Xanthomonas campestris ha-1 and a method for biosynthesis of hyaluronic acid

Abstract

The present application relates to a kind of xanthomonas campestris HA-1 and hyaluronic acid biosynthesis method.The present application provides a kind of xanthomonas campestris HA-1 capable of expressing hyaluronic acid in exogenous first, and the fermentation process of the strain is optimized, and a kind of segmented fermentation process is provided, and in fermentation stage, by regulating culture temperature, pH, ventilation ratio, dissolved oxygen (DO), effectively improve the hyaluronic acid fermentation yield, and the synthesis of hyaluronic acid has important significance.

Description

Technical Field

[0001] This invention belongs to the field of hyaluronic acid microbial fermentation technology, specifically relating to a species of Xanthomonas aeruginosa (…). Xanthomonas campestris HA-1, its application in the field of hyaluronic acid biosynthesis, and a method for biosynthesizing hyaluronic acid. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Hyaluronic acid (HA) is a functional glycosamine polymer widely found in living organisms, with broad application prospects in the daily chemical, medical, and food industries. After years of research, a clear understanding of the structure, physicochemical properties, physiological functions, and applications of hyaluronic acid has been achieved. Its molecular structure is a linear macromolecular acidic mucopolysaccharide polymerized from disaccharide units of β-1,3-N-acetyl-D-glucosamine (UDP-GlcNAc) and β-1,4-D-glucuronic acid (UDP-GlcUA). The molecular weight of the natural polysaccharide ranges from 1.0 to 40 × 10⁻⁶. 5 Hyaluronic acid possesses excellent moisturizing properties and is a naturally occurring molecule widely found in the skin and other tissues. Its superior performance has garnered significant attention in the cosmetics industry. Furthermore, hyaluronic acid is colorless, odorless, easily soluble in water, and does not alter the inherent properties of products. It has been approved as a new food ingredient and can be added to various applications, showing great promise.

[0004] The main methods for preparing hyaluronic acid include extraction from animal tissues and microbial fermentation. Animal tissue extraction was an early method, but its limited availability of raw materials, extremely low extraction rate, and complex process make it unsuitable for large-scale industrial production. Therefore, hyaluronic acid is currently mainly produced through microbial fermentation, typically using *Streptococcus vesicae* (a type of bacteria). Streptococcus zooepidemicus However, this bacterium is quite picky about the nutrients in the fermentation medium, such as its very demanding requirements for nitrogen sources. Different nitrogen sources must be used in combination to achieve good fermentation results, which makes its fermentation production cost and efficiency high compared to other microbial polysaccharides such as xanthan gum and pullulan. This has a significant constraint on the expansion of the hyaluronic acid industry. Summary of the Invention

[0005] To address the industrialization bottlenecks of low fermentation yield and high production cost in the current microbial fermentation production of hyaluronic acid, this invention first provides a strain of Xanthomonas javanica HA-1 capable of exogenously expressing hyaluronic acid, whose fermentation process has lower substrate requirements compared to existing strains. Furthermore, to realize the industrialization of hyaluronic acid biosynthesis and achieve hyaluronic acid production, this invention optimizes the fermentation process of the above-mentioned strain, providing a segmented fermentation method, the specific scheme of which is as follows: Firstly, provide a strain of Xanthomonas violaceus (…). Xanthomonas campestris HA-1, this strain was deposited on May 17, 2023 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO. M 2023763.

[0006] The morphological characteristics of the above-mentioned strain Xanthomonas javanica HA-1 are as follows: Bacterial characteristics: rod-shaped, Gram-negative.

[0007] Colony characteristics: yellow, moist, viscous, smooth surface, and easy to pick up.

[0008] The physiological and biochemical characteristics of Xanthomonas brassicae HA-1 are as follows: chemoheterotrophic, aerobic, capable of synthesizing amylase, and without special nutritional requirements.

[0009] Secondly, a microbial agent is provided, said microbial agent comprising Xanthomonas rapae HA-1 as described in the first aspect. Xanthomonas campestris (or a culture of that strain).

