Efficient production method of hyaluronic acid

Abstract

The invention belongs to the technical field of hyaluronic acid fermentation, and provides an efficient production method of hyaluronic acid, which comprises the following steps: step 1) inoculation, step 2) fermentation, step 3) filtration and impurity removal, and step 4) drying. The limitation of high-viscosity fermentation liquor on dissolved oxygen and matrix transfer is relieved through aeration, so that high-density thalli are obtained; and the hyaluronic acid synthetase is induced and synthesized, so that the high-density thallus recovers the capability of generating the capsule, and the purpose of improving the yield of hyaluronic acid is achieved.

Description

Technical Field

[0001] This invention belongs to the field of hyaluronic acid preparation technology, and specifically provides a method for efficient production of hyaluronic acid. Background Technology

[0002] Hyaluronic acid (HA) is a highly viscous, non-sulfated, natural acidic polysaccharide, also known as hyaluronic acid. Hyaluronic acid possesses various physiological functions such as moisturizing and lubrication, and is currently widely used in the food, pharmaceutical, and cosmetic industries, exhibiting a broad market potential and promising application prospects.

[0003] In traditional fermentation production of hyaluronic acid, problems such as increased fermentation broth viscosity leading to insufficient dissolved oxygen and hindered substrate transport are often encountered, affecting cell growth and hyaluronic acid synthesis. In addition, high-density cells may lose their ability to produce hyaluronic acid (i.e., capsule) during continuous growth, limiting the final yield of hyaluronic acid.

[0004] Application No. 2024115141222 discloses a method for improving the yield of hyaluronic acid fermentation. This method involves adding starch sugar and hyaluronidase solution at different fermentation stages. On the one hand, adding sugar prevents rapid viscosity buildup during fermentation, reducing the risk of microbial inactivation due to uneven alkali distribution caused by excessive viscosity. On the other hand, hyaluronidase breaks down the hyaluronic acid molecular chains, reducing the viscosity of the fermentation broth and improving mass transfer, thereby increasing the yield. This method offers high yield and conversion rate of hyaluronic acid fermentation broth, is simple, and suitable for large-scale production applications.

[0005] Due to the high viscosity of hyaluronic acid fermentation broth, the added materials cannot be quickly mixed, leading to issues such as bacterial growth and synthesis. Currently, there is an urgent need for a more efficient method for producing hyaluronic acid. Summary of the Invention

[0006] This invention provides a highly efficient method for producing hyaluronic acid. The preparation of hyaluronic acid based on the method of this invention can significantly improve the production efficiency of hyaluronic acid.

[0007] A method for efficient production of hyaluronic acid includes the following steps: 1) inoculation, 2) fermentation, 3) filtration and impurity removal, and 4) drying.

[0008] The specific steps are as follows: Step 1) Inoculation: Inoculate the Streptococcus ATCC39920 seed culture into the fermentation medium at an inoculation rate of 5-10% for fermentation culture. The fermentation medium consists of the following components: glucose 10-15 g / L, peptone 5-8 g / L, dipotassium hydrogen phosphate trihydrate 1-3 g / L, magnesium sulfate heptahydrate 1-1.5 g / L, potassium dihydrogen phosphate 0.5-1 g / L, dipotassium hydrogen phosphate 0.5-1 g / L, zinc sulfate heptahydrate 50-100 mg / L, agar 12-15 g / L, yeast extract powder 5-8 g / L, and water as the solvent. The pH is adjusted to 7-7.5 using sodium hydroxide solution. Step 2) Fermentation: During the initial fermentation process, aeration is carried out at a rate of 3-5 times the volume of the culture medium per hour for 0-8 hours. A 25-30 g / L glucose solution is added via atomization, a 5-15 g / L sodium chloride solution is added via atomization, and a 0.5-2 g / L hyaluronidase solution is added via atomization at regular intervals. During the later stage of fermentation, the aeration rate is 5-8 times the volume of the culture medium per hour, and a glucose solution with a concentration of 30-35 g / L is added by atomization, and a sodium chloride solution with a concentration of 1-5 g / L is added by atomization to obtain the fermentation broth. Step 3) Filtration to remove impurities: Centrifuge and filter the fermentation broth, take the supernatant, add 1-2 times the volume of water to dilute, then adsorb with activated carbon, and then filter through a resin column. (Step) Drying: Concentrate the modified solution and dry it with hot air to obtain hyaluronic acid powder.

[0009] Furthermore, in step 2) fermentation, aeration is carried out using an aeration disc, which is a nested disc. At the bottom of the fermentation tank, the aeration disc is connected to a pump unit and multiple atomizing pipes.

