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Method for improving fermentation yield of low-molecular hyaluronic acid (HA) by adding hyaluronidase

A technology of hyaluronidase and hyaluronic acid, applied in the field of bioengineering, can solve the problems of high price and unsuitable for small molecule HA, and achieve the effect of reducing viscosity and easy purification and recovery

Inactive Publication Date: 2014-10-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current commercialized hyaluronidase (prepared from animal tissue) is expensive and not suitable for industrial production of small molecule HA

Method used

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  • Method for improving fermentation yield of low-molecular hyaluronic acid (HA) by adding hyaluronidase
  • Method for improving fermentation yield of low-molecular hyaluronic acid (HA) by adding hyaluronidase
  • Method for improving fermentation yield of low-molecular hyaluronic acid (HA) by adding hyaluronidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1 Streptococcus zooepidemicus batch fermentation (control group)

[0023] The bacterial strain used in the present invention is Streptococcus zooepidemicus WSH-24 (screened by this laboratory), the bacterial strain is first streaked on the M17 medium plate, then transferred to 50ml seed medium, cultivated at 37°C for 16h, and press 10% The inoculum was transferred to a 3L fermenter (1.5L liquid volume) and cultured for 16 hours. Fermentation conditions: rotation speed 200rpm, ventilation rate 1.0vvm, temperature 37°C, pH 7.0. Seed medium (g / L): sucrose 20, yeast powder 20, magnesium sulfate heptahydrate 2.0, manganese sulfate tetrahydrate 0.1, potassium dihydrogen phosphate 2.0, calcium carbonate 20, trace elements 1ml / L, buffer 40ml / L, pH7.0. Trace elements (g / L): calcium chloride 2.0, zinc chloride 0.046, copper sulfate pentahydrate 0.019. Buffer (g / L): disodium hydrogen phosphate 36.76, sodium dihydrogen phosphate 15.98, sodium bicarbonate 12.5. Fermen...

Embodiment 2

[0025] Example 2 Add hyaluronidase for batch fermentation in the 8th hour during the fermentation of Streptococcus zooepidemicus

[0026] All operating conditions are the same as embodiment 1, add hyaluronidase liquid 7.5ml (10 6 U / ml), the content of hyaluronidase in the fermenter is 5×10 3 U / ml. The highest output of HA reaches 4.78g / L in 14h, the result is attached figure 2 shown. After the fermentation broth is collected, boil it in boiling water for 2 minutes to inactivate the hyaluronidase.

Embodiment 3

[0027] Example 3 Add hyaluronidase for fed-batch fermentation at the 8th hour during the fermentation of Streptococcus zooepidemicus

[0028] All operating conditions are the same as embodiment 1, add hyaluronidase liquid 7.5ml (10 6 U / ml), the content of hyaluronidase in the fermenter is 5×10 3 U / ml. At the same time, sucrose feeding was carried out at the 8th hour, and 50% sucrose was fed according to the following procedure:

[0029]

[0030]

[0031] After adding enzymes and supplementing sucrose in this fermentation process, the results are as attached image 3 As shown, the OD600 value of the thalline continued to increase, and the HA output reached 7.25g / L at 20h.

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Abstract

The invention discloses a method for preparing a low-molecular hyaluronic acid (HA) by adding a hyaluronidase liquid during microbial fermentation for production of the HA, and belongs to the technical field of bioengineering. The method comprises the following steps: adding the recombinant hyaluronidase obtained from leeches in fermentation of streptococcus zooepidemicus, and carrying out fed-batch fermentation so as to prepare the low-molecular HA and oligosaccharide (the molecular weight is lower than 105 Daltons) via a fermentation coupling enzymolysis method and improve the yield of the low-molecular HA at the same time. The method provides a certain foundation for the efficient preparation of the low-molecular HA and is suitable for industrial production.

Description

technical field [0001] The invention relates to a method for preparing small molecule hyaluronic acid by adding enzymes in a fermentation process, belonging to the technical field of bioengineering. Background technique [0002] Hyaluronic acid (Hyaluronic Acid, HA) is a kind of polymer viscous polysaccharide. With its unique molecular structure and physical and chemical properties, it has good moisture retention, viscoelasticity, permeability and extensibility. It is currently found in nature. It is the substance with the best moisturizing properties, and has no immunogenicity and toxicity. It is widely used in cosmetics, food and medicine and other industries. With its unique molecular structure and physical and chemical properties, hyaluronic acid shows a variety of 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 wo...

Claims

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Application Information

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IPC IPC(8): C12P19/26
Inventor 陈坚堵国成康振李江华金鹏
Owner JIANGNAN UNIV
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