Method for producing sodium hyaluronate by regulating and controlling plant peptone based on exogenous amino acid and exogenous amino acid culture medium

Abstract

The invention discloses a method for producing sodium hyaluronate by regulating and controlling plant peptone based on exogenous amino acid and an exogenous amino acid culture medium. The method for producing the sodium hyaluronate through the exogenous amino acid culture medium comprises the following steps: S1, preparing a seed solution, namely inoculating streptococcus zooepidemicus into the seed culture medium for culturing to obtain the seed solution; s2, fermentation culture: inoculating the seed liquid obtained in the step S1 into the fermentation culture medium for fermentation culture to obtain fermentation liquid; s3, extraction and purification: extracting and purifying the fermentation liquor obtained in the step S2 to obtain a sodium hyaluronate product. According to the method disclosed by the invention, a specific variety of amino acids are exogenously added to promote the growth of thalli, so that high yield and high molecular weight of sodium hyaluronate are realized on the premise of completely using a plant nitrogen source, and meanwhile, the stability, safety and repeatability of a production process are ensured.

Description

Technical Field

[0001] This invention relates to the technical field of sodium hyaluronate preparation, specifically a method for producing sodium hyaluronate based on the regulation of plant peptone by exogenous amino acids, and an exogenous amino acid culture medium. Background Technology

[0002] Sodium hyaluronate, the sodium salt of hyaluronic acid, is a glucuronic acid with excellent moisturizing, repairing, and anti-aging effects, and is therefore widely used in medicine, cosmetics, and food. Currently, sodium hyaluronate is mainly produced industrially using microbial fermentation, with Streptococcus veterinaria being the primary microbial strain.

[0003] Peptone, as the main raw material for microbial fermentation culture, provides the essential nitrogen source for microbial growth and development. It can be divided into three categories based on its source: animal, microbial, and plant peptone. Nowadays, animal peptone (bovine bone peptone) is commonly used as the core nitrogen source in the industrial fermentation production of sodium hyaluronate. However, this has brought several significant problems, such as (1) any changes in the species, age, health status, and processing conditions (such as degree of hydrolysis, temperature, and enzyme type) of animal peptone will cause fluctuations in the amino acid composition, vitamins, growth factors, peptide length, and trace element content of the peptone, ultimately leading to poor batch-to-batch repeatability during fermentation production and unstable molecular weight and yield of the final product. (2) Animal peptone carries the risk of introducing animal-derived pathogens (such as mad cow disease factor and viruses), which limits the production and use of pharmaceutical-grade or injectable sodium hyaluronate.

[0004] To address the aforementioned issues, existing technologies, such as patent CN108611387, first attempt to dissolve plant proteins at high temperatures, then enzymatically hydrolyze them to obtain peptone, and finally purify and use it for the fermentation production of sodium hyaluronate. However, while this technology solves the previous problem that plant peptone could not be utilized by sodium hyaluronate-producing bacteria, it still suffers from low expression levels, cumbersome production steps, and increased manufacturing costs related to labor and growth cycles. The fundamental reason lies in the significant differences in the amino acid composition and ratio between plant peptone and animal-derived peptone, which cannot fully support efficient bacterial growth and HA synthesis. Therefore, developing a fermentation method that overcomes the shortcomings of plant peptone and uses readily available plant peptone as a raw material to achieve efficient, stable, and safe production of high molecular weight sodium hyaluronate has become a pressing technical problem to be solved in this field. Summary of the Invention

[0005] This application provides a method for producing sodium hyaluronate based on the regulation of plant peptone using exogenous amino acids, as well as an exogenous amino acid culture medium. This application promotes bacterial growth by adding specific types of exogenous amino acids, thereby achieving high yield and high molecular weight of sodium hyaluronate while using only plant nitrogen sources, and ensuring the stability, safety, and reproducibility of the production process.