[0010] Furthermore, in step 2) fermentation, the aeration holes are 1-2 mm fine holes.

[0011] Furthermore, in step 2), the fermentation process involves timed atomization: atomization is stopped after 5-10 minutes, and atomization is repeated after an interval of 1-5 minutes.

[0012] Furthermore, in step 2), fermentation is atomized into ultrasonic atomization. Beneficial effects

[0013] By adding aeration, the ability of high-density cells to produce hyaluronic acid was successfully restored, significantly increasing the yield of hyaluronic acid and boosting hyaluronic acid fermentation production by 0.4%. The two-step fermentation process of this invention effectively solves the problems of dissolved oxygen and substrate transfer caused by high-viscosity fermentation broth by adding different carbon sources in stages, thus ensuring the smooth growth of high-density cells. This method simplifies the extraction process and reduces production costs. The two-step fermentation process provided by this patented technology is simple to operate and easy to control. During the fermentation process, by precisely controlling parameters such as carbon source concentration, fermentation time, temperature, and pH value at each stage, the stability and controllability of the fermentation process can be ensured, thereby improving the production efficiency and product quality of hyaluronic acid. The present invention employs a segmented carbon source supplementation method to address the limitations of dissolved oxygen and substrate transport during high-viscosity fermentation, induces the synthesis of hyaluronic acid synthase, and restores the ability of high-density cells to generate hyaluronic acid membranes, thereby increasing hyaluronic acid production. Detailed Implementation

[0014] To enable those skilled in the art to better understand the technical solutions in this application, the technical solutions will be fully described below in conjunction with specific embodiments of this application. Example

[0015] A method for efficient production of hyaluronic acid includes the following steps: 1) inoculation, 2) fermentation, 3) filtration and impurity removal, and 4) drying.

[0016] The specific steps are as follows: Step 1) Inoculation: Inoculate the Streptococcus ATCC39920 seed culture into the fermentation medium at an inoculation rate of 5-10% for fermentation culture. The fermentation medium consists of the following components: glucose 10-15 g / L, peptone 5-8 g / L, dipotassium hydrogen phosphate trihydrate 1-3 g / L, magnesium sulfate heptahydrate 1-1.5 g / L, potassium dihydrogen phosphate 0.5-1 g / L, dipotassium hydrogen phosphate 0.5-1 g / L, zinc sulfate heptahydrate 50-100 mg / L, agar 12-15 g / L, yeast extract powder 5-8 g / L, and water as the solvent. The pH is adjusted to 7-7.5 using sodium hydroxide solution. Step 2) Fermentation: During the initial fermentation process, aeration is carried out at a rate of 3-5 times the volume of the culture medium per hour for 0-8 hours. A 25-30 g / L glucose solution is added via atomization, a 5-15 g / L sodium chloride solution is added via atomization, and a 0.5-2 g / L hyaluronidase solution is added via atomization at regular intervals. During the later stage of fermentation, the aeration rate is 5-8 times the volume of the culture medium per hour, and a glucose solution with a concentration of 30-35 g / L is added by atomization, and a sodium chloride solution with a concentration of 1-5 g / L is added by atomization to obtain the fermentation broth. Step 3) Filtration to remove impurities: Centrifuge and filter the fermentation broth, take the supernatant, add 1-2 times the volume of water to dilute, then adsorb with activated carbon, and then filter through a resin column. (Step) Drying: Concentrate the modified solution and dry it with hot air to obtain hyaluronic acid powder.

[0017] In step 2) fermentation, aeration is carried out using an aeration disc, which is a nested disc. At the bottom of the fermentation tank, the aeration disc is connected to a pump unit and multiple atomizing pipes.

[0018] In step 2) fermentation, the aeration is carried out through fine pores of 1-2 mm.

[0019] Step 2) Fermentation, timed atomization: atomize for 5-10 minutes, stop, and atomize again after an interval of 1-5 minutes.

[0020] Step 2) involves fermentation, followed by atomization using ultrasonic atomization. Example

[0021] A method for efficient production of hyaluronic acid includes the following steps: 1) inoculation, 2) fermentation, 3) filtration and impurity removal, and 4) drying.

