A strain of Streptococcus equi subspecies zooepidemicus and its application in producing hyaluronic acid

Abstract

The invention belongs to the field of biochemical engineering technology, and discloses a Streptococcus equi subsp. zooepidemicus strain and its application in producing hyaluronic acid. The Streptococcus equi subsp. zooepidemicus strain provided by the invention is a KM-32 strain, which is based on Streptococcus equi subsp. zooepidemicus GDMCC No: 60146 as a starting strain, and is purified by N + Ion implantation mutagenesis and screening improved the fermentation yield of hyaluronic acid and significantly reduced the raw material cost of hyaluronic acid. Under shake flask fermentation conditions, the hyaluronic acid fermentation yield of this strain can reach 1.16g / L, which is 38.6% higher than that of the starting strain. The yield of fermentation in a fermenter can reach 6.87g / L.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical engineering, and in particular to a Streptococcus equi subspecies zooepidemicus strain and application thereof in producing hyaluronic acid. Background Art

[0002] As a medical auxiliary agent, hyaluronic acid has been widely used in ophthalmology, orthopedics, dermatology, plastic surgery, endoscopy, burns and other fields, and the demand for hyaluronic acid in the domestic and foreign markets is relatively strong. In particular, on January 7, 2021, the National Health Commission of China approved sodium hyaluronate as a "new food ingredient", allowing the addition of hyaluronic acid to ordinary foods, including milk and dairy products, beverages, alcohol, cocoa products, chocolate and chocolate products, as well as candy, frozen drinks, etc. Since then, the market demand for hyaluronic acid has risen rapidly.

[0003] The medical and beauty industries have high requirements for the purity of hyaluronic acid, especially the protein content, which is generally less than 0.1%. However, whether it is animal tissue extraction or microbial fermentation to produce hyaluronic acid, the cost of separation and purification accounts for a large proportion of the total production cost of the product. This is also the bottleneck that restricts many companies from producing high-standard hyaluronic acid. The price of the end product is naturally high when using high-purity hyaluronic acid raw materials to produce food or feed. The food and feed industry does not have to have too high requirements for the purity of hyaluronic acid, especially the protein content. As long as the pollutants and hygiene indicators in the product meet the requirements for food raw materials, low-purity hyaluronic acid can also be used as a raw material for food and feed. In other words, the purity requirements of hyaluronic acid raw materials should be based on the needs of hyaluronic acid end products, rather than blindly pursuing high purity. This is crucial to reducing the price of hyaluronic acid products.

[0004] At present, there are two main methods for the production of hyaluronic acid. One is the extraction method from the tissues of higher animals; the other is the microbial fermentation method. Animal tissues that can be used to prepare hyaluronic acid include vitreous body of eyes, combs, umbilical cords and synovial fluid, among which the highest content is combs, followed by synovial fluid. Based on the consideration of material sources and costs, animal vitreous body of eyes and combs are mainly used as raw materials in production. Although animal raw materials with wide sources and low costs have been used as much as possible, the animal tissue extraction method also has the disadvantages of high raw material requirements, complex separation and purification processes, low extraction yields, and high production costs. The microbial fermentation method uses certain species of streptococci to secrete capsules with hyaluronic acid as the main component to the extracellular space during growth and reproduction. Compared with the animal tissue extraction method, the microbial fermentation method has the advantages of low cost, no material restrictions, simple separation and purification, and suitability for large-scale production, and has gradually become the mainstream method for producing hyaluronic acid. Even so, research on improving the fermentation yield of hyaluronic acid and reducing production costs is still advancing, especially in terms of strains. Fermentation strains with fast growth, low nutritional requirements and high yields are the key to reducing the production of hyaluronic acid by fermentation. Summary of the invention

[0005] The purpose of the present invention is to overcome the problem of high cost of hyaluronic acid raw materials used in the food and feed industries, provide a strain of Streptococcus equi subsp. zooepidemicus and its application in producing hyaluronic acid in a fermentation method, improve the fermentation yield of hyaluronic acid, and significantly reduce the raw material cost of hyaluronic acid.

[0006] To achieve the purpose of the present invention, the present invention provides a new hyaluronic acid fermentation strain, which is a Streptococcus equi subsp. zooepidemicus KM-32 strain, and the KM-32 strain is deposited in the Guangdong Microbiological Culture Collection Center, with a deposit number of GDMCC No: 64426, a deposit date of March 18, 2024, and an address of No. 100, Xianlie Middle Road, Guangzhou, China, Postal Code: 510070.

