Method for improving fermentation yield of hyaluronic acid

Abstract

The invention belongs to the technical field of microbial fermentation, and provides a method for improving the fermentation yield of hyaluronic acid, starch sugar and hyaluronidase liquid are fed in different fermentation stages, on one hand, the fed-batch sugar can prevent rapid viscosity generation in the fermentation process, and the condition of strain inactivation caused by uneven distribution of alkali liquor due to too high viscosity is reduced; on the other hand, hyaluronic acid molecular chains are split by hyaluronidase, the viscosity of hyaluronic acid fermentation liquor is reduced, the mass transfer effect of materials in the fermentation process is improved, and therefore the yield is increased. The method is high in yield and conversion rate of the hyaluronic acid fermentation liquor, simple in process and suitable for large-scale production and application.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for improving the fermentation yield of hyaluronic acid. Background Art

[0002] Hyaluronic acid (HA) is a high molecular weight viscous polysaccharide. With its unique molecular structure and physical and chemical properties, it has good moisturizing, viscoelasticity, permeability and ductility. It is the best moisturizing substance found in nature. It is also non-immunogenic and non-toxic. It is widely used in cosmetics, food, medicine and other industries. With its unique molecular structure and physical and chemical properties, HA has many important physiological functions in the body, such as lubricating joints, regulating the permeability of blood vessel walls, regulating protein, water and electrolyte diffusion and operation, and promoting wound healing.

[0003] Due to the physiological and metabolic characteristics of the hyaluronic acid production strain itself, the increase in the production of sodium hyaluronate is restricted by many factors, such as: high fermentation viscosity, low mass transfer and oxygen transfer, competition between bacterial growth and hyaluronic acid biosynthesis for carbon sources and energy, etc. At present, the fermentation yield of hyaluronic acid is generally 12-13g / L, which is relatively low. Therefore, how to increase the yield of hyaluronic acid produced by microbial fermentation and reduce production costs has become an urgent issue of concern. Summary of the invention

[0004] The purpose of the present invention is to provide a method for improving the fermentation yield of hyaluronic acid. The method of the present invention can significantly improve the yield of hyaluronic acid.

[0005] The technical solution of the present invention is achieved through the following technical solution.

[0006] A method for improving the yield of hyaluronic acid fermentation, characterized in that starch sugar and hyaluronidase solution are added at different fermentation stages to reduce the viscosity of the fermentation liquid, improve the mass transfer effect of materials in the fermentation process, and thus improve the yield.

[0007] Preferably, hyaluronidase is added for the first time when the hyaluronic acid fermentation liquid just produces viscosity, and the viscosity is 50-200 mpas. The first enzyme addition amount is 0.1-0.2 mL hyaluronidase per 100 L fermentation liquid.

[0008] Preferably, when the viscosity of the hyaluronic acid fermentation liquid reaches 2000-3000 mpas, hyaluronidase is added for the second time, and the amount of the second enzyme addition is 0.1-0.2 mL of hyaluronidase per 100 L of fermentation liquid.

[0009] Preferably, when the viscosity of the hyaluronic acid fermentation liquid reaches 5000-6000 mpas, hyaluronidase is added for the third time, and the amount of enzyme added for the third time is 0.1-0.2 mL hyaluronidase per 100 L fermentation liquid.

[0010] Preferably, the base sugar concentration is controlled at 2.5-3.0 g / L by adding starch sugar 8 h before fermentation.

[0011] Preferably, during fermentation for 8-16 hours, the residual sugar concentration is controlled at 1.5-2.0 g / L by adding starch sugar.

[0012] Preferably, the amount of starch sugar added is gradually reduced during fermentation for 16-24 hours, and the residual sugar is maintained at 1.0-1.5 g / L.

[0013] Preferably, the residual sugar is maintained at 0.5-1.0 g / L during fermentation for 24-32 hours, and the residual sugar is controlled to below 0.5 g / L at the end of fermentation.

[0014] Preferably, the enzyme activity of the hyaluronidase is 1500 U / ml; the total fermentation time is 32-36 h; and the starch sugar is sucrose.