[0006] On one hand, this application provides a method for producing sodium hyaluronate based on the regulation of plant peptone using exogenous amino acids. The method for producing sodium hyaluronate through the exogenous amino acid culture medium includes the following steps: S1, seed culture preparation: inoculating Streptococcus vesiculosus into the seed culture medium for culture to obtain seed culture; S2, fermentation culture: inoculating the seed culture medium obtained in step S1 into the fermentation culture medium for fermentation culture to obtain fermentation broth; S3, extraction and purification: extracting and purifying the fermentation broth obtained in step S2 to obtain sodium hyaluronate product. The exogenous amino acids include at least two of L-arginine, L-threonine, L-cysteine, and L-proline.

[0007] Preferably, the preparation of the seed culture specifically includes the following steps: inoculating the preserved Streptococcus veterinaria strain into the seed culture medium according to the inoculation amount, activating and culturing for a certain period of time to obtain the primary seed culture; inoculating the primary seed culture into the seed culture medium according to the inoculation amount, expanding and culturing for a certain period of time to obtain the secondary seed culture.

[0008] Preferably, the fermentation culture specifically includes the following steps: inoculating the secondary seed liquid into a fermenter according to the inoculation amount, fermenting for a certain period of time to obtain fermentation broth, wherein the fermenter contains the fermentation culture medium.

[0009] Preferably, the extraction and purification specifically includes the following steps: coarse sedimentation, dissolution, degradation, filtration, secondary precipitation, centrifugation, washing and drying of the fermentation broth collected after fermentation, and finally obtaining sodium hyaluronate product.

[0010] On the other hand, this application provides an exogenous amino acid culture medium, comprising a seed culture medium for seed liquid preparation and a fermentation culture medium for seed liquid fermentation; the seed culture medium is formulated with plant peptone, yeast powder, glucose monohydrate, dipotassium hydrogen phosphate trihydrate, sodium glutamate monohydrate, magnesium sulfate heptahydrate and exogenous amino acids; the fermentation culture medium is formulated with plant peptone, yeast powder, glucose monohydrate, dipotassium hydrogen phosphate trihydrate, sodium glutamate monohydrate, magnesium sulfate heptahydrate, defoamer and exogenous amino acids.

[0011] Preferably, the raw materials for the plant peptone include one or more of wheat, soybeans, and corn.

[0012] Preferably, in the seed culture medium, the amount of exogenous amino acids added is 1‰ to 5‰ of the total mass of the seed culture medium. Preferably, in the fermentation medium, the amount of exogenous amino acids added is 1‰ to 5‰ of the total mass of the fermentation medium. One or more technical solutions provided in this application have at least the following technical effects or advantages: 1. This application optimizes and guides the metabolic flow of *Streptococcus vesiculosus* using plant peptone as a nitrogen source by exogenously adding specific amino acids (at least two of L-arginine, L-threonine, L-cysteine, and L-proline), promoting cell growth and achieving or even exceeding the yield and molecular weight of traditional animal nitrogen sources. This method shortens the fermentation cycle, improves production efficiency and economy, resulting in higher yield per unit time and reduced energy consumption and equipment occupancy costs.

[0013] 2. This invention uses plant-derived peptones (such as wheat and soybean peptones), completely eliminating the risk of pathogens such as mad cow disease factors that may be introduced by using animal peptones. This makes the product more suitable for high-requirement fields such as pharmaceutical and injectable applications. Simultaneously, by adding exogenously sourced amino acids with clearly defined components and stable quality, the inherent differences in amino acid composition between different batches and from different sources of plant peptones are effectively compensated for, thereby ensuring batch-to-batch stability and repeatability of the fermentation process. Detailed Implementation

[0014] This application provides a method for producing sodium hyaluronate based on the regulation of plant peptone using exogenous amino acids, as well as an exogenous amino acid culture medium. This application promotes bacterial growth by adding specific types of exogenous amino acids, thereby achieving high yield and high molecular weight of sodium hyaluronate while using only plant nitrogen sources, and ensuring the stability, safety, and reproducibility of the production process.