[0010] The active ingredient in the bacterial agent includes Xanthomonas hygroscopicus HA-1, and feasible dosage forms include liquid preparations, powders, or granules. The liquid preparation can be a culture broth or solution containing the above-mentioned strain. The powder is the bacterial powder of the above-mentioned strain, and feasible preparation methods include expansion fermentation followed by drying. The granules include the above-mentioned strain and a carrier. Feasible carrier components include inorganic materials such as zeolite powder, diatomaceous earth, vermiculite, and bentonite, as well as inert organic materials such as peat moss, weathered coal, and phycolipids.

[0011] The culture represents the active product of the strain after fermentation under suitable conditions. In the solution provided by the present invention, the active product mainly represents hyaluronic acid.

[0012] Thirdly, the application of Xanthomonas rapae HA-1 described in the first aspect or the bacterial agent described in the second aspect in the field of hyaluronic acid biosynthesis.

[0013] One specific form of the application is: using Xanthomonas hyaluronic acid HA-1 as an industrial fermentation strain to synthesize hyaluronic acid.

[0014] In existing research, Xanthomonas violaceum ( Xanthomonas campestris This strain, a standard strain for xanthan gum production, has milder requirements for substrate nutrients compared to Streptococcus vesiculosus. It can be fermented using low-cost carbon and nitrogen sources such as corn starch, potato starch, soybean flour, corn steep liquor, fish meal, and peanut meal. To achieve large-scale industrial production of hyaluronic acid, this invention focuses on optimizing the fermentation process of the above-mentioned strain. The hyaluronic acid fermentation process generally consists of a cell growth phase and a polysaccharide synthesis phase. Furthermore, as fermentation progresses, the viscosity of the fermentation broth increases rapidly, while the pH decreases rapidly. Therefore, the preferred technical solution is to perform phased control of culture temperature, pH, aeration ratio, and dissolved oxygen (DO) at different fermentation stages to effectively improve fermentation yield and the molecular weight of the hyaluronic acid product. The above-mentioned segmented fermentation control method is as follows: Fourthly, a method for biosynthesizing hyaluronic acid includes the following steps: after the Xanthomonas hygroscopicus HA-1 described in the first aspect is cultured on a solid slant and in seed liquid, it is inoculated into the fermentation culture broth at a volume of 5-10% of the fermentation culture liquid fraction, the pH of the fermentation culture broth is adjusted, and aeration and stirring are carried out for fermentation culture. By controlling the fermentation conditions in stages, hyaluronic acid products are produced.

[0015] 0~16h: Temperature 32~35°C, pH at rest, ventilation 1.2~1.5vvm, tank pressure 0.05~0.08MPa, dissolved oxygen above 30%; Fermentation time: 16 hours to 40-50 hours in total, with a temperature of 26-28°C, UMP added, alkali solution added every hour to adjust the pH of the fermentation broth to 6.5, ventilation rate of 1.8-2.0 vvm, tank pressure of 0.05-0.08 MPa, and dissolved oxygen of 5-15%.

[0016] Preferably, the amount of UMP added is 0.5~1.5ppm.

[0017] Preferably, the fermentation broth comprises the following components in parts by weight: corn starch 3.5~5.0 g / 100 mL; soybean flour 0.2~0.4 g / 100 mL; NaCl 0.2~0.4 g / 100 mL; kanamycin 100 ng / mL; trace elements 2.0~5.0 ppm (final concentration); polyether defoamer 0.02~0.05 g / 100 mL; the remainder being softened water; pH 7.0~7.2. The trace elements are one or a combination of several of manganese sulfate, ferrous sulfate, copper sulfate, zinc chloride, cobalt chloride, and boric acid.

[0018] In one embodiment of the present invention, an activation process for the above-mentioned strain is also provided: The bacterial strain preserved at -80°C was aseptically inoculated onto an agar slant culture medium and incubated statically at 28-32°C for 1-2 days to obtain an activated strain, which was then used for the preparation of seed culture for fermentation production. Further, the agar slant culture medium was LB medium containing 100 ng / mL kanamycin.