[0015] Specifically, the steps include: The seed liquid of Streptococcus zooepidemicus ATCC39920 was inoculated into the fermentation medium at an inoculum rate of 10% for fermentation culture; the components of the fermentation medium were: sucrose 70 g / L, peptone 2.0 g / L, magnesium sulfate heptahydrate 1.2 g / L, potassium dihydrogen phosphate 1 g / L, potassium hydrogen phosphate 1 g / L, zinc sulfate heptahydrate 100 mg / L, pH 7.0; When the hyaluronic acid fermentation liquid just produces viscosity, when the viscosity is 50-200mpas, add hyaluronidase for the first time, and the first enzyme addition amount is 0.1-0.2mL / 100L fermentation liquid; when the viscosity of the hyaluronic acid fermentation liquid reaches 2000-3000mpas, add hyaluronidase for the second time, and the second enzyme addition amount is 0.1-0.2mL / 100L fermentation liquid. When the viscosity of the hyaluronic acid fermentation liquid reaches 6000mpas, add hyaluronidase for the third time, and the third enzyme addition amount is 0.1-0.2mL / 100L fermentation liquid.

[0016] In the first 8 hours of fermentation, the base sugar concentration is controlled to 2-3g / L by adding sugar continuously; when fermenting for 8-16 hours, the residual sugar is controlled to 1-2g / L by adding sugar continuously; when fermenting for 16-24 hours, the residual sugar is controlled to be maintained at 1.0-1.5g / L; when fermenting for 24-32 hours, the residual sugar is maintained at 0.5g / L. The total fermentation time is 32-36 hours, and the residual sugar is controlled to below 0.5g / L at the end of fermentation.

[0017] The beneficial effects achieved by the present invention mainly include but are not limited to the following aspects: The present invention can significantly increase the yield of hyaluronic acid, with the yield of hyaluronic acid fermentation liquid reaching 19.5 g / L and the conversion rate reaching 23%.

[0018] The present invention controls the viscosity of the hyaluronic acid fermentation liquid by adding sugar in a flow manner, thereby preventing the rapid generation of viscosity during the fermentation process; the high-concentration alkali solution inactivates the seeds, and the present invention reduces the situation in which the seed activity is reduced due to the uneven distribution of the alkali solution due to excessively high viscosity.

[0019] (3) The present invention utilizes the characteristics of hyaluronidase to cleave the hyaluronic acid molecular chain, thereby reducing the viscosity of hyaluronic acid and improving the mass transfer effect between materials, thereby achieving the purpose of increasing production.

[0020] (4) The present invention can control the molecular weight of hyaluronic acid between 60-100 WDa by controlling the amount and frequency of adding hyaluronidase.

[0021] (5) The process control of the present invention is simple and suitable for large-scale production and application. DETAILED DESCRIPTION

[0022] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The products and methods of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the products and methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention. In order to further understand the present invention, the present invention is described in detail below in conjunction with embodiments.

[0023] Example 1 A method for improving the fermentation yield of hyaluronic acid, comprising the following steps: The seed liquid of Streptococcus zooepidemicus ATCC39920 (OD600 is about 16) is inoculated into the fermentation medium at an inoculum of 10% for fermentation culture; the components of the fermentation medium are: sucrose 70g / L, peptone 2.0g / L, magnesium sulfate heptahydrate 1.2g / L, potassium dihydrogen phosphate 1g / L, potassium hydrogen phosphate 1g / L, zinc sulfate heptahydrate 100mg / L, pH 7.0; When the hyaluronic acid fermentation liquid just produces viscosity and the viscosity is 50mpas, add hyaluronidase (1500U / ml) for the first time, and the first enzyme addition amount is 0.15mL / 100L fermentation liquid. When the viscosity of the hyaluronic acid fermentation liquid reaches 3000mpas, add hyaluronidase for the second time, and the second enzyme addition amount is 0.15mL / 100L fermentation liquid. When the viscosity of the hyaluronic acid fermentation liquid reaches 6000mpas, add hyaluronidase for the third time, and the third enzyme addition amount is 0.15mL / 100L fermentation liquid.