[0015] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0016] It should be noted that the terms "first," "second," etc., used in the specification and claims of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that embodiments of this application can be implemented in a sequence other than that described. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or server that includes a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or modules not explicitly listed or inherent to these processes, methods, products, or devices. Example

[0017] (1) Seed culture preparation: The preserved Streptococcus vesicular strain was inoculated into 1L of seed culture medium at an inoculation rate of 0.2% (v / v) and activated at 35℃ and 200rpm for 16h to obtain primary seed culture. The above primary seed culture was inoculated into 1L of seed culture medium at an inoculation rate of 2% (v / v) and expanded at 35℃ and 200rpm for 10h to obtain secondary seed culture.

[0018] (2) Fermentation: The secondary seed culture was inoculated into a 5L fermenter at an inoculation rate of 5% (v / v) and cultured for 24h. The culture conditions were DO 30%, stirring rate 600rpm, and aeration rate 6L / m³. 3 The tank pressure was 0.05 MPa, the temperature was 35℃, and the pH was adjusted to 7 with ammonia. The viscosity of the fermentation broth was measured when it remained constant or began to decrease.

[0019] (3) Extraction and purification: Fermentation was terminated after 24 hours when the viscosity of the fermentation broth no longer increased. After fermentation, the fermentation broth was collected and subjected to coarse sedimentation, dissolution, degradation, filtration, secondary precipitation, centrifugation, washing and drying to finally obtain high-purity sodium hyaluronate product.

[0020] Examples 2-5 The difference between Examples 2-5 and Example 1 lies in the different amino acids added during the preparation of the seed culture and the fermentation stage in the process of producing sodium hyaluronate by streptococcal fermentation. Finally, high-purity sodium hyaluronate was obtained through extraction and purification. The data are summarized in tables, as shown in Tables 1-4.

[0021] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that no additional amino acids were added during the seed culture preparation and fermentation stages of the Streptococcus fermentation process to produce sodium hyaluronate. High-purity sodium hyaluronate was then obtained through extraction and purification. The data are presented in Tables 1-4.

[0022] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that, in the process of producing sodium hyaluronate by streptococcal fermentation, bovine bone peptone was used in the culture medium, and no additional amino acids were added during the seed culture preparation and fermentation stages. High-purity sodium hyaluronate was then obtained through extraction and purification. The data are summarized in tables, as shown in Tables 1-4.

[0023] The seed and fermentation medium formulations are summarized in the following tables, Table 1 and Table 2. In the tables below, "concentration" refers to "volume percentage (w / v)%".

[0024] Table 1. Composition of seed culture medium in the examples and comparative examples

[0025] Table 2. Composition of fermentation medium in the examples and comparative examples

[0026] The peptone used was wheat peptone, and the data were compiled into the following table, Table 3.

[0027] Table 3. Types of peptones in the Examples and Comparative Examples

[0028] The amino acids used are a combination of L-arginine, L-threonine, L-cysteine, and L-proline, and the data are compiled into the following table, see Table 4.

[0029] Table 4. Information on exogenously added amino acids in the examples and comparative examples.

[0030] Determining the state of microorganisms by measuring cell growth time and concentration is a core method for precise control in fermentation processes, directly reflecting the metabolic activity and stability of microorganisms. Meanwhile, hyaluronic acid is a high-molecular-weight polysaccharide, and its viscosity is directly related to its molecular weight; the higher the molecular weight, the higher the viscosity. Therefore, to further investigate the effect of exogenously adding specific amino acids to plant peptone on the fermentation production of sodium hyaluronate, a comprehensive assessment is needed using parameters such as the culture time to reach the fermentation standard, the final cell concentration, and the yield. Furthermore, this invention conducted the following tests on the seed culture, and the data and test results are summarized in Table 5.

[0031] Table 5. Detection results of seed solutions in the examples and comparative examples.