[0019] In another embodiment, the present invention also provides a method for preparing the above-mentioned strain seed liquid: Select 2-3 loops of Xanthomonas haplocalyx HA-1 strain activated by slant culture, inoculate it into the fermentation seed liquid, and culture at 32-35°C for 16-20 h to obtain mature seed liquid.

[0020] Preferably, the seed culture medium is composed of the following components in parts by weight: corn starch 1.0~2.0 g / 100 mL; soybean flour 0.3~0.5 g / 100 mL; NaCl 0.2~0.4 g / 100 mL; kanamycin 100 ng / mL; trace elements 0.5~2.0 ppm (final concentration); polyether defoamer 0.02~0.05 g / 100 mL; the rest is softened water; pH 7.0~7.2; the trace elements are one or a combination of several of manganese sulfate, ferrous sulfate, copper sulfate, zinc chloride, cobalt chloride, and boric acid.

[0021] The beneficial effects of one or more of the above technical solutions are: (1) The present invention first provides Xanthomonas hyaluronic acid HA-1, which has the ability to express hyaluronic acid exogenously. Compared with existing hyaluronic acid microbial fermentation strains, the above strain has higher requirements for substrate temperature and can achieve efficient synthesis by using low-cost carbon and nitrogen sources, laying a good technical foundation for the expansion of hyaluronic acid biosynthesis process.

[0022] (2) The present invention optimizes the fermentation process of the above-mentioned strains and provides a segmented fermentation process. Verification has shown that, based on the optimized fermentation process of the present invention, the strains… Xanthomonas campestris The results of HA-1 fermentation for the preparation of hyaluronic acid are as follows: a. The yield of hyaluronic acid was 1.8~2.3 g / 100 g fermentation broth; b. The viscosity of the hyaluronic acid fermentation broth is 12450~18860 mPa·s; c. The molecular weight of hyaluronic acid is 1.35 ± 0.12 × 10⁻⁶. 6 Da, and commercially available hyaluronic acid products (1.33±0.15 ×10) 6 There was no significant difference in molecular weight between Da and Da. Detailed Implementation

[0023] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0025] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0026] Example 1 In this embodiment, a hyaluronic acid fermentation production process based on Xanthomonas brassicae HA-1 is provided: (1) Activation of strain: Xanthomonas hygroscopicus HA-1, stored at -80°C, was aseptically inoculated onto LB slant medium containing 100 ng / mL kanamycin and incubated at 32°C for 1 day to obtain activated strains for fermentation production of seed liquid.

[0027] (2) Preparation of seed liquid for production: Select 2-3 loops of Xanthomonas haplocalyx HA-1 strain activated by slant culture, inoculate it into the fermentation seed liquid, and culture at 32°C for 16-20 h to obtain mature seed liquid.

[0028] The seed culture medium is composed of the following components by mass fraction (g / 100mL): corn starch 2.0; soybean flour 0.3; NaCl 0.2; trace elements 0.5 ppm (final concentration); kanamycin 100 ng / mL; polyether defoamer 0.05; the rest is softened water; pH 7.0~7.2.

[0029] The trace elements are one or a combination of several of the following: manganese sulfate, ferrous sulfate, copper sulfate, zinc chloride, cobalt chloride, and boric acid.

[0030] (3) Hyaluronic acid fermentation production: The seed culture of mature Xanthomonas hyaluronic acid strain HA-1 was inoculated into a sterilized fermentation broth. The pH of the fermentation broth was adjusted, and the fermentation was carried out with aeration and stirring. Hyaluronic acid was produced by controlling the fermentation conditions in stages.

[0031] The fermentation broth has the following composition by mass fraction (g / 100mL): corn starch 5.0; soybean flour 0.4; NaCl 0.4; trace elements 5.0ppm (final concentration); kanamycin 100ng / mL; polyether defoamer 0.05; the rest is softened water; pH 7.0~7.2.

