CN111172222A - Method for producing hyaluronic acid by fermentation and application thereof - Google Patents

Method for producing hyaluronic acid by fermentation and application thereof Download PDF

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CN111172222A
CN111172222A CN201911379590.2A CN201911379590A CN111172222A CN 111172222 A CN111172222 A CN 111172222A CN 201911379590 A CN201911379590 A CN 201911379590A CN 111172222 A CN111172222 A CN 111172222A
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hyaluronic acid
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glucose
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李斌
徐飞
祝俊
余允东
马晶晶
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Jiangsu Chengxin Pharmaceutical Co ltd
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Abstract

The invention provides a method for producing hyaluronic acid by fermentation and application thereof. The method comprises the following steps: and (3) activating and expanding streptococcus zooepidemicus, fermenting, supplementing a primary carbon source in the fermentation process to obtain hyaluronic acid fermentation liquor, 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 concentration of glucose in the fermentation liquor can be maintained, the fermentation period is shortened, the bacterial contamination risk caused by adding the carbon source for multiple times is avoided, the normal growth and metabolism of thalli are facilitated, the problem of imbalance between the self metabolism and the product expression of streptococcus zooepidemicus is solved, and the obtained hyaluronic acid has high yield and relatively 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 pharmaceutical-grade hyaluronic acid by fermentation and application thereof.
Background
Hyaluronic Acid (HA) is also called hyaluronic acid, and is a macromolecular mucopolysaccharide, which is a chain-shaped high molecular polymer formed by repeatedly and alternately connecting glucuronic acid and N-acetylglucosamine through β -1,3 and β -1,4 glycosidic bonds, based on the high viscosity, lubricity, water retention, good biocompatibility and special physiological action of hyaluronic acid, the hyaluronic acid HAs wide application in the fields of medicine, beauty treatment, health food and the like, HA with different purposes HAs different requirements on molecular weight, HA with medium molecular weight (molecular weight range of 1,000-1,800KDa) is mainly used in cosmetics, while HA with high molecular weight (molecular weight range of 1,800-2,200KDa) can be used in ophthalmological adhesive surgery, joint disease treatment, soft tissue repair, drug carriers and the like, and particularly HAs high application value in preventing and reducing postoperative tissue adhesion.
The hyaluronic acid production method comprises 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 HA in medicines and cosmetics is limited. Compared with an animal tissue extraction method, 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, the separation and purification are easy, the 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 a hyaluronic acid fermentation broth, wherein a trace element liquid is added in a culture medium, the trace element liquid is composed of calcium chloride, zinc chloride, manganese sulfate and copper sulfate, and water for injection is used for preparing a seed culture medium and a fermentation culture medium, so that the problem of hyaluronic acid yield and quality reduction caused by nutrient substance deficiency or improper blending of various nutrient substances in the process of producing hyaluronic acid by bacterial fermentation is solved, however, the yield of hyaluronic acid in the invention is 6-7g/L, and the yield is still low.
CN109536550A discloses a preparation method of sodium hyaluronate, which improves the yield of sodium hyaluronate, realizes the control of the molecular weight of sodium hyaluronate and expands the application field of sodium hyaluronate by reasonably planning a fermentation process and optimally preparing a fermentation culture medium. According to the method, a carbon source is supplemented by adopting a fed-batch method during fermentation, and the carbon source is supplemented for five times at different times respectively, so that the yield of the obtained hyaluronic acid is high.
Therefore, it is required to provide a fermentation method for preparing hyaluronic acid with high yield and high molecular weight of hyaluronic acid to meet the market demand.
Disclosure of Invention
Aiming at the defects 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 and increase the yield of the hyaluronic acid, and the obtained hyaluronic acid has higher molecular weight. In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for the fermentative production of hyaluronic acid, comprising the steps of: and (3) activating and expanding streptococcus zooepidemicus, fermenting, supplementing a primary carbon source in the fermentation process to obtain hyaluronic acid fermentation liquor, and purifying to obtain hyaluronic acid.
