Method for improving viscosity of hyaluronic acid fermentation liquor

Abstract

The invention discloses a method for improving the viscosity of hyaluronic acid fermentation liquor, which is characterized in that the fermentation is two-stage fermentation, the initial stirring speed of a fermentation tank is set to be 60rpm, the initial ventilation capacity is set to bubble, the pH is more than or equal to 0.02 / min, the ventilation ratio is 1: 0.4 V / V.m, the stirring speed is 100rpm, the pH is more than or equal to 0.04 / min, the ventilation ratio is 1: 1.0 V / V.m, the stirring speed is 200rpm, the pH is more than or equal to 0.08 / min, the ventilation ratio is 1: 2.0 V / V.m, the stirring speed is maximum 1000rpm, the glucose content is less than or equal to 0.2 g / L, and the pH drop point is less than or equal to 0.02 / min. In the fermentation process, the ventilatory capacity and the rotating speed are adjusted and controlled while the PH is increased, compressed air can be remarkably saved, energy consumption is low, the obtained fermentation liquid is high in fermentation viscosity, and the method is particularly suitable for industrial production.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial fermentation, and particularly relates to a method for improving the viscosity of hyaluronic acid fermentation liquor. BACKGROUND

[0002] Hyaluronic acid (HA) is a naturally occurring mucopolysaccharide in the human body, which is widely distributed in tissues such as skin, joints and eyeballs, and has the effects of strong moisturizing, lubricating buffering and repairing regeneration. Because of its high safety and good biocompatibility, it is widely used in medical cosmetology, skin care products and orthopedic treatment fields.

[0003] At present, HA is mostly produced by fermentation method, but the existing technology only controls the pH value in a certain range, has high energy consumption and is difficult to control the viscosity. The application adjusts the ventilation and rotation speed for control when the pH value is improved, can save compressed air, has low energy consumption and high fermentation viscosity, and is particularly suitable for industrial production of sodium hyaluronate. SUMMARY

[0004] The purpose of the application is to overcome the problems of high energy consumption and low viscosity in the production of hyaluronic acid fermentation liquor in the prior art, and provide a method for improving the viscosity of hyaluronic acid fermentation liquor. The method can significantly save compressed air, has low energy consumption and high hyaluronic acid fermentation viscosity.

[0005] The purpose of the application is to be prepared by the following way:

[0006] A method for improving the viscosity of hyaluronic acid fermentation liquor, the method is secondary fermentation, and specifically comprises the following steps:

[0007] a) Dissolution: the seed tank is weighed with raw and auxiliary materials according to the medium formula, and is dissolved to a total volume of 80% of the tank volume. After sterilization, the circulating water is started to reduce the temperature, and the temperature is controlled at 37℃;

[0008] b) Inoculation: the seed tank is inoculated with OD: 1.3-1.5, and is inoculated with a flame inoculation amount of 0.5-1.0%, the fermentation culture temperature is controlled at 35℃, the pH value is controlled at 6.8-7.3 during the culture process, the ventilation amount is a small amount of bubbles, and the culture period is 10-14h; when the seed tank ΔpH is greater than or equal to 0.03 / min, the seed is moved to the fermentation tank for continuous culture;

[0009] c) fermentation: the fermentation tank is weighed with raw and auxiliary materials according to the medium formula, dissolved in water to a total volume of 80% of the volume of the fermentation tank, the culture temperature is 36-37℃, the process control pH is 6.8-7.3; the initial stirring of the fermentation tank is set to 60rpm, the initial aeration amount is to bubble, △PH≥0.02 / min, the ventilation ratio is 1:0.4V / V·m, the stirring is 100rpm, △pH≥0.04 / min, the ventilation ratio is 1:1.0V / V·m, the stirring is 200rpm, △pH≥0.08 / min, the ventilation ratio is 1:2.0V / V·m, the stirring is to the maximum 1000rpm, and when the glucose content is ≤0.2g / L and the pH drop point is ≤0.02 / min, the tank is discharged.

[0010] Preferably, the sterilization of step a) is to open the tank body interlayer, heat to 90℃, and then sterilize with steam for 25-30min.

[0011] Preferably, the fermentation tank culture period of step c) is 33-39h.