[0022] The specific steps are as follows: Step 1) Inoculation: Inoculate the Streptococcus ATCC39920 seed culture into the fermentation medium at an inoculation rate of 5-10% for fermentation culture. The fermentation medium consists of: 10 g / L glucose, 5 g / L peptone, 1 g / L dipotassium hydrogen phosphate trihydrate, 1 g / L magnesium sulfate heptahydrate, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L dipotassium hydrogen phosphate, 50 mg / L zinc sulfate heptahydrate, 12 g / L agar, 5 g / L yeast extract, and water as the solvent. The pH is adjusted to 7.5 using sodium hydroxide solution. Step 2) Fermentation: During the early fermentation process, aeration is carried out at a rate of 3 times the volume of the culture medium per hour for 0-8 hours. A 25 g / L glucose solution is added via atomization, a 5 g / L sodium chloride solution is added via atomization, and a 0.5 g / L hyaluronidase solution is added via atomization at regular intervals. During the later stage of fermentation, the aeration rate was 5 times the volume of the culture medium per hour, and a 30 g / L glucose solution and a 1 g / L sodium chloride solution were added by atomization from 8 to 36 hours to obtain the fermentation broth. Step 3) Filtration to remove impurities: Centrifuge and filter the fermentation broth, take the supernatant, add 2 times the volume of water to dilute, then adsorb with activated carbon, and then filter through a resin column. (Step) Drying: Concentrate the modified solution and dry it with hot air to obtain hyaluronic acid powder. Example

[0023] According to weighing, the hyaluronic acid recovery rate of the fermentation broth in Example 2 was 15.5%.

[0024] In Example 2, the atomization aeration in step 2 was replaced by titration. After weighing, the hyaluronic acid recovery rate of the fermentation broth was 15.1%.

[0025] Based on the above comparison, aeration allows the additives and fermentation liquid to mix thoroughly, increasing the product recovery rate by 0.4 percentage points.

[0026] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for efficient production of hyaluronic acid, comprising, Step 1) Inoculation, Step 2) Fermentation, Step 3) Filtration to remove impurities, Step 4) Drying.

2. The efficient hyaluronic acid production method according to claim 1, characterized in that: The specific steps are as follows: Step 1) Inoculation: Inoculate the Streptococcus ATCC39920 seed culture into the fermentation medium at an inoculation rate of 5-10% for fermentation culture. The fermentation medium consists of the following components: glucose 10-15 g / L, peptone 5-8 g / L, dipotassium hydrogen phosphate trihydrate 1-3 g / L, magnesium sulfate heptahydrate 1-1.5 g / L, potassium dihydrogen phosphate 0.5-1 g / L, dipotassium hydrogen phosphate 0.5-1 g / L, zinc sulfate heptahydrate 50-100 mg / L, agar 12-15 g / L, yeast extract powder 5-8 g / L, and water as the solvent. The pH is adjusted to 7-7.5 using sodium hydroxide solution. Step 2) Fermentation: During the initial fermentation process, aeration is carried out at a rate of 3-5 times the volume of the culture medium per hour for 0-8 hours. A 25-30 g / L glucose solution is added via atomization, a 5-15 g / L sodium chloride solution is added via atomization, and a 0.5-2 g / L hyaluronidase solution is added via atomization at regular intervals. During the later stage of fermentation, the aeration rate is 5-8 times the volume of the culture medium per hour, and a glucose solution with a concentration of 30-35 g / L is added by atomization, and a sodium chloride solution with a concentration of 1-5 g / L is added by atomization to obtain the fermentation broth. Step 3) Filtration to remove impurities: Centrifuge and filter the fermentation broth, take the supernatant, add 1-2 times the volume of water to dilute, then adsorb with activated carbon, and then filter through a resin column. (Step) Drying: Concentrate the modified solution and dry it with hot air to obtain hyaluronic acid powder.

3. The efficient hyaluronic acid production method according to claim 1, characterized in that: In step 2) fermentation, aeration is carried out using an aeration disc, which is a nested disc. At the bottom of the fermentation tank, the aeration disc is connected to a pump unit and multiple atomizing pipes.

4. The efficient hyaluronic acid production method according to claim 1, characterized in that: In step 2) fermentation, the aeration is carried out through fine pores of 1-2 mm.

5. The efficient hyaluronic acid production method according to claim 1, characterized in that: Step 2) Fermentation, timed atomization: atomize for 5-10 minutes, stop, and atomize again after an interval of 1-5 minutes.

6. The efficient hyaluronic acid production method according to claim 1, characterized in that: Step 2) involves fermentation, followed by atomization using ultrasonic atomization.

7. The efficient hyaluronic acid production method according to claim 1, characterized in that: The pH of the fermentation broth was controlled at 7.0 using NaOH, and the rotation speed was 200 r / min.