[0032] The trace elements are one or a combination of several of the following: manganese sulfate, ferrous sulfate, copper sulfate, zinc chloride, cobalt chloride, and boric acid.

[0033] Fermentation control conditions are as follows: 0~16h: Temperature 32°C, pH at rest, ventilation 1.2vvm, tank pressure 0.05~0.08MPa, dissolved oxygen above 30%; 16h ~ 50h: Temperature 28°C, add 1.0ppm UMP, add alkali solution every 1h to adjust the pH of fermentation broth to 6.5, ventilation rate 2.0vvm, tank pressure 0.05~0.08MPa, dissolved oxygen 5~15%.

[0034] A hyaluronic acid fermentation broth was obtained, and the content of fermented hyaluronic acid was determined to be 2.13±0.08 g / 100 g fermentation broth; the apparent viscosity of the fermentation broth was 17680±160 mPa·s; and the molecular weight of the product was 1.31±0.11×10⁻⁶. 6 Da.

[0035] Example 2 In this embodiment, another hyaluronic acid fermentation production process based on Xanthomonas brassicae HA-1 is provided: (1) Activation of microbial strains: Xanthomonas hygroscopicus HA-1, stored at -80°C, was aseptically inoculated onto LB slant medium containing 100 ng / mL kanamycin and incubated at 28°C for 2 days to obtain activated strains, which were then used for the preparation of seed culture for fermentation production.

[0036] (2) Preparation of seed liquid for production: Select 2-3 loops of Xanthomonas haplocalyx HA-1 strain activated by slant culture, inoculate it into the fermentation seed liquid, and culture at 32-35°C for 16-20 h to obtain mature seed liquid.

[0037] The seed culture medium is composed of the following components by mass fraction (g / 100mL): corn starch 1.0; soybean flour 0.3; NaCl 0.2; trace elements 2.0ppm (final concentration); kanamycin 100ng / mL; polyether defoamer 0.02; the rest is softened water; pH 7.0~7.2.

[0038] The trace elements are one or a combination of several of the following: manganese sulfate, ferrous sulfate, copper sulfate, zinc chloride, cobalt chloride, and boric acid.

[0039] (3) Hyaluronic acid fermentation production: The seed culture of mature Xanthomonas hyaluronic acid strain HA-1 was inoculated into a sterilized fermentation broth. The pH of the fermentation broth was adjusted, and the fermentation was carried out with aeration and stirring. Hyaluronic acid was produced by controlling the fermentation conditions in stages.

[0040] The fermentation broth has the following composition by mass fraction (g / 100mL): corn starch 3.5; soybean flour 0.2; NaCl 0.2; trace elements 2.0ppm (final concentration); kanamycin 100ng / mL; polyether defoamer 0.02; the rest is softened water; pH 7.0~7.2.

[0041] The trace elements are one or a combination of several of the following: manganese sulfate, ferrous sulfate, copper sulfate, zinc chloride, cobalt chloride, and boric acid.

[0042] Fermentation control conditions are as follows: 0~16h: Temperature 35°C, pH at rest, ventilation 1.5vvm, tank pressure 0.05~0.08MPa, dissolved oxygen above 30%; 16h ~ 40h: Temperature 28°C, add 0.5ppm UMP, add alkali solution every 1h to adjust the pH of fermentation broth to 6.5, ventilation rate 1.8vvm, tank pressure 0.05~0.08MPa, dissolved oxygen 5~15%.

[0043] A hyaluronic acid fermentation broth was obtained, and the content of fermented hyaluronic acid was determined to be 1.83±0.09 g / 100 g fermentation broth; the apparent viscosity of the fermentation broth was 13680±180 mPa·s; and the molecular weight of the product was 1.34±0.11×10⁻⁶. 6 Da.

[0044] Comparative Example 1 In this embodiment, another method for hyaluronic acid fermentation is provided: The difference from Example 1 is that step (3) "hyaluronic acid fermentation production" does not use a segmented fermentation process, and the fermentation control conditions are as follows: 0h ~ 40h: Temperature 28°C, add 0.5ppm UMP, add alkali solution every 1h to adjust the pH of fermentation broth to 6.5, ventilation 1.8vvm, tank pressure 0.05~0.08MPa, dissolved oxygen above 5%.