In the fermentation process, the initial glucose concentration is too high, so that the growth inhibition is caused to the growth of strains, the fermentation period is prolonged, and the yield of the hyaluronic acid is influenced by the too low glucose concentration.
Preferably, the content of hyaluronic acid in the hyaluronic acid fermentation liquor is 10-15.5g/L, and can be, for example, 10g/L, 10.2g/L, 10.5g/L, 11g/L, 11.5g/L, 12g/L, 13g/L, 14g/L, 14.5g/L, 15g/L or 15.5g/L, etc.
Preferably, the molecular weight of the hyaluronic acid is 2200 to 4500KDa, such as 2200KDa, 2500KDa, 2800KDa, 3000KDa, 3500KDa, 4000KDa or 4500KDa, etc.
The hyaluronic acid obtained by the invention has high yield which can basically reach 10-15.5g/L, and the product has high molecular weight, can be used as a pharmaceutical grade high molecular weight raw material, can be further developed into orthopedic injection, eye drops, ophthalmic surgery viscoelastic agent, injection beauty filling product and surgery anti-adhesion product, and has wide application range and high additional value.
As a preferred technical scheme of the invention, the carbon source is glucose.
Preferably, the carbon source is supplemented in an amount of 10-20g/L fermentation broth, such as 10g, 11g, 12g, 13g, 14g, 15g, 16g, 17g, 18g, 19g, 20g, etc.
Preferably, the carbon source is supplemented for 4 to 18 hours after the transfer of the strain to the fermentation medium after the amplification, 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 fermentation medium used in the fermentation comprises 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, or 100g/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, C, 16g/L, 18g/L, 19g/L, 20g/L, etc.), peptone, magnesium sulfate, potassium dihydrogen phosphate, sodium glutamate, and sodium glutamate, wherein the peptone is 0.5-2g/L (for example, 0.5g/L, 0.8g/L, 1g/L, 1.2g/L, 1.5g/L, 1.8g/L, 2g/L, etc.), magnesium sulfate, 0.5-2g/L (for example, 0.5g/L, 0.8g/L, 1g/L, 1.2g/L, 1.5g/L, 1.8g/L, 2g/L, etc.), potassium dihydrogen phosphate, 10-20g/L (for example, 10g/L, 12g/L, 14g/L, 15g/L, 16g/L, 18g/L, 19g/L, 20g/L, etc.), sodium glutamate, and 1-5g/L (for example, 1g/L, 1, 2g/L, 1.5g/L, 2g/L, 2.5g/L, 3g/L, 3.5g/L, 4g/L, 4.5g/L, or 5g/L, etc.) arginine hydrochloride.
Preferably, the transfer amount of the strain to the fermentation medium after the propagation is 3 to 5%, for example, 3%, 3.2%, 3.5%, 3.8%, 4%, 4.2%, 4.5%, 4.8%, or 5%.
Preferably, the stirring speed during the fermentation is 100 to 500rpm, and may be, for example, 100rpm, 150rpm, 200rpm, 250rpm, 300rpm, 350rpm, 400rpm, 450rpm or 500rpm, etc.
Preferably, the fermentation temperature is 33-37 ℃, for example, 33 ℃, 33.5 ℃, 34 ℃, 34.5 ℃, 35 ℃, 35.5 ℃, 36 ℃, 36.5 ℃ or 37 ℃.
Preferably, the fermentation time is 20-24h, for example 20h, 20.5h, 21h, 21.5h, 22h, 22.5h, 23h, 23.5h or 24h, etc.
Preferably, the aeration rate during the fermentation is 1 to 3vvm, and may be, for example, 1vvm, 1.2vvm, 1.5vvm, 2vvm, 2.2vvm, 2.5vvm, 2.6vvm, 2.8vvm, 3vvm, or the like.
As a preferable technical scheme of the invention, the fermentation process also comprises a step of adjusting the pH of the fermentation liquor by using a pH regulator.
Preferably, the pH adjusting agent 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, or the like.