[0032] The data analysis of the examples and comparative examples in Table 5 shows that when exogenous amino acid compositions were added during the seed culture preparation stage of Examples 1-5, the culture time to reach the standard for loading into the fermentation tank was shorter than that of Comparative Example 1 (using only wheat peptone), and the final bacterial concentration and yield were higher than those of Comparative Example 1 (using only wheat peptone). Compared with Comparative Example 2 (using only bovine bone peptone), the culture time, final bacterial concentration, and yield to reach the standard for loading into the fermentation tank in Examples 1-5 were comparable to or even higher than those in Comparative Example 2 (using only bovine bone peptone). Among them, the final bacterial concentration and yield of Example 1 were higher than those of Comparative Example 2, indicating that the addition of exogenous amino acids effectively solved the problem of low molecular weight of plant nitrogen source products.

[0033] Examples 6-8 To further investigate the effect of the amount of exogenous amino acids added to plant peptone on the fermentation production of sodium hyaluronate, this application further conducted verification through examples. Examples 6-8 differ from Example 1 in that the amount of exogenous amino acids added during the seed culture preparation and fermentation stages of the streptococcal fermentation production of sodium hyaluronate differs. Finally, high-purity sodium hyaluronate was obtained after extraction and purification. The seed culture was tested as follows, and the test results are summarized in Table 6.

[0034] Table 6. Results of exogenous amino acid addition and seed culture detection in Examples 6-8

[0035] The data in Table 6 show that the culture time, final bacterial concentration and yield of Examples 6-8 that reached the standard for the upper tank were comparable to or even higher than those of Comparative Example 2, indicating that the optimal amount of exogenous amino acids added to plant peptone was 1‰.

[0036] In summary, Example 7 is the optimal example.

[0037] The present invention further provides the detection results of the fermentation broth, and the test results are shown in Table 7.

[0038] Table 7. Detection results of fermentation broth in the examples.

[0039] The test results in Table 7 show that the fermentation cycle of Example 7 is shorter than that of Comparative Example 1 and Comparative Example 2, and the viscosity and yield of the fermentation broth of Example 7 are significantly higher than those of Comparative Example 1 (using only wheat peptone) and Comparative Example 2 (using only bovine bone peptone). Example

[0040] The difference between Example 9 and Example 7 is that, in the process of producing sodium hyaluronate by streptococcal fermentation, the peptone used in the seed culture preparation and fermentation stages is soybean peptone, and finally, high-purity sodium hyaluronate product is obtained after extraction and purification. The seed culture was tested as follows, and the data are compiled into a table, as shown in Table 8.

[0041] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that, in the process of producing sodium hyaluronate by streptococcal fermentation, soybean peptone was used in the culture medium, and no additional amino acids were added during the seed culture preparation and fermentation stages. High-purity sodium hyaluronate was then obtained through extraction and purification. The data were compiled into a table, as shown in Table 8.

[0042] Table 8. Detection results of seed solutions from the examples and comparative examples.

[0043] Generally, plant peptones have significantly lower levels of many amino acids essential for microbial growth than animal peptones. For example, plant peptones are generally deficient in sulfur amino acids (such as cysteine) and threonine. This imbalance in amino acid composition is one of the fundamental reasons that limits the efficient growth and synthesis of target products (such as hyaluronic acid) of microorganisms, especially bacteria like Streptococcus vesicaceae that require abundant nitrogen sources, in plant nitrogen sources.

[0044] The test results in Table 8 show that the culture time for Example 9 to reach the standard for fermentation was longer than that of Comparative Example 3 (using only soybean peptone) and comparable to that of Comparative Example 2 (using only bovine bone peptone). However, the final bacterial concentration and seed culture viscosity were higher than those of Comparative Examples 2 and 3, indicating that although plant-derived peptones differ in their specific amino acid composition, they generally have a disadvantage in overall amino acid balance compared to animal-derived peptones. This application achieves stable high yields and high molecular weights by exogenously adding specific types of amino acids to promote cell growth.

[0045] The present invention further provides the detection results of the fermentation broth, and the test results are shown in Table 9.

[0046] Table 9. Detection results of fermentation broth in the examples.

[0047] The test results in Table 9 show that the fermentation cycle of Example 9 is shorter than that of Comparative Example 2 and Comparative Example 3, and the viscosity and yield of the fermentation broth of Example 9 are significantly higher than those of Comparative Example 3 (using only soybean peptone) and Comparative Example 2 (using only bovine bone peptone).