[0007] Further, in some embodiments of the present invention, the KM-32 strain is based on Streptococcus equi subsp. zooepidemicus SXY36 strain (deposit number GDMCC No: 60146, deposited in Guangdong Microbiological Culture Collection Center on February 27, 2017) as the starting strain, + The strain was bred and screened after ion implantation mutagenesis. The yield of hyaluronic acid produced by shake flask fermentation was significantly higher than that of Streptococcus equi subspecies zooepidemicus GDMCC No:60146.

[0008] For detailed information such as the deposit certificate of the starting strain Streptococcus equi subsp. zooepidemicus SXY36 strain described in the present invention, please refer to the patent application with application number CN201710485218.4 filed on June 23, 2017, and the invention name is "Streptococcus equi subsp. zooepidemicus SXY36 and its application in the fermentation production of hyaluronic acid".

[0009] On the other hand, the present invention also provides an application of the Streptococcus equi subspecies Zooepidemicus KM-32 strain in the fermentation production of hyaluronic acid, wherein the application is to inoculate the Streptococcus equi subspecies Zooepidemicus KM-32 strain into a culture medium for fermentation to obtain a fermentation liquid containing hyaluronic acid.

[0010] Furthermore, in some embodiments of the present invention, the fermentation is carried out at 35-38°C; preferably, in some embodiments of the present invention, the fermentation is carried out at 36.5-37.5°C.

[0011] Furthermore, in some embodiments of the present invention, the fermentation time is 18 to 24 hours; preferably, in some embodiments of the present invention, the fermentation time is 19 to 21 hours.

[0012] Furthermore, in some embodiments of the present invention, the inoculation into culture medium for fermentation is inoculation into shake flask fermentation medium for fermentation or inoculation into a mechanically stirred fermenter for fermentation.

[0013] Further, in some embodiments of the present invention, the composition of the shake flask fermentation medium is: glucose 10-25 g / L, beef extract 8-15 g / L, peptone 6-15 g / L, yeast extract powder 3-8 g / L, K2HPO4 2.5-7.0 g / L, MgSO4 1.5-3.5 g / L, the solvent is tap water, pH 6.8-7.2; preferably, in some embodiments of the present invention, the composition of the shake flask fermentation medium is: glucose 19-21 g / L, beef extract 11-13 g / L, peptone 9-11 g / L, yeast extract powder 4-6 g / L, K2HPO4 4-6 g / L, MgSO4 2-3 g / L, the solvent is tap water, pH 6.9-7.1.

[0014] Further, in some embodiments of the present invention, the fermentation medium of the mechanically stirred fermenter is composed of: sucrose 35-50 g / L, glucose 10-25 g / L, beef extract 8-15 g / L, peptone 6-12 g / L, yeast extract powder 3-7 g / L, K2HPO4 2.5-7.0 g / L, MgSO4 1.5-3.5 g / L, the solvent is tap water, pH 6.8-7.2; preferably, in some embodiments of the present invention, the fermentation medium of the mechanically stirred fermenter is composed of: sucrose 38-42 g / L, glucose 18-22 g / L, beef extract 11-13 g / L, peptone 9-11 g / L, yeast extract powder 4-6 g / L, K2HPO4 4-6 g / L, MgSO4 2-3 g / L, the solvent is tap water, pH 6.9-7.1.

[0015] Further, in some embodiments of the present invention, the Streptococcus equi subsp. zooepidemicus KM-32 strain needs to be firstly activated by slant culture before being inoculated into the culture medium for fermentation, and then cultured by seeds to obtain seed liquid, and then inoculated into the fermentation medium for fermentation. The specific steps are as follows:

[0016] (1) inoculating Streptococcus equi subspecies zooepidemicus KM-32 strain into a slant culture medium, activating the slant culture, and obtaining activated slant cells;

[0017] (2) inoculating the activated slant cells in step (1) into a seed culture medium, and performing seed culture to obtain a seed solution;

[0018] (3) transplanting the seed solution obtained in step (2) into a fermentation medium, and fermenting and culturing the solution in a shake flask or in a fermentation tank to obtain a fermentation solution containing hyaluronic acid.

