Method for producing hyaluronic acid

Abstract

The present invention provides a method for producing hyaluronic acid. The method includes fermenting Streptococcus zooepidemicus to obtain hyaluronic acid, wherein the fermentation is carried out under the following conditions: within 0 to 6 hours after the start of the fermentation, the fermentation is carried out at a pH of 6.7 to 7.2; within 6 to 12 hours after the start of the fermentation, the fermentation is carried out at a pH of 7.4 to 7.6; after 12 hours from the start of the fermentation, the fermentation is carried out at a pH of 6.5 to 9.0. Compared with the prior art, the yield of hyaluronic acid is significantly increased by this method.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically, the present invention relates to a method for producing hyaluronic acid. Background Art

[0002] Hyaluronic acid (hyaluronic acid or hyaluronan, HA) is a polysaccharide composed of disaccharide repeating units of glucuronic acid and glucosamine, and is widely distributed in cartilage tissue, synovial fluid, and the dermis and epidermis of skin tissue, where it plays physiological roles such as moisturizing, nourishing, repairing, and preventing damage. The production methods of hyaluronic acid include animal tissue extraction method and fermentation method. Due to the high preparation cost and complex separation and purification of the animal tissue extraction method, it has gradually been replaced by the fermentation method.

[0003] Fermentation yield is the key to cost control in the production of hyaluronic acid by fermentation method. During the production of hyaluronic acid by microbial fermentation, pH is an important environmental parameter for cell growth and product synthesis, and is a comprehensive index of metabolic activities. Most of the pH regulations reported in current patent literatures for hyaluronic acid fermentation processes are constant pH control. For example, in a method for preparing hyaluronic acid disclosed in CN101649337A, the entire fermentation process is at a constant pH of 7.0. However, the optimal pH for the growth of hyaluronic acid bacteria is often not the best pH value for product synthesis, and constant pH is not conducive to cell growth or product accumulation. There are also patent reports on technical solutions for variable pH regulation. For example, in a method for increasing the yield of hyaluronic acid in fermentation production by an intermittent high pH stress strategy disclosed in patent CN 1978658 A, the yield of hyaluronic acid can be increased from 5.0 g / L to 6.7 g / L. However, this technical solution has cumbersome process control and is not conducive to scale-up production, and maintaining a high pH for a long time in this technical solution will affect the viability of bacteria, and to a large extent will affect the growth of bacteria and is not conducive to the synthesis of hyaluronic acid.

[0004] Therefore, finding a pH regulation that can improve product synthesis without affecting the viability of bacteria is the key to the control of hyaluronic acid fermentation process. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems in the related technologies to some extent.

[0006] To this end, in the first aspect of the present invention, the present invention proposes a method for producing hyaluronic acid. According to an embodiment of the present invention, the method includes fermenting Streptococcus zooepidemicus to obtain hyaluronic acid, wherein the fermentation treatment is carried out under the following conditions: within 0 to 6 hours after the start of the fermentation treatment, the fermentation treatment is carried out under the condition that the pH is 6.7 to 7.2; within 6 to 12 hours after the start of the fermentation treatment, the fermentation treatment is carried out under the condition that the pH is 7.4 to 7.6; after 12 hours from the start of the fermentation treatment, the fermentation treatment is carried out under the condition that the pH is 6.5 to 9.0.

[0007] According to the method for producing hyaluronic acid according to the embodiment of the present invention, during the early stage of fermentation, the pH is reasonably regulated according to the growth characteristics of the bacterial cells and the secretion characteristics of hyaluronic acid, which can ensure the best growth vitality of the bacterial cells. In the initial stage of fermentation (0 to 6 hours), according to the growth characteristics of Streptococcus zooepidemicus bacterial cells, the optimal pH suitable for the growth of the bacterial cells is selected, which is beneficial for the bacterial cells to adapt to the environment and grow rapidly; in the middle stage of fermentation (6 to 12 hours), according to the growth and product secretion characteristics of the bacterial cells, controlling the pH to be 7.4 to 7.6 helps to prevent the actual pH of the fermentation broth from being too low and affecting the growth of the bacterial cells; controlling the fermentation to be carried out under the condition that the pH is 6.5 to 9.0 in the middle and late stages of fermentation can, on the premise of ensuring that the activity of the bacterial cells is not affected, help to reduce the production of by-products and promote the secretion of hyaluronic acid by the bacterial cells. According to the method of the embodiment of the present invention, compared with the prior art, the yield of hyaluronic acid is significantly increased.