[0048] It should be noted that the order of the embodiments described above is merely for descriptive purposes and does not represent the superiority or inferiority of the embodiments. Furthermore, specific embodiments have been described above. Other embodiments are within the scope of the appended claims. In some cases, the actions or steps described in the claims can be performed in a different order than those shown in the embodiments and still achieve the desired results. In some implementations, multitasking and parallel processing are also possible or may be advantageous.

[0049] The above description is only a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

[0050] This specification is merely an illustrative description of this application and is intended to cover any and all modifications, variations, combinations, or equivalents within the scope of this application. Clearly, those skilled in the art can make various alterations and modifications to this application without departing from its scope. Therefore, if such modifications and modifications fall within the scope of this application and its equivalents, this application intends to include such modifications and modifications.

Claims

1. A method for regulating production of sodium hyaluronate by a plant proteus based on an exogenous amino acid, characterized by, The method comprises the following steps: S1, seed liquid preparation: inoculating Streptococcus zooepidemicus into a seed culture medium for culture to obtain a seed liquid; S2, fermentation culture: inoculating the seed liquid obtained in step S1 into a fermentation culture medium for fermentation culture to obtain a fermentation liquid; S3, extraction and purification: extracting and purifying the fermentation liquid obtained in step S2 to obtain a sodium hyaluronate product; The seed culture medium and the fermentation culture medium are added with exogenous amino acids; The exogenous amino acids include at least two of L-arginine, L-threonine, L-cysteine and L-proline.

2. The method for regulating production of sodium hyaluronate by plant proteoglycans using exogenous amino acids according to claim 1, wherein, The seed liquid preparation specifically comprises the following operation steps: A preserved Streptococcus zooepidemicus strain is inoculated into a seed culture medium according to an inoculation amount, and activated culture is performed for a certain time to obtain a first-stage seed liquid; The first-stage seed liquid is inoculated into a seed culture medium according to an inoculation amount, and enlarged culture is performed for a certain time to obtain a second-stage seed liquid.

3. The method for regulating production of sodium hyaluronate by plant proteoglycans based on exogenous amino acids according to claim 2, characterized in that, The fermentation culture specifically comprises the following operation steps: The second-stage seed liquid is inoculated into a fermentation tank according to an inoculation amount, and fermentation is performed for a certain time to obtain a fermentation liquid, and the fermentation tank is the fermentation culture medium.

4. The method for regulating production of sodium hyaluronate by plant proteoglycans using exogenous amino acids according to claim 3, wherein The extraction and purification specifically comprise the following operation steps: The fermentation liquid collected after fermentation is subjected to rough sedimentation, dissolution, degradation, filtration, secondary precipitation, centrifugal washing and drying to finally obtain a sodium hyaluronate product.

5. An exogenous amino acid culture medium, characterized by, The seed culture medium used for the seed liquid preparation and the fermentation culture medium used for the fermentation culture in the method for producing sodium hyaluronate based on exogenous amino acid regulation of plant peptone according to any one of claims 1-4; The formula of the seed culture medium comprises plant peptone, yeast powder, monohydrate glucose, trihydrate dipotassium hydrogen phosphate, monohydrate sodium glutamate, magnesium sulfate heptahydrate and exogenous amino acids; The formula of the fermentation culture medium comprises plant peptone, yeast powder, monohydrate glucose, trihydrate dipotassium hydrogen phosphate, monohydrate sodium glutamate, magnesium sulfate heptahydrate, defoaming agent and exogenous amino acids.

6. The exogenous amino acid medium of claim 5, wherein, The raw material of the plant peptone comprises one or more of wheat, soybean and corn.

7. The exogenous amino acid medium of claim 5, wherein, In the seed culture medium, the addition amount of the exogenous amino acids is 1‰-5‰ of the total mass of the seed culture medium.

8. The exogenous amino acid medium of claim 5, wherein, In the fermentation culture medium, the addition amount of the exogenous amino acids is 1‰-5‰ of the total mass of the fermentation culture medium.

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