[0019] Further, in some embodiments of the present invention, the composition of the slant culture medium in step (1) is: glucose 1.5-4 g / L, beef extract 3-7 g / L, peptone 5-8 g / L, yeast extract powder 1-4 g / L, K2HPO4 1.5-5.0 g / L, MgSO4 1.0-2.0 g / L, agar 15-25 g / L, the solvent is tap water, pH 7.0-7.2; preferably, in some embodiments of the present invention, the composition of the slant culture medium in step (1) is: glucose 1.7-2.3 g / L, beef extract 4-6 g / L, peptone 4-6 g / L, yeast extract powder 1.5-2.5 g / L, K2HPO4 2-3 g / L, MgSO4 1.2-1.8 g / L, agar 18-22 g / L, the solvent is tap water, pH 6.9-7.1.

[0020] Furthermore, in some embodiments of the present invention, the slant activation culture is carried out at 35-38°C; preferably, in some embodiments of the present invention, the slant activation culture is carried out at 36.5-37.5°C.

[0021] Furthermore, in some embodiments of the present invention, the slant activation culture time is 20 to 40 hours; preferably, in some embodiments of the present invention, the slant activation culture time is 24 to 36 hours.

[0022] Further, in some embodiments of the present invention, the composition of the seed culture medium in step (2) is: 2-6 g / L glucose, 3-7 g / L beef extract, 3-8 g / L peptone, 1-4 g / L yeast extract powder, 1.5-5.0 g / L K2HPO4, 1.0-2.0 g / L MgSO4, and the solvent is tap water, pH 7.0-7.2; preferably, in some embodiments of the present invention, the composition of the seed culture medium in step (2) is: 3.5-4.5 g / L glucose, 4-6 g / L beef extract, 4-6 g / L peptone, 1.5-2.5 g / L yeast extract powder, 2-3 g / L K2HPO4, 1.2-1.8 g / L MgSO4, and the solvent is tap water, pH 6.9-7.1.

[0023] Furthermore, in some embodiments of the present invention, the seed culture conditions are culturing at 35-37°C and 150-300 r / min shaking conditions for 20-26 h; preferably, in some embodiments of the present invention, the seed culture conditions are culturing at 36.5-37.5°C and 200-250 r / min for 23-25 ​​h.

[0024] Furthermore, in some embodiments of the present invention, in step (3), the seed solution obtained in step (2) is transplanted into the fermentation medium at an inoculation amount of 2% to 5% by volume.

[0025] Furthermore, in some embodiments of the present invention, the shaking speed of the fermentation culture in the shake flask in step (3) is 150 to 300 r / min, and the preferred speed is 200 to 300 r / min; the conditions for the fermentation culture in the fermenter in step (3) are: controlling the temperature to 37±0.5°C, the pH to 7.0±0.2, the ventilation volume to 0.5 to 1.0 v / v·min, the stirring speed to 400 to 650 r / min, and the dissolved oxygen saturation to be greater than 60%.

[0026] Compared with the prior art, the present invention uses Streptococcus equi subsp. zooepidemicus SXY36 strain as the starting strain, and +Through ion implantation mutagenesis and screening, we obtained a high-yield and genetically stable hyaluronic acid fermentation strain - Streptococcus equi subsp. zooepidemicus KM-32 strain. Under shake flask fermentation conditions, the hyaluronic acid fermentation yield can reach 1.16 g / L, which is 38.6% higher than the starting strain. The yield of fermentation in a fermenter can reach 6.87 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a glucuronic acid-A 530 Standard curve. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Additional aspects and advantages of the present invention will be partially given in the following description, partially become apparent from the following description, or be understood through the practice of the present invention. It should be understood that the following description is only used to explain the present invention and is not intended to limit the present invention.

[0029] As used herein, the terms "comprises," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0030] When amount, concentration or other value or parameter is expressed as range, preferred range or a series of upper preferred value and lower preferred value limit range, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value and any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when disclosing range "1 to 5", described range should be interpreted as including range "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5" etc. When numerical range is described in this article, unless otherwise stated, the range is intended to include its end value and all integers and fractions within the range.