[0008] According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

[0009] According to an embodiment of the present invention, within 6 to 12 hours after the start of the fermentation treatment, first increase the pH by 0.1 to 0.2 every 1 hour. After the pH rises to 7.4 to 7.6, control the fermentation treatment to be carried out under the condition that the pH is 7.4 to 7.6. The inventors found that in the middle stage of fermentation (6 to 12 hours), hyaluronic acid accumulates slowly, the viscosity of the fermentation broth gradually increases, and the pH electrode detection of the fermentation tank will show the phenomenon of lag in the feedback of the detection value. By slowly increasing the pH and increasing the pH by 0.1 to 0.2 every 1 hour, on the one hand, it helps to prevent the actual pH of the fermentation broth from being too low and affecting the growth of the bacterial cells, and on the other hand, it avoids the impact of rapidly increasing the pH on the vitality of the bacterial cells.

[0010] According to an embodiment of the present invention, within 12 to 30 hours after the start of fermentation treatment, the pH maintains a continuously fluctuating state up and down, the lower limit of the fluctuating pH is 6.5 to 7.0, and the upper limit of the pH is 8.0 to 9.0. The inventors found that within 12 to 30 hours in the middle and late stages of fermentation, adopting a pH volatility regulation strategy can take into account the growth of bacteria and product synthesis, and bring the fermentation synthesis of hyaluronic acid into the best state. The pH volatility control is an automatic feedback regulation based on the viability of the bacteria. Since the viability of the bacteria varies in different periods of the fermentation process, the cycle of pH fluctuation can be changed accordingly. For example, when the viability of the bacteria is the highest in the middle stage of fermentation, the pH fluctuation cycle is the shortest, and then as the viability of the bacteria weakens, the corresponding pH fluctuation cycle becomes longer. Automatically adjusting according to the viability of the bacteria is beneficial to reducing the production of by-products and promoting the secretion of hyaluronic acid by the bacterial cells without affecting the activity of the bacterial cells.

[0011] According to an embodiment of the present invention, within 0 to 6 hours after the start of fermentation treatment, first, the pH of the fermentation medium is allowed to naturally drop to 6.7 to 7.2, and then the fermentation treatment is controlled to be carried out under the condition that the pH is 6.7 to 7.2.

[0012] According to an embodiment of the present invention, the fermentation treatment is controlled to be carried out under the condition that the pH is 6.7 to 7.2 by adding alkali. The inventors found that during the processes of bacterial growth and synthetic metabolism, organic acids such as acetic acid, lactic acid, and pyruvic acid are continuously secreted, which will cause the pH of the fermentation broth to drop. Therefore, the pH of the fermentation broth is controlled by adding alkali to make the pH stable at 6.7 to 7.2.

[0013] According to an embodiment of the present invention, the alkali added is ammonia water, a NaOH or KOH solution with a concentration of 20 to 30%.

[0014] According to an embodiment of the present invention, the fermentation treatment is carried out for 29 to 31 hours under the conditions of an aeration rate of 0.5 to 1 vvm, a rotation speed of 100 to 600 rpm, and 37°C.

[0015] According to an embodiment of the present invention, the fermentation medium for the fermentation treatment includes 77 g / L of glucose, 40 g / L of peptone, 1.3 g / L of KH 2 PO 4 4, 0.4 g / L of MgSO 4 and 4 g / L of monosodium glutamate.

[0016] According to an embodiment of the present invention, the classification and naming of the Streptococcus zooepidemicus is Streptococcus equisubsp. zooepidemicus HEC-SE01, Streptococcus equi subsp. zooepidemicus HEC-SE01, which is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M2020231, the deposit date being June 22, 2020, and the deposit address being: Wuhan University, Bayi Road, Hongshan District, Wuhan City, Hubei Province, China. Furthermore, according to the method of the embodiment of the present invention, the yield of hyaluronic acid is further improved.