[0045] A hyaluronic acid fermentation broth was obtained, and the content of fermented hyaluronic acid was determined to be 1.66±0.09 g / 100 g fermentation broth; the apparent viscosity of the fermentation broth was 12080±180 mPa·s; and the molecular weight of the product was 1.14±0.11×10⁻⁶. 6 Da.

[0046] The comparison of the above production data shows that the staged fermentation method can effectively increase the production of hyaluronic acid.

[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A species of Xanthomonas aurea ( Xanthomonas campestris HA-1, this strain was deposited on May 17, 2023 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO. M 2023763.

2. A microbial agent comprising Xanthomonas hygroscopicus HA-1 or a culture of the bacteria as described in claim 1.

3. The microbial agent as described in claim 2, characterized in that, The dosage form of the microbial agent is selected from liquid preparations, powders, or granules.

4. The application of Xanthomonas hyaluronic acid HA-1 as described in claim 1 or the bacterial agent as described in claim 2 or 3 in the field of hyaluronic acid biosynthesis.

5. A method for the biosynthesis of hyaluronic acid, characterized in that, The process includes the following steps: After the Xanthomonas hygroscopicus HA-1 of claim 1 is cultured on a solid slant and in seed liquid, it is inoculated into the fermentation broth at a volume of 5-10% of the fermentation broth volume, the pH of the fermentation broth is adjusted, and the fermentation is carried out with aeration and stirring. By controlling the fermentation conditions in stages, hyaluronic acid products are produced. The staged fermentation control method is as follows: 0~16h: Temperature 32~35°C, pH at rest, ventilation 1.2~1.5vvm, tank pressure 0.05~0.08MPa, dissolved oxygen above 30%; 16h ~ Release from tank: Temperature 26~28°C, add UMP, add alkali solution every 1h to adjust the pH of fermentation broth to 6.5, ventilation rate 1.8~2.0vvm, tank pressure 0.05~0.08MPa, dissolved oxygen 5~15%.

6. The method for biosynthesizing hyaluronic acid as described in claim 5, characterized in that, The amount of UMP to be added is 0.5~1.5 ppm.

7. The method for biosynthesizing hyaluronic acid as described in claim 5, characterized in that, The fermentation broth consists of the following components at the following mass concentrations: corn starch 3.5~5.0 g / 100 mL; soybean flour 0.2~0.4 g / 100 mL; NaCl 0.2~0.4 g / 100 mL; kanamycin 100 ng / mL; trace elements at a final concentration of 2.0~5.0 ppm; polyether defoamer 0.02~0.05 g / 100 mL; the remainder is softened water; pH 7.0~7.2; wherein the trace elements are one or a combination of several of manganese sulfate, ferrous sulfate, copper sulfate, zinc chloride, cobalt chloride, and boric acid.

8. The method for biosynthesizing hyaluronic acid as described in claim 5, characterized in that, The activation process of the strain is as follows: The strain stored at -80°C was aseptically inoculated onto an agar slant culture medium and incubated at 28-32°C for 1-2 days to obtain an activated strain, which was then used for the preparation of seed liquid for fermentation production.

9. The method for biosynthesizing hyaluronic acid as described in claim 5, characterized in that, The method for preparing the seed liquid of the strain is as follows: Select 2-3 loops of Xanthomonas HA-1 strain of rapeseed that has been activated by slant culture and inoculate it into the fermentation seed liquid. Incubate at 32-35°C for 16-20 h to obtain mature seed liquid. The fermented seed liquid is composed of the following components at the following mass concentrations: corn starch 1.0~2.0g / 100mL; soybean flour 0.3~0.5g / 100mL; NaCl 0.2~0.4g / 100mL; kanamycin 100ng / mL; trace elements final concentration 0.5~2.0ppm; polyether defoamer 0.02~0.05g / 100mL; the rest is softened water; pH 7.0~7.2.