[0031] Singular forms include plural references unless the context clearly indicates otherwise. "Optional" or "either" means that the subsequently described event or incident may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

[0032] The indefinite articles "a" and "an" before the elements or components of the present invention have no limitation on the quantity requirements (i.e. the number of occurrences) of the elements or components. Therefore, "a" or "an" should be interpreted as including one or at least one, and the elements or components in the singular form also include the plural form, unless the quantity obviously refers to the singular form only.

[0033] In addition, the descriptions of the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" described below mean that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0034] In the embodiment of the present invention, the composition of the slant culture medium and the plate culture medium is: glucose 2g / L, beef extract 5g / L, peptone 5g / L, yeast extract powder 2g / L, K2HPO42.5 g / L, MgSO41.5g / L, agar 20g / L, the solvent is tap water, pH 7.0; the preparation method is: boiling until the agar is dissolved, or filling in 15mm×150mm test tubes, each filling 4mL, sterilizing at 121°C with high-pressure steam for 20min, and putting the slant on the slant before solidification to make a slant culture medium; or filling in a conical flask, tying the mouth with 8 layers of gauze, sterilizing at 121°C with high-pressure steam for 20min, and pouring into a sterile culture dish with a diameter of 9cm before solidification, 15-20mL per dish, to make a plate culture medium.

[0035] In an embodiment of the present invention, the composition of the seed culture medium is: 4 g / L glucose, 5 g / L beef extract, 5 g / L peptone, 2 g / L yeast extract powder, 2.5 g / L K2HPO4, 1.5 g / L MgSO4, the solvent is tap water, pH 7.0; the preparation method is: 50 mL of seed culture medium is filled in a 250 mL conical bottle, tied with 8 layers of gauze, and sterilized with high-pressure steam at 121°C for 20 min.

[0036] In the embodiment of the present invention, the composition of the shake flask fermentation medium is: glucose 20g / L, beef extract 12g / L, peptone 10g / L, yeast extract powder 5g / L, K2HPO45 g / L, MgSO42.5 g / L, the solvent is tap water, pH 7.0. The preparation method is: 80mL of fermentation medium is placed in a 250mL triangular bottle, 8 layers of gauze are tied, and high-pressure steam sterilization is performed at 121°C for 20min.

[0037] In the embodiment of the present invention, the fermentation medium of the mechanical stirring fermenter is composed of: sucrose 40g / L, glucose 20g / L, beef extract 12g / L, peptone 10g / L, yeast extract powder 5g / L, K2HPO45g / L, MgSO42.5 g / L, the solvent is tap water, pH 7.0.

[0038] In the embodiment of the present invention, the sterile physiological saline is a 0.9% NaCl aqueous solution, which is sterilized by high-pressure steam at 121° C. for 15 minutes.

[0039] In the embodiment of the present invention, the hyaluronic acid content analysis method is:

[0040] (1) Preparation of test samples: ① Preparation of fermentation broth test samples: After 5 mL of fermentation broth is fully shaken, centrifuge at 8000 r / min for 10 min. Take the supernatant after centrifugation, add 10 mL of anhydrous ethanol, shake well and let stand for 20 min to allow HA to precipitate. After centrifugation at 8000 r / min for 10 min, add 10 mL of deionized water to dissolve the precipitate to prepare a hyaluronic acid test sample; ② Preparation of solid hyaluronic acid test samples: 0.1 g of sample is placed in a test tube, add 5 mL of deionized water to dissolve, add 10 mL of anhydrous ethanol, shake well and let stand for 20 min to allow hyaluronic acid to precipitate. The subsequent steps are the same as ① Preparation of fermentation broth test samples.

[0041] (2) Determination of hyaluronic acid content in the test sample: After the hyaluronic acid sample to be tested is diluted appropriately, 1 mL is taken into a test tube and the test tube is placed in an ice water bath. Take out the sodium tetraborate sulfuric acid solution (concentration of 9.54 g / L) that has been stored in a 4°C refrigerator for 2 hours, and slowly add 5 mL to each test tube while stirring. After adding, stir evenly, put into boiling water and boil for 15 minutes, and cool in an ice water bath. Add 0.2 mL of carbazole ethanol solution (concentration of 1.25 g / L) to each test tube, shake well, put into boiling water and boil for 15 minutes, and cool to room temperature. Use 1 mL of distilled water instead of the same treatment of the sample to be tested as a reference, and measure the absorbance (A) at 530 nm. 530 ).