[0017] In the second aspect of the present invention, the present invention proposes a method for producing hyaluronic acid. According to an embodiment of the present invention, the method includes: inoculating the Streptococcus zooepidemicus strain into a seed medium at a volume ratio of 1%, culturing at 36.5 °C and 200 - 220 rpm for 17.5 - 18 h. Before the end of the culture, Gram staining microscopy is performed. After no abnormalities are found, it is inoculated into the fermentation medium. In a 15 L fermenter, the aeration rate is 0.5 - 1 vvm, the stirring speed is 100 - 600 rpm, and fermentation culture is carried out at 37 °C for 30 h. Among them, the seed medium includes 10 g / L of glucose, 15 g / L of peptone, 5 g / L of yeast extract powder, 1.5 g / L of K 2 HPO 4 , 0.35 g / L of MgSO 4 , and the pH is 6.5; during the fermentation process, the following pH control method is adopted for the fermenter: In the early stage of fermentation (0 - 6 h): After inoculation, when the pH of the fermentation broth naturally drops to 6.7 - 7.2, 20 - 30% concentration of NaOH is used for alkali control to maintain the pH of the fermentation broth at 6.7 - 7.2; In the middle stage of fermentation (6 - 12 h): Starting from 6 h, the pH is increased by 0.1 - 0.2 every 1 h. When the pH rises to 7.4 - 7.6, the pH is constantly controlled at 7.4 - 7.6; In the middle and late stages of fermentation (12 - 30 h): By changing the parameters of the alkali control automatic control program, the constant rate of the peristaltic pump, and the opening time of the solenoid valve, the pH is maintained in a continuously fluctuating state. The lower limit of the pH fluctuation is 6.5 - 7.0, and the upper limit is 8.0 - 9.0 until the end of fermentation. According to the method of the embodiment of the present invention, compared with the prior art, the yield of hyaluronic acid is significantly increased.

[0018] The additional aspects and advantages of the present invention will be partially given in the following description, partially become obvious from the following description, or be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The above and / or additional aspects and advantages of the present invention will become obvious and easy to understand from the description of the embodiments in conjunction with the following drawings, where:

[0020] Figure 1 is the pH control curve according to Embodiment 2 of the present invention;

[0021] Figure 2 is the pH control curve according to Comparative Example 1 of the present invention; and

[0022] Figure 3 is the pH control curve according to Comparative Example 3 of the present invention. Detailed Description of the Invention

[0023] Embodiments of the present invention will be described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below by reference to the drawings are exemplary and are intended to explain the present invention, and should not be construed as limiting the present invention.

[0024] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include at least one of such features. In the description of the present invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0025] According to an embodiment of the present invention, the pH regulation method in the method for producing hyaluronic acid proposed by the present invention is specifically as follows:

[0026] 1) In the early stage of fermentation (0 - 6 h): After inoculation, the pH of the fermentation broth naturally drops to 6.7 - 7.2, and then the pH of the fermentation broth is maintained at 6.7 - 7.2 by alkali control;

[0027] 2) In the middle stage of fermentation (6 - 12 h), starting from 6 h, the pH is increased by 0.1 - 0.2 every 1 h. When the pH rises to 7.4 - 7.6, the pH is constantly controlled at 7.4 - 7.6;

[0028] 3) In the middle and late stages of fermentation (12 - 30 h), by changing the parameters of the alkali control and self-control program, the constant rate of the peristaltic pump, and the opening time of the solenoid valve, the pH during the fermentation process is maintained in a continuously fluctuating state. The lower limit of the fluctuation is set to 6.5 - 7.0, and the upper limit is set to 8.0 - 9.0 until the end of fermentation.

[0029] The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0030] The strain used in the following examples or comparative examples is Streptococcus equi subsp. zooepidemicus, with the preservation number of CCTCC NO: M2020231 and the preservation date of June 22, 2020, which is referred to as Streptococcus equisubsp. zooepidemicus HEC-SE01 in the embodiments of the present application. However, it is by no means limited to this Streptococcus equi subsp. zooepidemicus.

[0031] Seed culture medium (g / L): Glucose 10, peptone 15, yeast extract powder 5, K 2 HPO 4 1.5, MgSO 4 0.35, pH 6.5;

[0032] Fermentation medium (g / L): Glucose 77, peptone 40, KH 2 PO 4 1.3, MgSO 4 0.4, monosodium glutamate 4.

[0033] Example 1

[0034] Take out the glycerol tube strain from the ultra-low temperature refrigerator, melt it in a 37°C water bath or in the palm of the hand, inoculate it into the seed culture medium at a volume ratio of 1%, and culture it at 36.5°C with 200 - 220 rpm for 17.5 - 18 h. Before the end of the culture, conduct Gram staining microscopy. After no abnormalities are found, inoculate a 15 L fermenter with an aeration rate of 0.5 - 1 vvm and stirring at 100 - 600 rpm, and ferment and culture at 37°C for 30 h.