[0012] Preferably, the seed tank and the fermentation tank are sterilized before fermentation, cooled to 36-37℃, and then adjusted to pH 7.3-7.5 with 30-35% liquid caustic soda, respectively, for inoculation and seed transfer.

[0013] Preferably, the pH 6.8-7.3 control in steps b) and c) is achieved by using 30% NaOH solution to automatically supplement alkali, and the alkali supplementing process is mixed evenly with stirring.

[0014] Preferably, the medium formula in the seed tank is 15-20g / L of proteose peptone, 1.5-2.0g / L of yeast extract powder, 1.5-2.0g / L of sodium glutamate, 1.5-2.0g / L of potassium phosphate dibasic, 0.5-1.0g / L of magnesium sulfate, 0.1-0.5g / L of defoaming agent, and 10-15g / L of glucose. Most preferably, the medium formula in the seed tank is 17g / l of proteose peptone, 2.0g / l of yeast extract powder, 2.0g / l of sodium glutamate, 2.0g / l of potassium phosphate dibasic, 0.8g / l of magnesium sulfate, 0.4g / l of defoaming agent, and 12.0g / l of glucose.

[0015] Preferably, the medium formula in the fermentation tank is 18-20g / l of proteose peptone, 7-9g / l of yeast extract powder, 4-6g / l of sodium glutamate, 1-3g / l of potassium phosphate dibasic, 0.5-1.0g / l of magnesium sulfate, 0.1-0.5g / l of defoaming agent, and 70-90g / l of glucose. Most preferably, the medium formula in the fermentation tank is 19.8g / l of proteose peptone, 7.9g / l of yeast extract powder, 5.6g / l of sodium glutamate, 1.9g / l of potassium phosphate dibasic, 0.9g / l of magnesium sulfate, 0.4g / l of defoaming agent, and 82.6g / l of glucose.

[0016] Preferably, the volume of the fermenter is 8-10 times the volume of the seed tank.

[0017] The present application has the following advantages compared with the prior art: the present application uses specific secondary fermentation, and the effect is obviously superior to single-stage fermentation; no contamination occurs during the cultivation process, and the cultivation period is short, the viscosity of the fermentation broth is high, and the volume of the fermenter is large; at the same time, the ventilation volume and stirring speed are controlled according to the change of pH during the cultivation process in the fermenter, which can significantly save compressed air, has low energy consumption, and obtain high viscosity of the fermentation, and is particularly suitable for industrialized production of sodium hyaluronate. DETAILED DESCRIPTION

[0018] The specific embodiments of the present application are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0019] In the specific embodiments of the present application, unless otherwise specified, the reagents and materials used are conventional commercially available products in the art, which can be obtained through conventional channels.

[0020] In the specific embodiments of the present application, unless otherwise specified, the reagents and materials used are conventional commercially available products in the art, which can be obtained through conventional channels. Among them, the strain used is Streptococcus equi subsp. Zooepidemicus, which was deposited in the China General Microbiological Culture Collection Center on June 4, 2025, and the deposit number is CGMCC No. 34761.

[0021] Example 1

[0022] Secondary fermentation: 10L tank as seed tank, 100L tank as fermenter.

[0023] The medium formula is shown in Table 1

[0024] Table 1

[0025]

[0026] a) Dissolution: the seed tank is weighed according to the medium formula, and the raw and auxiliary materials are dissolved in water to a total volume of 80% of the tank volume. After opening the tank interlayer steam to warm up to 90℃, sterilize for 25-30min, open the circulating water to cool down, and control the temperature at 37℃; adjust the pH to 7.3-7.5 with 30-35% liquid alkali, and prepare the inoculum respectively.