[0042] According to the concentration of glucuronic acid (GA) - A 530 The standard curve is obtained by measuring the A 530 Calculate the corresponding GA content, and then calculate the hyaluronic acid content according to formula 1.

[0043] Hyaluronic acid (g / L) = 2.07 × n × GA content (g / L) Formula 1

[0044] In formula 1, n is the sample dilution factor; 2.07 is theoretically obtained by dividing the relative molecular mass of the repeating disaccharide unit of hyaluronic acid, 401.3, by the relative molecular mass of GA, 194.1.

[0045] (3) Preparation of GA standard curve: Prepare aqueous solutions of standard GA with concentrations of 0, 10, 20, 30, 40, and 50 μg / mL, respectively, and then place them in an ice water bath. Take out the sodium tetraborate sulfuric acid solution (concentration of 9.54 g / L) that has been stored in a 4°C refrigerator for 2 hours, and slowly add 5 mL to each test tube while stirring. Stir evenly after adding, boil in boiling water for 15 minutes, and cool in an ice water bath. Add 0.2 mL of carbazole ethanol solution (concentration of 1.25 g / L) to each test tube, shake well, boil in boiling water for 15 minutes, and cool to room temperature. Use test tube No. 0 as a reference and measure the absorbance of each group at 530 nm. Take the measured A 530 The vertical axis is GA concentration, and the horizontal axis is fitted to obtain a standard curve. The standard curve is shown in the attached Figure 1 .

[0046] The raw materials, instruments and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials, instruments and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0047] Example 1

[0048] The Streptococcus equi subspecies zooepidemicus KM-32 strain described in the present invention is obtained by mutagenesis screening according to the following method.

[0049] (1) Preparation of bacteria: The starting strain was Streptococcus equi subsp. zooepidemicus SXY36 strain (GDMCC No: 60146, deposited on February 27, 2017, at Guangdong Microbiological Culture Collection Center, 100 Xianlie Middle Road, Guangzhou, China, zip code: 510070). Fresh slant culture medium was inoculated and cultured at 37°C for 24 h. Then, the bacteria were picked and placed in sterile saline to prepare a cell concentration of 1.53 × 10 7 Take 0.1 mL of bacterial suspension and spread it on a sterile culture dish. Air-dry it on a clean bench and observe under a microscope to see if there is any overlap between cells.

[0050] (2) Mutagenesis: The bacteria coated in the sterile culture dish in step (1) were subjected to a mutagenesis test at an operating voltage of 10 keV and a vacuum degree of 8 to 9 × 10 -3 Pa,N + The ion implantation dose is 1.56×10 15 ions / cm 2 Mutagenesis. After mutagenesis, wash out the bacteria from the culture dish with sterile saline, dilute with sterile saline, spread on the plate culture medium and culture until the number of colonies no longer increases.

[0051] (3) Preliminary screening: The colonies on the plate culture medium in step (2) were selected and transferred to the slant culture medium for 36 hours to obtain 57 mutant strains. Two loops of slant bacteria of each strain were picked with an inoculation loop and directly inoculated into 80 mL of shake flask fermentation medium. After constant temperature shaking and culturing at 37°C and 250 r / min for 24 hours, the hyaluronic acid content in the fermentation broth was analyzed, and mutant strains with a hyaluronic acid fermentation yield greater than 10% of the starting strain were selected to obtain 11 mutant strains.

[0052] (4) Rescreening: The 11 mutant strains obtained in step (3) were re-inoculated into the slant medium, and after culturing at 37°C for 24 hours, 2 loops of bacteria of each strain were picked with an inoculation loop and inoculated into 50 mL of seed medium, and cultured at 37°C and 200 r / min for 24 hours to obtain the seed solution of each mutant strain. At a volume fraction of 5%, the seed solution of each strain was inoculated into 80 mL of shake flask fermentation medium, and after constant temperature culturing at 37°C and 250 r / min for 24 hours, the hyaluronic acid content in the fermentation broth of each mutant strain was analyzed.

[0053] The hyaluronic acid yields and improvement rates of the 11 mutant strains obtained are shown in Table 1.