[0024] In the first 8 hours of fermentation, the base sugar concentration is controlled to 2.5g / L by adding sugar. During the 8-16 hours of fermentation, the residual sugar is controlled to 1.5g / L by adding sugar. During the 16-24 hours of fermentation, the amount of added sugar is gradually reduced, and the residual sugar is maintained at 1.0g / L. During the 24-32 hours of fermentation, the residual sugar is maintained at 0.5g / L, the total fermentation time is 36h, and the residual sugar is controlled to below 0.5g / L at the end of the final fermentation. The sugar used in the above-mentioned addition is sucrose.

[0025] Under these conditions, the yield of hyaluronic acid fermentation broth reached 19.5 g / L, the conversion rate reached 23%, and the molecular weight of hyaluronic acid was 60-100 WDa.

[0026] Example 2 A method for improving the fermentation yield of hyaluronic acid, comprising the following steps: The seed liquid of Streptococcus zooepidemicus ATCC39920 (OD600 is about 16) is inoculated into the fermentation medium at an inoculum of 10% for fermentation culture; the components of the fermentation medium are: sucrose 70g / L, peptone 2.0g / L, magnesium sulfate heptahydrate 1.2g / L, potassium dihydrogen phosphate 1g / L, potassium hydrogen phosphate 1g / L, zinc sulfate heptahydrate 100mg / L, pH 7.0; When the hyaluronic acid fermentation liquid just produces viscosity and the viscosity is 100mpas, add hyaluronidase for the first time, and the first enzyme addition amount is 0.2mL / 100L fermentation liquid. When the viscosity of the hyaluronic acid fermentation liquid reaches 3000mpas, add hyaluronidase for the second time, and the second enzyme addition amount is 0.2mL / 100L fermentation liquid. When the viscosity of the hyaluronic acid fermentation liquid reaches 6000mpas, add hyaluronidase for the third time, and the third enzyme addition amount is 0.2mL / 100L fermentation liquid.

[0027] In the first 8 hours of fermentation, the base sugar concentration was controlled to 3g / L by adding sugar. During the 8-16 hours of fermentation, the residual sugar was controlled to 2g / L by adding sugar. During the 16-24 hours of fermentation, the amount of added sugar was gradually reduced, and the residual sugar was maintained at 1.5g / L. During the 24-32 hours of fermentation, the residual sugar was maintained at 0.5g / L, and the total fermentation time was 36 hours. At the end of the fermentation, the residual sugar was controlled to below 0.5g / L.

[0028] Under these conditions, the yield of hyaluronic acid fermentation broth reached 17.6 g / L, the conversion rate reached 20.9%, and the molecular weight of hyaluronic acid was between 60-100 WDa.

[0029] Example 3 A method for improving the fermentation yield of hyaluronic acid, comprising the following steps: The seed liquid of Streptococcus zooepidemicus ATCC39920 (OD600 is about 16) is inoculated into the fermentation medium at an inoculum of 10% for fermentation culture; the components of the fermentation medium are: sucrose 70g / L, peptone 2.0g / L, magnesium sulfate heptahydrate 1.2g / L, potassium dihydrogen phosphate 1g / L, potassium hydrogen phosphate 1g / L, zinc sulfate heptahydrate 100mg / L, pH 7.0; When the hyaluronic acid fermentation liquid just produces viscosity and the viscosity is 60mpas, add hyaluronidase for the first time, and the first enzyme addition amount is 0.15mL / 100L fermentation liquid. When the viscosity of the hyaluronic acid fermentation liquid reaches 2500mpas, add hyaluronidase for the second time, and the second enzyme addition amount is 0.15mL / 100L fermentation liquid. When the viscosity of the hyaluronic acid fermentation liquid reaches 5000mpas, add hyaluronidase for the third time, and the third enzyme addition amount is 0.15mL / 100L fermentation liquid.

[0030] In the first 8 hours of fermentation, the base sugar concentration was controlled to 3g / L by adding sugar. During the 8-16 hours of fermentation, the residual sugar was controlled to 1.5g / L by adding sugar. During the 16-24 hours of fermentation, the amount of added sugar was gradually reduced, and the residual sugar was maintained at 1.0g / L. During the 24-32 hours of fermentation, the residual sugar was maintained at 0.5g / L, and the total fermentation time was 36 hours. At the end of the fermentation, the residual sugar was controlled to below 0.5g / L.

[0031] Under these conditions, the yield of hyaluronic acid fermentation broth reached 19.5 g / L, the conversion rate reached 23.4%, and the molecular weight of hyaluronic acid was between 60-100 WDa.