[0027] b) Inoculation: Seed tank inoculation OD: 1.3-1.5, flame inoculation, inoculation amount: 0.5-1.0%, control fermentation culture temperature 35°C, culture process pH 6.8-7.3, aeration amount is a small amount of bubbling, culture period 10-14h; when culture to △pH≥0.03 / min, move to fermentation tank for further culture;

[0028] c) Fermentation: fermentation tank according to medium formula, weigh raw and auxiliary materials, dissolve in water to total volume of 80% of the volume of the fermentation tank, culture temperature 36-37°C, process control pH 6.8-7.3; set initial stirring of the fermentation tank 60rpm, initial aeration amount to bubbling, △PH≥0.02 / min, ventilation ratio 1:0.4V / V·m, stirring 100rpm, △pH≥0.04 / min, ventilation ratio 1:1.0V / V·m, stirring 200rpm, △pH≥0.08 / min, ventilation ratio 1:2.0V / V·m, stirring to maximum 1000rpm, when glucose content≤0.2g / L, pH drop point≤0.02 / min, discharge the tank.

[0029] The above control of pH 6.8-7.3 is automatic alkali supplement using 30% NaOH solution, during the alkali supplement process, open the stirring to mix uniformly.

[0030] During the culture process, no contamination occurred, the hyaluronic acid content in the fermentation broth determined was more than 95%.

[0031] Comparative Example 1

[0032] Single-stage fermentation: 30L as fermentation tank.

[0033] Table 2

[0034]

[0035] Fermentation method

[0036] According to the medium formula, weigh raw and auxiliary materials, dissolve in water to total volume of 80% of the volume of the sugar dissolving tank, check the airtightness of the tank body and each valve, start sterilization, first pass interlayer steam to warm up to above 90°C, then open the bottom valve, sampling valve and tank inlet valve three-way steam to warm up, maintain 121-123°C sterilization for 30min, then open the circulating water to cool down, control the temperature at 36-37°C, adopt flame inoculation of shake flask strain to start fermentation culture. The fermentation culture conditions are as follows: inoculation of seed liquid OD600=1.3-1.5, flame inoculation, inoculation amount: 0.5%, control culture temperature 36-37°C, culture process pH control 7.00, aeration amount 1:1.2-1.5.

[0037] Example 2 Two-stage fermentation step is the same as Example 1, except that pH and fermentation period are shown in Table 3.

[0038] Comparative Example 2-3 Single-stage fermentation step same as Comparative Example 1, except for pH and fermentation period as shown in Table 3.

[0039] Comparative Example 4-5 In the two-stage fermentation process, the aeration amount and stirring speed were not adjusted.

[0040] Comparative Example 6-8 Two-stage fermentation step same as Example 1, except for pH and fermentation period as shown in Table 3.

[0041] Table 3

[0042] Fermentation batch Stage Fermentation period pH Reducing sugar Viscosity at tank discharge (mPa-S) Example 1 Two-stage 33h 7.12 ≤100 mg / dL 77478 Example 2 Two-stage 39h 6.83 ≤100 mg / dL 99186 Comparative Example 1 Single-stage 58h 7.00 ≤100 mg / dL 33909 Comparative Example 2 Single-stage 31h 7.20 ≤100 mg / dL 51152 Comparative Example 3 Single-stage 28h 7.02 0 mg / dL 48000 Comparative Example 4 Two-stage 64h 7.16 ≤100 mg / dL 43541 Comparative Example 5 Two-stage 51h 7.13 ≤100 mg / dL 42868 Comparative Example 6 Two-stage 39h 7.39 ≤100 mg / dL 55950 Comparative Example 7 Two-stage 31h 7.80 ≤100 mg / dL 62808 Comparative Example 8 Two-stage 38h 7.39 ≤100 mg / dL 88808

[0043] Analysis of fermentation results:

[0044] (1) The cultivation period of Example 1 is less than 35 h, and the seed tank expansion effect is ideal. After being transferred to the fermentation tank, the high-density bacterial balls can be observed in the field of view by process sampling microscopy, and the growth rate and viscosity of the fermentation broth increase rapidly;

[0045] (2) In single-stage fermentation, the highest viscosity after discharge is 51152 mPa·S, and the average viscosity is 44353 mPa·S. In Comparative Example 1, the cultivation period is longer, and the viscosity of the fermentation broth after discharge is lower. In the early stage of cultivation, when the pH gradually increases, the aeration amount and stirring speed of the tank cannot be increased in time, which cannot meet the oxygen demand of the large number of bacterial colonies, and the best growth period of the bacterial colonies is missed. The growth and reproduction speed slows down in the later stage, and the viscosity increase rate decreases;