[0035] The following pH control strategy is adopted for the fermenter:

[0036] Early stage of fermentation (0 - 6 h): After inoculation, when the pH of the fermentation broth naturally drops to 6.7, control the pH of the fermentation broth at 6.7 with 20 - 30% concentration of NaOH. In the middle stage of fermentation (6 - 12 h), starting from 6 h, increase the pH by 0.1 every 1 h. When the pH rises to 7.4, keep the pH constant at 7.4. In the middle and late stages of fermentation (12 - 30 h), maintain the pH of the fermentation process in a continuous fluctuating state by changing the parameters of the alkali control automatic control program, the constant rate of the peristaltic pump, and the opening time of the solenoid valve (the specific operation is as follows: ① There are two parameters in the alkali control program. One is the "cycle", which refers to the time interval from when the alkali control system opens the peristaltic pump or solenoid valve to the next opening (provided that at the end of each cycle, the actual measured pH value is lower than the automatic control value); the other is the "maximum time", which refers to the maximum time for controlling the opening of the peristaltic pump or solenoid valve within a cycle according to the difference between the actually measured pH value and the set pH value at the start of each new cycle (not exceeding the cycle time). The larger the maximum time setting value, the corresponding increase in the alkali addition time each time. The combined action of the two parameters can cause large fluctuations in the control process. ② Change the flow rate of NaOH, that is, increase the rate of the peristaltic pump and the thickness of the loading tube (sterile hose or stainless steel tube).) Set the lower limit of the fluctuation to 6.5 and the upper limit to 8.0 until the end of fermentation. The results show that the cell concentration (OD660) of the intermediate sample at 12 h is 5.16, and the hyaluronic acid production at the end of fermentation is 9.20 g / L.

[0037] Example 2

[0038] The fermenter adopts the following pH control strategy:

[0039] In the early stage of fermentation (0 - 6 h): After inoculation, when the pH of the fermentation broth naturally drops to 7.2, alkali control is used to maintain the pH of the fermentation broth at 7.2. In the middle stage of fermentation (6 - 12 h), starting from 6 h, the pH is increased by 0.2 every 1 h. When the pH rises to 7.6, the pH is constantly controlled at 7.6. In the middle and late stages of fermentation (12 - 30 h), by changing the parameters of the alkali control automatic control program, the constant rate of the peristaltic pump, and the opening time of the solenoid valve, the pH during the fermentation process is maintained in a continuously fluctuating state. The lower limit of the fluctuation is set at 7.0, and the upper limit is 9.0 until the end of fermentation. The pH control curve is as Figure 1 shown. The results show that the cell concentration (OD660) of the intermediate sample at 12 h is 5.42, and the hyaluronic acid yield at the end of fermentation is 8.52 g / L.

[0040] Comparative Example 1 adopts constant pH control

[0041] The fermenter adopts the following pH control strategy:

[0042] The pH is constantly controlled at 7.0 throughout the fermentation process. The pH control curve is as Figure 2 shown, and other operations are the same as in Example 1. The results show that the cell concentration (OD660) of the intermediate sample at 12 h is 4.42, and the HA yield at the end of fermentation is 6.54 g / L.

[0043] Comparative Example 2 adopts repeated intermittent high pH stress control

[0044] The fermenter adopts the following pH control strategy:

[0045] From 0 to 12 h, the pH regulation is the same as in Example 1. Starting from 12 h, repeated intermittent high pH stress control is adopted, that is, at 12 h, the pH is first raised to 8.5 and maintained for 1 h, then lowered to 7.0 and maintained for 1 h, then the pH is raised to 8.5 and maintained for 1 h,... and so on until the end of fermentation. The results show that the cell concentration of the intermediate sample at 12 h is 5.25, and the HA yield at the end of fermentation is 7.82 g / L.

[0046] Comparative Example 3 adopts constant pH control in the early stage and repeated intermittent high pH stress control in the middle and late stages

[0047] The fermenter adopts the following pH control strategy:

[0048] From 0 to 12 h, the pH is constantly controlled at 7.0. Starting from 12 h, repeated intermittent high pH stress control is adopted, that is, at 12 h, the pH is first raised to 8.5 and maintained for 1 h, then lowered to 7.0 and maintained for 1 h,... and so on until the end of fermentation. The pH control curve is as Figure 3As shown. The results show that the cell concentration of the intermediate sample at 12 h is 4.14, and the HA production at the end of fermentation is 7.23 g / L.