[0032] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for improving the fermentation yield of hyaluronic acid, characterized in that: Starch sugar and hyaluronidase solution are added at different fermentation stages to reduce the viscosity of the fermentation liquid, improve the mass transfer effect of materials in the fermentation process, and thus increase the yield.

2. A method for improving the fermentation yield of hyaluronic acid according to claim 1, characterized in that: When the hyaluronic acid fermentation liquid just starts to produce viscosity, and the viscosity is between 50-200mpas, hyaluronidase is added for the first time. The first enzyme addition amount is 0.1-0.2mL hyaluronidase per 100L fermentation liquid.

3. A method for improving the fermentation yield of hyaluronic acid according to claim 1, characterized in that: When the viscosity of the hyaluronic acid fermentation liquid reaches 2000-3000mpas, hyaluronidase is added for the second time, and the amount of enzyme added for the second time is 0.1-0.2mL hyaluronidase per 100L fermentation liquid.

4. A method for improving the fermentation yield of hyaluronic acid according to claim 1, characterized in that: When the viscosity of the hyaluronic acid fermentation liquid reaches 5000-6000mpas, hyaluronidase is added for the third time, and the amount of enzyme added for the third time is 0.1-0.2mL hyaluronidase per 100L fermentation liquid.

5. A method for improving the fermentation yield of hyaluronic acid according to claim 1, characterized in that: 8h before fermentation, the base sugar concentration was controlled at 2.5-3.0g / L by adding starch sugar.

6. A method for improving the fermentation yield of hyaluronic acid according to claim 1, characterized in that: When fermenting for 8-16 hours, the residual sugar concentration is controlled at 1.5-2.0 g / L by adding starch sugar.

7. A method for improving the fermentation yield of hyaluronic acid according to claim 1, characterized in that: During the fermentation period of 16-24 hours, the amount of starch sugar added was gradually reduced to maintain the residual sugar at 1.0-1.5 g / L.

8. A method for improving the fermentation yield of hyaluronic acid according to claim 1, characterized in that: During the fermentation for 24-32 hours, the residual sugar was maintained at 0.5-1.0g / L, and at the end of the fermentation, the residual sugar was controlled to below 0.5g / L.

9. A method for improving the fermentation yield of hyaluronic acid according to any one of claims 1 to 8, characterized in that: The enzyme activity of the hyaluronidase is 1500U / ml; the total fermentation time is 32-36h; and the starch sugar is sucrose.

10. A method for improving the fermentation yield of hyaluronic acid according to any one of claims 1 to 9, characterized in that: The specific steps include: The seed liquid of Streptococcus zooepidemicus ATCC39920 was inoculated into the fermentation medium at an inoculum rate of 10% for fermentation culture; the components of the fermentation medium were: sucrose 70 g / L, peptone 2.0 g / L, magnesium sulfate heptahydrate 1.2 g / L, potassium dihydrogen phosphate 1 g / L, potassium hydrogen phosphate 1 g / L, zinc sulfate heptahydrate 100 mg / L, pH 7.0; When the hyaluronic acid fermentation liquid just produces viscosity, when the viscosity is 50-200mpas, add hyaluronidase for the first time, and the first enzyme addition amount is 0.1-0.2mL / 100L fermentation liquid; when the viscosity of the hyaluronic acid fermentation liquid reaches 2000-3000mpas, add hyaluronidase for the second time, and the second enzyme addition amount is 0.1-0.2mL / 100L fermentation liquid. When the viscosity of the hyaluronic acid fermentation liquid reaches 6000mpas, add hyaluronidase for the third time, and the third enzyme addition amount is 0.1-0.2mL / 100L fermentation liquid; In the first 8 hours of fermentation, the base sugar concentration is controlled to 2-3g / L by adding sugar continuously; when fermenting for 8-16 hours, the residual sugar is controlled to 1-2g / L by adding sugar continuously; when fermenting for 16-24 hours, the residual sugar is controlled to be maintained at 1.0-1.5g / L; when fermenting for 24-32 hours, the residual sugar is maintained at 0.5g / L. The total fermentation time is 32-36 hours, and the residual sugar is controlled to below 0.5g / L at the end of fermentation.