g/L potassium dihydrogen phosphate, 10-20g/L sodium glutamate and 1-5g/L arginine hydrochloride;

the carbon source is glucose;

the mass of the carbon source added into each liter of fermentation liquor is 10-20g;

the time for supplementing the carbon source is 12-18 hours after transferring the strain to the fermentation culture medium after the expansion culture;

the content of hyaluronic acid in the hyaluronic acid fermentation liquor is 10-15.5g/L;

the molecular weight of the hyaluronic acid is 2200-4500kDa.

2. The method of claim 1, wherein the time for supplementing the carbon source is 16 hours after transferring the strain to the fermentation medium after the expanding culture.

3. The method of claim 1, wherein the transfer of the strain to the fermentation medium after the propagation is 3-5%.

4. The method according to claim 1, wherein the stirring speed during fermentation is 100-500rpm.

5. The method of claim 1, wherein the fermentation temperature is 33-37 ℃.

6. The method of claim 1, wherein the fermentation time is 20-24 hours.

7. The method of claim 1, wherein the aeration during fermentation is 1-3vvm.

8. The method of claim 1, further comprising the step of adjusting the pH of the fermentation broth using a pH adjuster.

9. The method of claim 8, wherein the pH adjuster is sodium hydroxide.

10. The method of claim 8, wherein the pH of the fermentation broth is maintained between 7.0 and 8.0.

11. The method according to claim 1, wherein the temperature at which the activation is performed is 33-37 ℃ for a period of 10-14 hours.

12. The method of claim 1, wherein the medium used in the activation is shake flask seed medium.

13. The method of claim 12, wherein the shake flask seed medium comprises 1-10g/L glucose, 5-10g/L yeast powder, 10-20g/L peptone, 0.5-2g/L magnesium sulfate, and 0.5-2g/L potassium dihydrogen phosphate.

14. The method according to claim 1, wherein the temperature during the propagation is 33-37 ℃ and the time is 8-12 hours.

15. The method of claim 1, wherein the culture medium used in the expanding culture is a seed tank seed culture medium.

16. The method of claim 15, wherein the seed tank seed medium comprises 1-10g/L glucose, 5-10g/L yeast powder, 10-20g/L peptone, 0.5-2g/L magnesium sulfate, 0.5-2g/L potassium dihydrogen phosphate, and 10-20g/L sodium glutamate.

17. The method of claim 1, wherein the method used to purify the hyaluronic acid fermentation broth is an ethanol precipitation method.

18. The method according to claim 1, characterized in that it comprises the steps of:

(3) Transferring the strain to a fermentation medium, adding a carbon source for 12-18 hours, wherein the fermentation time is 20-24 hours, obtaining hyaluronic acid fermentation liquor after fermentation, and purifying by an ethanol precipitation method to obtain hyaluronic acid.

19. Use of a process according to any one of claims 1-18 for the preparation of pharmaceutical grade hyaluronic acid and/or hyaluronate.