In a preferred embodiment of the present invention, the temperature at the time of activation in the above-mentioned method is 33 to 37 ℃ and may be, for example, 33 ℃, 33.5 ℃, 34 ℃, 34.5 ℃, 35 ℃, 35.5 ℃, 36 ℃, 36.5 ℃ or 37 ℃; the time is 10 to 14 hours, for example, 10 hours, 10.5 hours, 11 hours, 11.5 hours, 12 hours, 12.5 hours, 13 hours or 13.5 hours.
Preferably, the medium used in the activation is a shake flask seed medium.
Preferably, the shake flask seed medium comprises 1-10g/L (e.g., can be 1g/L, 2g/L, 4g/L, 5g/L, 6g/L, 8g/L, 9g/L, or 10g/L, etc.) glucose, 5-10g/L (e.g., can be 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., can be 10g/L, 12g/L, 14g/L, 15g/L, 16g/L, 18g/L, 19g/L, or 20g/L, etc.) peptone, 0.5-2g/L (for example, 0.5g/L, 0.8g/L, 1g/L, 1.2g/L, 1.5g/L, 1.8g/L, 2g/L, etc.) magnesium sulfate and 0.5-2g/L (for example, 0.5g/L, 0.8g/L, 1g/L, 1.2g/L, 1.5g/L, 1.8g/L, 2g/L, etc.) potassium dihydrogen phosphate.
In a preferred embodiment of the present invention, the temperature during the amplification in the method is 33 to 37 ℃ and may be, for example, 33 ℃, 33.5 ℃, 34 ℃, 34.5 ℃, 35 ℃, 35.5 ℃, 36 ℃, 36.5 ℃ or 37 ℃; the time is 8 to 12 hours, and may be, for example, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours, 12 hours, or the like.
Between the seed culture and fermentation culture stages of streptococcus zooepidemicus, a seed tank expanding culture step is added, so that the strain is quickly adapted to the nutrient environment provided by a fermentation culture medium, and enters the fermentation stage, the nutrition in the fermentation culture can be quickly utilized for growth, and the fermentation period is shortened.
Preferably, the culture medium used in the expanding culture is a seed tank seed culture medium.
Preferably, the seedpot seed medium comprises 1-10g/L (e.g., can be 1g/L, 2g/L, 4g/L, 5g/L, 6g/L, 8g/L, 9g/L, or 10g/L, etc.) glucose, 5-10g/L (e.g., can be 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., can be 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, 1g/L, 1.2g/L, 1.5g/L, 1.8g/L, 2g/L, etc.) magnesium sulfate, 0.5-2g/L (e.g., 0.5g/L, 0.8g/L, 1g/L, 1.2g/L, 1.5g/L, 1.8g/L, 2g/L, etc.) potassium dihydrogen phosphate, and 10-20g/L (e.g., 10g/L, 12g/L, 14g/L, 15g/L, 16g/L, 18g/L, 19g/L, 20g/L, etc.) sodium glutamate.
Preferably, the method used for purifying the hyaluronic acid fermentation broth is ethanol precipitation.
The ethanol precipitation method is a commonly used method for separating various polysaccharides, can effectively dehydrate and decolor hyaluronic acid so as to improve the product quality of the hyaluronic acid, and in order to ensure that the hyaluronic acid is completely precipitated, about 1 percent of NaCl or NaAc is added to achieve proper ionic concentration due to the fact that the solution has enough ionic strength, and the adding 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 33-37 deg.C for 10-14 hr, transferring to seed tank seed culture medium, and culturing at 33-37 deg.C for 8-12 hr;
(2) transferring streptococcus zooepidemicus in seed tank seed culture medium into fermentation culture medium at 3-5%, fermenting at 33-37 deg.C under stirring at 100-500rpm, and maintaining pH of the fermentation liquid at 7.0-8.0 and ventilation amount of 1-3 vvm;
(3) and (3) transferring the strain to a fermentation culture medium, adding a primary carbon source for 4-16 hours, wherein the fermentation time is 20-24 hours, obtaining hyaluronic acid fermentation liquor after the fermentation is finished, and purifying by using an ethanol precipitation method to obtain hyaluronic acid.