[0049] The concentrations of the bacteria after fermentation, as well as the contents of lactic acid by-products and HA in the above examples and comparative examples are shown in Table 1 below.

[0050] Table 1:

[0051]

[0052] It can be seen from Table 1 that under the pH regulation strategy of this application, the concentration of bacteria is higher, the lactic acid by-product is lower, and the HA production is higher within the same time.

[0053] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0054] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for producing hyaluronic acid, characterized in that, Streptococcus zooepidemicus is subjected to fermentation treatment to obtain hyaluronic acid, and the preservation number of the Streptococcus zooepidemicus is CCTCC NO: M2020231; wherein, the fermentation treatment is carried out under the following conditions: Within 0 to 6 h after the start of the fermentation treatment, the fermentation treatment is carried out under the condition that the pH is 6.7 to 7.2; Within 6 to 12 h after the start of the fermentation treatment, first increase the pH by 0.1 to 0.2 every 1 h, and after the pH rises to 7.4 to 7.6, control the fermentation treatment to be carried out under the condition that the pH is 7.4 to 7.6; After 12 h from the start of the fermentation treatment, the fermentation treatment is carried out under the condition that the pH is 6.5 to 9.0; wherein within 12 to 30 h after the start of the fermentation treatment, the pH maintains a continuously fluctuating state, the lower limit of the fluctuating pH is 6.5 to 7.0, and the upper limit of the pH is 8.0 to 9.

0.

2. The method according to claim 1, characterized in that, Within 0 to 6 h after the start of the fermentation treatment, first allow the pH of the fermentation medium to naturally drop to 6.7 to 7.2, and then control the fermentation treatment to be carried out under the condition that the pH is 6.7 to 7.

2.

3. The method according to claim 2, characterized in that, Control the fermentation treatment to be carried out under the condition that the pH is 6.7 to 7.2 by adding alkali.

4. The method according to claim 3, characterized in that, The alkali added is ammonia water, a NaOH or KOH solution with a concentration of 20% to 30%.

5. The method according to claim 1, characterized in that, The fermentation treatment is carried out under the conditions of an aeration rate of 0.5 to 1 vvm, a rotation speed of 100 to 600 rpm, and 37 °C for 29 to 31 h.

6. The method according to claim 1, characterized in that, The fermentation medium for the fermentation treatment comprises 77 g / L of glucose, 40 g / L of peptone, 1.3 g / L of KH 2 PO 4 , 0.4 g / L of MgSO 4 and 4 g / L of monosodium glutamate.

7. A method for producing hyaluronic acid, characterized in that, comprising: Inoculate the Streptococcus zooepidemicus strain into the seed medium at a volume ratio of 1%, and culture it at 36.5 °C and 200 to 220 rpm for 17.5 - 18 h. Before the end of the culture, perform Gram staining microscopy. After no abnormality is found, inoculate it into the fermentation medium, and ferment and culture it in a 15 L fermenter under the conditions of an aeration rate of 0.5 to 1 vvm, a stirring speed of 100 to 600 rpm, and 37 °C for 30 h. The preservation number of the Streptococcus zooepidemicus is CCTCC NO: M2020231; Among them, the seed culture medium includes 10 g / L of glucose, 15 g / L of peptone, 5 g / L of yeast extract powder, 1.5 g / L of K 2 HPO 4 , 0.35 g / L of MgSO 4 , pH 6.5, During the fermentation process, the following pH control method is adopted for the fermenter: In the early stage of fermentation (0 - 6 h): After inoculation, when the pH of the fermentation broth naturally drops to 6.7 - 7.2, control the pH of the fermentation broth to 6.7 - 7.2 by alkali control with 20% - 30% concentration NaOH; In the middle stage of fermentation (6 - 12 h): Starting from 6 h, increase the pH by 0.1 - 0.2 every 1 h. After the pH rises to 7.4 - 7.6, keep the pH constant at 7.4 - 7.6; During the middle and late stages of fermentation, within 12 to 30 hours: By changing the parameters of the alkali control and automatic control program, the constant rate of the peristaltic pump, and the opening time of the solenoid valve, the pH is maintained in a continuous fluctuating state, with the lower limit of pH fluctuation being 6.5 to 7.0 and the upper limit being 8.0 to 9.0 until the end of fermentation.

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