[0054] Table 1 Hyaluronic acid production and improvement rate of 11 mutant strains

[0055]

[0056]

[0057] Among the 11 mutant strains screened, strain KM-32 had the highest fermentation yield of hyaluronic acid, reaching 1.16 g / L, which was 38.6% higher than the 0.837 g / L of the starting strain GDMCC No: 60146. The strain was subcultured for 5 times, and the yield of hyaluronic acid in each generation of shake flask fermentation is shown in Table 2. There was no significant difference in the yield of hyaluronic acid between the generations (P>0.05), indicating that the genetic performance of the strain in producing hyaluronic acid was stable.

[0058] Table 2 The yield of hyaluronic acid in each generation of KM-32 after 5 consecutive generations

[0059]

[0060] In summary, Streptococcus equi subspecies zooepidemicus GDMCC No: 60146 was used as the starting strain and N +Ion implantation mutagenesis was used to screen and obtain the mutant strain KM-32, namely, Streptococcus equi subsp. zooepidemicus KM-32 strain, which was deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC No: 64426 and the deposit date March 18, 2024. Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province; Postal Code 510070.

[0061] Example 2

[0062] Using KM-32 strain as the bacterial strain, shake flask fermentation was performed to prepare hyaluronic acid in the following steps:

[0063] (1) Inoculate the KM-32 strain stored in a frozen glycerol tube into a slant culture medium and culture at 37°C for 24 h to obtain an activated slant of the KM-32 strain;

[0064] (2) Inoculation loop picking step (1) After activation culture, two loops of KM-32 bacteria were inoculated into 50 mL of seed culture medium and cultured at 37° C. and 200 rpm for 24 h to obtain a seed solution with a dry bacterial cell concentration of 1.62 g / L;

[0065] (3) The seed solution obtained in step (2) is inoculated at a volume concentration of 5% (i.e., 4 mL) into 80 mL of shake flask fermentation medium in a 250 mL Erlenmeyer flask, and cultured at 37° C. and 250 r / min shaking conditions for 20 h to obtain a fermentation liquid containing hyaluronic acid.

[0066] After analysis, the fermentation liquid prepared by the above method had a dry bacterial content of 3.65 g / L and a hyaluronic acid content of 1.15 g / L.

[0067] Example 3

[0068] The Streptococcus equi subspecies zooepidemicus KM-32 strain was used as the fermentation strain for producing hyaluronic acid, and hyaluronic acid was produced by fermentation in a 5L mechanical stirring fermentation tank according to the following steps:

[0069] (1) Inoculate the KM-32 cells stored in a slant at 4°C into a fresh slant medium and culture at 37°C for 30 h to obtain activated slant cells;

[0070] (2) Inoculation loop picking step (1) After activation culture, two loops of KM-32 cells were inoculated into two bottles of 100 mL seed culture medium, and cultured at 37° C. and 200 rpm for 20 h to obtain a seed solution with a dry cell concentration of 1.43 g / L;

[0071] (3) The seed solution obtained in step (2) was inoculated with 140 mL of the seed solution at a volume concentration of 4% into a 3.5 L mechanical stirring fermentation medium in a 5 L fermentation tank; the fermentation temperature was set to 37 ± 0.5 ° C, the ventilation was adjusted to 0.6-1.0 v / v·min, the stirring speed was 400-600 r / min, so that the dissolved oxygen saturation was above 60%, and the pH of the medium was controlled to 7.0 ± 0.2 by adding 2 mol / L NaOH aqueous solution or 2 mol / L HCl aqueous solution. The fermentation was run for 18 h and the tank was released to obtain a fermentation liquid containing hyaluronic acid.

[0072] After analysis, the fermentation liquid prepared by the above method had a dry cell content of 6.17 g / L and a hyaluronic acid content of 5.84 g / L.

[0073] Example 4

[0074] The Streptococcus equi subspecies zooepidemicus KM-32 strain was used as the fermentation strain for producing hyaluronic acid, and hyaluronic acid was produced by fermentation in a 10L mechanical stirring fermentation tank according to the following steps:

[0075] (1) Inoculate the KM-32 cells stored in a slant at 4°C into fresh slant culture medium and culture at 37°C for 36 h to obtain activated slant cells;

[0076] (2) Inoculation loop picking step (1) After activation culture, two loops of the bacterial cells of strain KM-32 were inoculated into three bottles of 100 mL seed culture medium, and cultured at 37° C. and 200 rpm for 24 h to obtain a seed solution with a dry bacterial cell concentration of 1.57 g / L;