In a second aspect, the present invention provides the use of a method as described in the first aspect for the preparation of pharmaceutical grade hyaluronic acid and/or a hyaluronic acid salt.
The recitation of numerical ranges herein includes not only the above-recited numerical values, but also any numerical values between non-recited numerical ranges, and is not intended to be exhaustive or to limit the invention to the precise numerical values encompassed within the range for brevity and clarity.
Compared with the prior art, the invention has the beneficial effects that:
(1) the method for producing hyaluronic acid by fermentation provided by the invention can maintain the concentration of glucose in the fermentation liquor, is beneficial to normal growth and metabolism of thalli, shortens the fermentation period, avoids the risk of contamination caused by supplementing a carbon source for many times, and solves the problem that the yield and the molecular weight of hyaluronic acid are small due to imbalance between the self-metabolism and product expression of streptococcus zooepidemicus;
(2) the hyaluronic acid prepared by the method has high yield, the content of hyaluronic acid in hyaluronic acid fermentation liquor can reach 15.5g/L, the molecular weight is high, the application value of hyaluronic acid with high molecular weight is high, and meanwhile, the method is simple to operate, short in fermentation period, high in production efficiency and beneficial to industrial production.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
In the following examples, the formulation of the seed solid medium used was: 10g/L of glucose, 5g/L of yeast powder, 10g/L of peptone, 1g/L of magnesium sulfate, 1g/L of monopotassium phosphate and 20g of agar; the sterilization temperature after the culture medium is configured is 115 ℃, and the sterilization time is 20 minutes.
In the following examples, the formulation of the shake flask seed medium used was: 10g/L of glucose, 5g/L of yeast powder, 10g/L of peptone, 1g/L of magnesium sulfate and 1g/L of monopotassium phosphate; the sterilization temperature after the culture medium is configured is 115 ℃, and the sterilization time is 20 minutes.
In the following examples, the formulation of the seed tank seed medium used was: 10g/L of glucose, 5g/L of yeast powder, 10g/L of peptone, 1g/L of magnesium sulfate, 1g/L of monopotassium phosphate and 20g/L of sodium glutamate; the sterilization temperature after the culture medium is configured is 115 ℃, and the sterilization time is 20 minutes.
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 5 g/L; the sterilization temperature is 121 ℃ after the culture medium is prepared, and the sterilization time is 30 min.
Example 1
The embodiment provides a method for producing hyaluronic acid by fermentation, which comprises the following steps:
1. inoculating streptococcus zooepidemicus to a seed solid culture medium by taking streptococcus zooepidemicus as a fermentation strain, culturing at 33 ℃ for 16 h;
2. inoculating streptococcus zooepidemicus to a shake flask seed culture medium for culture, and carrying out shake culture at 33 ℃ for 12h to obtain a streptococcus zooepidemicus shake flask seed culture solution;
3. inoculating the streptococcus zooepidemicus shake-flask seed culture solution to a seed tank culture medium for culture, adjusting the pH of the culture solution with 30% sodium hydroxide at the culture temperature of 33 ℃, maintaining the pH at 7.5, and culturing for 12h to obtain a 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 the temperature of 33 ℃, the pH value of 7.5 and the ventilation volume of 1vvm, supplementing 10g of glucose into each liter of fermentation broth, continuously culturing, adjusting the pH value of the fermentation broth by using 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 broth;
5. and adding 58L of ethanol into 200g of NaCl in the obtained fermentation liquor to precipitate sodium hyaluronate to obtain sodium hyaluronate, converting the amount of hyaluronic acid according to the yield of sodium hyaluronate, measuring the intrinsic viscosity by using a Hill's viscometer, and converting the relative molecular weight, wherein the yield of hyaluronic acid is 10.2g/L, and the molecular weight of hyaluronic acid is 2200 KDa.