[0046] (3) In two-stage fermentation, the highest viscosity after discharge can reach 99186 mPa·S, and the average viscosity is 67234 mPa·S. The cultivation period of Comparative Example 4 and Comparative Example 5 is more than 50 h, and the viscosity after discharge is less than 50000 mPa·S. The fermentation period is too long, the strain growth enters the decline phase, and the bacterial autolysis occurs. The viscosity of the fermentation broth after discharge of Comparative Example 6 is only 55950 mPa·S. The change of pH in this batch of fermentation process is relatively slow, the bacterial ball density is relatively low by process sampling microscopy, and the viscosity increase rate of the fermentation broth is small.

Claims

1. A method for increasing the viscosity of hyaluronic acid fermentation broth, wherein the method is a two-stage fermentation, specifically including the following steps: a) Dissolving: Weigh the raw materials and auxiliary materials according to the culture medium formula in the seed tank, add water to dissolve them until the total volume is 80% of the tank volume, sterilize and then cool to 37℃; b) Inoculation: The OD of the seed tank is 1.3-1.5, and flame inoculation is used. The inoculation amount is 0.5-1.0%. The fermentation temperature is controlled at 35℃, the pH is 6.8-7.3 during the culture process, the aeration rate is small bubbles, and the culture cycle is 10-14h. When the ΔpH of the seed tank reaches ≥0.03 / min, the seed tank is transferred to the fermenter for further culture. c) Fermentation: Weigh the raw materials and auxiliary materials according to the culture medium formula in the fermenter, add water to dissolve them to a total volume of 80% of the fermenter volume, and culture at 36-37℃. Control the pH at 6.8-7.3 during the process. Set the initial stirring speed in the fermenter to 60 rpm, the initial aeration rate to bubble, ΔpH≥0.02 / min, and the ventilation ratio to 1:0.4V / V·m. Stir at 100 rpm, ΔpH≥0.04 / min, and the ventilation ratio to 1:1.0V / V·m. Stir at 200 rpm, ΔpH≥0.08 / min, and the ventilation ratio to 1:2.0V / V·m. Stir to a maximum of 1000 rpm until the glucose content is ≤0.2g / L and the pH drop point is ≤0.02 / min, then discharge the fermenter.

2. The method for increasing the viscosity of hyaluronic acid fermentation broth according to claim 1, characterized in that, The sterilization process described in step a) involves heating the tank jacket to 90°C with steam and then sterilizing with steam for 25-30 minutes.

3. The method for increasing the viscosity of hyaluronic acid fermentation broth according to claim 1, characterized in that, Before fermentation, the seed tank and fermentation tank are sterilized and cooled to 36-37°C, and then the pH is adjusted to 7.3-7.5 with 30-35% alkali solution to prepare for inoculation and transplanting, respectively.

4. The fermentation method for reducing energy consumption and sodium hyaluronate content according to claim 1, characterized in that, In steps b) and c), the pH is controlled at 6.8-7.3 by automatically replenishing the alkali with a 30% NaOH solution, and stirring is turned on during the alkali replenishment process to ensure uniform mixing.

5. The method for increasing the viscosity of hyaluronic acid fermentation broth according to claim 1, characterized in that, The culture medium formula in the seed tank is as follows: peptone 15-20 g / L, yeast extract 1.5-2.0 g / L, monosodium glutamate 1.5-2.0 g / L, dipotassium hydrogen phosphate 1.5-2.0 g / L, magnesium sulfate 0.5-1.0 g / L, antifoaming agent 0.1-0.5 g / L, and glucose 10-15 g / L.

6. The method for increasing the viscosity of hyaluronic acid fermentation broth according to claim 1, characterized in that, The culture medium formula in the fermenter is as follows: peptone 18-20 g / L, yeast extract 7-9 g / L, sodium glutamate 4-6 g / L, dipotassium hydrogen phosphate 1-3 g / L, magnesium sulfate 0.5-1.0 g / L, defoamer 0.1-0.5 g / L, and glucose 70-90 g / L.

7. The method for increasing the viscosity of hyaluronic acid fermentation broth according to claim 1, characterized in that, The volume of the fermentation tank is 8-10 times that of the seed tank.

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