Example 2
The same as in example 1, except that 10g of glucose per liter of fermentation broth was fed in 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 2400 kDa.
Example 3
The same as in example 1, except that 10g of glucose per liter of fermentation broth was fed 12 hours after transfer to the fermentation medium; the yield of the obtained hyaluronic acid is 10.7g/L, and the molecular weight of the hyaluronic acid is 3000 KDa.
Example 4
The same as in example 1, except that 10g of glucose per liter of fermentation broth was fed in at 16 hours after transfer to the fermentation medium; the yield of the obtained hyaluronic acid is 12.4g/L, and the molecular weight of the hyaluronic acid is 3600 KDa.
Example 5
The same as in example 1, except that 10g of glucose per liter of fermentation broth was fed 18 hours after transfer to the fermentation medium; the yield of the obtained hyaluronic acid is 11.2g/L, and the molecular weight of the hyaluronic acid is 3400 KDa.
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 supplied per liter of the fermentation broth at 16 hours after transfer to the fermentation medium; the yield of the obtained hyaluronic acid is 12.8g/L, and the molecular weight of the hyaluronic acid is 3800 KDa.
Example 7
The same as in example 1, except that at 16 hours after transfer to the fermentation medium, 5g of glucose was fed per liter of fermentation broth; the yield of the obtained hyaluronic acid is 9.3g/L, and the molecular weight of the hyaluronic acid is 3200 KDa.
Example 8
The same as in example 1, except that 8g of glucose per liter of fermentation broth was fed in at 16 hours after transfer to the fermentation medium; the yield of the obtained hyaluronic acid is 10.6g/L, and the molecular weight of the hyaluronic acid is 3400 KDa.
Example 9
The same as in example 1, except that 15g of glucose per liter of fermentation broth was fed in at 16 hours after transfer to the fermentation medium; the yield of the obtained hyaluronic acid is 14.2g/L, and the molecular weight of the hyaluronic acid is 4000 KDa.
Example 10
The same as in example 1, except that 20g of glucose per liter of fermentation broth was fed in at 16 hours after transfer to the fermentation medium; the yield of the obtained hyaluronic acid is 15.5g/L, and the molecular weight of the hyaluronic acid is 4200 KDa.
Example 11
The same as in example 1, except that 25g of glucose per liter of fermentation broth was fed in at 16 hours after transfer to the fermentation medium; the yield of the obtained hyaluronic acid is 15.0g/L, and the molecular weight of the hyaluronic acid is 4200 KDa.
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 supplied per liter of the fermentation broth at 16 hours after transfer to the fermentation medium; the yield of the obtained hyaluronic acid is 14.4g/L, and the molecular weight of the hyaluronic acid is 4500 KDa.
Comparative example 1
A method for producing a hyaluronic acid fermentation liquid by using 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: 10g/L of glucose, 5g/L of yeast powder, 10g/L of peptone, 1g/L of magnesium sulfate and 1g/L of monopotassium phosphate; the sterilization temperature after the culture medium is configured is 115 ℃, and the sterilization time is 20 minutes.
Inoculating streptococcus zooepidemicus to a seed culture medium for culture, and performing shaking culture at 33 ℃ for 12 hours to obtain a streptococcus zooepidemicus seed culture solution.
3. Preparing a seed culture medium of a seeding tank: 10g/L of glucose, 5g/L of yeast powder, 10g/L of peptone, 1g/L of magnesium sulfate, 1g/L of monopotassium phosphate, 20g/L of sodium glutamate, 115 ℃ of sterilization temperature and 20 minutes of sterilization time.
Inoculating Streptococcus zooepidemicus into seed tank culture medium, culturing at 33 deg.C, adjusting pH to 6.0-8.0 with 30% sodium hydroxide, and culturing for 8-12 hr to obtain Streptococcus zooepidemicus seed culture solution.
4. Preparation of fermenter 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 20 g/L; arginine hydrochloride 5g/L, sterilizing at 121 deg.C for 30 min.
Inoculating a streptococcus zooepidemicus seed culture solution to a fermentation culture solution, adjusting the pH value of the fermentation solution by using 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 volume of 1vvm to keep the pH value at 7.5 +/-0.5, and culturing for 24 hours to obtain hyaluronic acid fermentation solution;
5. 200g NaCl is added into the obtained fermentation liquor, 58L ethanol is added to precipitate sodium hyaluronate, the yield of the obtained hyaluronic acid is 8.1g/L after conversion, and the molecular weight of the hyaluronic acid is 1800 KDa.
Comparative example 2
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: 10g/L of glucose, 5g/L of yeast powder, 10g/L of peptone, 1g/L of magnesium sulfate and 1g/L of monopotassium phosphate; the sterilization temperature after the culture medium is configured is 115 ℃, and the sterilization time is 20 minutes.
Inoculating streptococcus zooepidemicus to a seed culture medium for culture, and performing shaking culture at 33 ℃ for 12 hours to obtain a streptococcus zooepidemicus seed culture solution.
3. Preparing a seed culture medium of a seeding tank: 10g/L of glucose, 5g/L of yeast powder, 10g/L of peptone, 1g/L of magnesium sulfate, 1g/L of monopotassium phosphate, 20g/L of sodium glutamate, 115 ℃ of sterilization temperature and 20 minutes of sterilization time.
Inoculating Streptococcus zooepidemicus into seed tank culture medium, culturing at 33 deg.C, adjusting pH to 8.0 with 30% sodium hydroxide, and culturing for 8-12 hr to obtain Streptococcus zooepidemicus seed culture solution.
4. Preparation of fermenter 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 20 g/L; arginine hydrochloride 5g/L, sterilizing at 121 deg.C for 30 min.
Inoculating a streptococcus zooepidemicus seed culture solution to a fermentation culture solution, culturing for 4 hours under the conditions of stirring rotation speed of 200rpm, temperature of 33 ℃, pH value of 7.5 and ventilation volume of 1vvm, supplementing 2g of glucose into each liter of fermentation liquor in the 8 th hour, supplementing 2g of glucose into each liter of fermentation liquor in the 16 th hour, and supplementing 6g of glucose into each liter of fermentation liquor in the 8 th hour; adjusting the pH value of the fermentation liquor by using 30% sodium hydroxide in the fermentation process to keep the pH value 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 liquid, and 72L ethanol was added to precipitate sodium hyaluronate, and the obtained hyaluronic acid yield was 12.1g/L, and the molecular weight of hyaluronic acid was 3500 KDa.
The glucose addition time, the amount added, and the hyaluronic acid production and hyaluronic acid molecular weight obtained in examples 1 to 12 and comparative examples 1 to 2 above are shown in table 1.
TABLE 1
Figure BDA0002341919310000131
According to the data in Table 1, as can be seen from the comparison between example 4 and comparative examples 1-2, compared with comparative example 1 in which the carbon source content in the fermentation medium is directly increased without supplementing a carbon source, and comparative example 2 in which an equal amount of carbon source is supplemented for a plurality of times, the hyaluronic acid obtained in example 4 has a higher yield and a larger molecular weight; as can be seen from the comparison between examples 4 and 6 and between examples 9 and 12, the stirring forms of the 500L tank and the 50L tank are different, the 500L tank is stirred more sufficiently, the aeration and dissolved oxygen states are better, the growth of the bacterial cells is facilitated, and the yield and the molecular weight of hyaluronic acid can be improved; however, when the addition amount of glucose is further increased in the amplification experiment, the hyaluronic acid yield and the molecular weight obtained by the 500L tank and the 50L tank are more and more similar; as is clear from comparison of example 4 with example 5, the optimum time point for carbon source addition was found to be 16 hours and 18 hours, respectively, in which the yield decreased and the molecular weight no longer increased; as is clear from comparison of example 4 with examples 7 to 11, the production of hyaluronic acid increases with the amount of carbon source supplementation, and the higher the amount of carbon source supplementation, the higher the production of hyaluronic acid and the larger the molecular weight, but when the amount of carbon source supplementation exceeds 20g/L, for example, when the amount added is 25g/L in example 11, the production of hyaluronic acid does not increase any more, indicating that the production of hyaluronic acid is limited when the concentration of carbon source exceeds a certain range.
In conclusion, the fermentation method provided by the invention can maintain the glucose concentration in the fermentation liquid, shorten the fermentation period, avoid the risk of contamination caused by supplementing carbon sources for many times, is beneficial to normal growth and metabolism of thalli, solves the problem of imbalance between the metabolism of streptococcus zooepidemicus and product expression, and has high yield of the obtained hyaluronic acid and larger molecular weight.
The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.

Claims (10)

1. A method for producing hyaluronic acid by fermentation, which is characterized by comprising the following steps:
and (3) activating and expanding streptococcus zooepidemicus, fermenting, supplementing a primary carbon source in the fermentation process to obtain hyaluronic acid fermentation liquor, and purifying to obtain hyaluronic acid.
2. The method according to claim 1, wherein the content of hyaluronic acid in the hyaluronic acid fermentation broth is 10-15.5 g/L;
preferably, the molecular weight of the hyaluronic acid is 2200 to 4500 KDa.
3. The method of claims 1-2, wherein the carbon source is glucose;
preferably, the mass of the carbon source supplemented in each liter of fermentation liquor is 10-20 g;
preferably, the time for supplementing the carbon source is 4 to 18 hours, preferably 16 hours after the strain is transferred to the fermentation medium after the amplification culture;
preferably, the fermentation medium used in the fermentation comprises 50-100g/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.
4. The method according to any one of claims 1 to 3, wherein the transfer amount of the strain to the fermentation medium after the propagation is 3 to 5%;
preferably, the stirring speed during fermentation is 100-500 rpm;
preferably, the fermentation temperature is 33-37 ℃;
preferably, the fermentation time is 20-24 h;
preferably, the aeration during the fermentation is 1-3 vvm.
5. The method according to any one of claims 1 to 4, wherein the fermentation process further comprises the step of adjusting the pH of the fermentation broth using a pH adjusting agent;
preferably, the pH adjuster is sodium hydroxide;
preferably, the pH of the fermentation broth is maintained between 7.0 and 8.0.
6. The method according to any one of claims 1 to 5, wherein the temperature at the activation in the method is 33 to 37 ℃ and the time is 10 to 14 hours;
preferably, the culture medium used in the activation is a shake flask seed culture medium;
preferably, the shake flask seed culture 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.
7. The method according to any one of claims 1 to 6, wherein the temperature during propagation is 33 to 37 ℃ and the time is 8 to 12 hours;
preferably, the culture medium used in the expanding culture is a seed tank seed culture medium;
preferably, the seed tank seed culture 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.
8. The method according to any one of claims 1 to 7, wherein the method used for purifying the hyaluronic acid fermentation broth is ethanol precipitation.
9. Method according to any of claims 1-8, characterized in that the method comprises the steps of:
(1) inoculating Streptococcus zooepidemicus into shake flask seed culture medium, culturing at 33-37 deg.C for 10-14 hr, transferring to seed tank seed culture medium, and culturing at 33-37 deg.C for 8-12 hr;
(2) transferring streptococcus zooepidemicus in seed tank seed culture medium into fermentation culture medium at 3-5%, fermenting at 33-37 deg.C under stirring at 100-500rpm, and maintaining pH of the fermentation liquid at 7.0-8.0 and ventilation amount of 1-3 vvm;
(3) and (3) transferring the strain to a fermentation culture medium, adding a primary carbon source for 4-18 hours, wherein the fermentation time is 20-24 hours, obtaining hyaluronic acid fermentation liquor after the fermentation is finished, and purifying by using an ethanol precipitation method to obtain hyaluronic acid.
10. Use of a method according to any one of claims 1 to 9 for the preparation of pharmaceutical grade hyaluronic acid and/or hyaluronate.
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