[0077] (3) The seed solution obtained in step (2) is inoculated with 300 mL of the seed solution at a volume concentration of 2% into a 6L mechanical stirring fermentation medium in a 10L fermentation tank; the fermentation temperature is set to 37±0.5°C, the ventilation volume is adjusted to 0.6-1.0 v / v·min, and the stirring speed is 400-600 r / min, so that the dissolved oxygen saturation is above 60%, and the pH of the medium is controlled to 7.0±0.2 by adding 2 mol / L NaOH aqueous solution or 2 mol / L HCl aqueous solution. The fermentation is run for 20 hours and the tank is released to obtain a fermentation liquid containing hyaluronic acid.

[0078] After analysis, the fermentation liquid prepared by the above method had a dry cell content of 7.38 g / L and a hyaluronic acid content of 6.87 g / L.

[0079] It will be easily understood by those skilled in the art that the above descriptions are only partial embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Streptococcus equi subsp. zooepidemicus, characterized in that: The Streptococcus equi zooepidemicus strain is the Streptococcus equi zooepidemicus KM-32 strain, and the KM-32 strain is preserved in the Guangdong Microbiological Culture Collection Center, with the preservation number: GDMCC No: 64426, the preservation date is March 18, 2024, and the address is No. 100 Xianlie Middle Road, Guangzhou, China, Postal Code: 510070.

2. The Streptococcus equi subsp. zooepidemicus strain according to claim 1, characterized in that The KM-32 strain is a strain of Streptococcus equi subspecies zooepidemicus SXY36. + The SXY36 strain was bred and screened after ion implantation mutagenesis. The deposit number of the strain is GDMCC No: 60146, and it was deposited in Guangdong Microbiological Culture Collection Center on February 27, 2017.

3. Use of the Streptococcus equi subspecies zooepidemicus KM-32 strain according to any one of claims 1 to 2 in the fermentation production of hyaluronic acid, characterized in that: The application is to inoculate the Streptococcus equi subspecies zooepidemicus KM-32 strain into a culture medium for fermentation to obtain a fermentation liquid containing hyaluronic acid.

4. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 3, characterized in that: The fermentation is carried out at 35-38°C.

5. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 4, characterized in that: The fermentation is carried out at 36.5-37.5°C.

6. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 3, characterized in that: The fermentation time is 18 to 24 hours.

7. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 6, characterized in that: The fermentation time is 19 to 21 hours.

8. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 3, characterized in that: The inoculation into culture medium fermentation is inoculation into shake flask fermentation culture medium fermentation or inoculation into mechanical stirring fermentation tank fermentation.

9. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 8, characterized in that: The shake flask fermentation medium is composed of: 10-25 g / L glucose, 8-15 g / L beef extract, 6-15 g / L peptone, 3-8 g / L yeast extract powder, 2.5-7.0 g / L K2HPO4, 1.5-3.5 g / L MgSO4, the solvent is tap water, and the pH value is 6.8-7.

2.

10. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 8, characterized in that: The shake flask fermentation medium is composed of: 19-21 g / L glucose, 11-13 g / L beef extract, 9-11 g / L peptone, 4-6 g / L yeast extract powder, 4-6 g / L K2HPO4, 2-3 g / L MgSO4, the solvent is tap water, and the pH is 6.9-7.

1.

11. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 8, characterized in that: The fermentation medium of the mechanical stirring fermenter is composed of: 35-50 g / L sucrose, 10-25 g / L glucose, 8-15 g / L beef extract, 6-12 g / L peptone, 3-7 g / L yeast extract powder, 2.5-7.0 g / L K2HPO4, 1.5-3.5 g / L MgSO4, the solvent is tap water, and the pH value is 6.8-7.

2.

12. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 8, characterized in that: The composition of the fermentation medium of the mechanical stirring fermenter is: 38-42 g / L sucrose, 18-22 g / L glucose, 11-13 g / L beef extract, 9-11 g / L peptone, 4-6 g / L yeast extract powder, 4-6 g / L K2HPO4, 2-3 g / L MgSO4, the solvent is tap water, and the pH is 6.9-7.

1.

13. The use of the Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 3, characterized in that: Before the Streptococcus equi subspecies zooepidemicus KM-32 strain is inoculated into the culture medium for fermentation, it needs to be firstly activated by slant culture, and then cultured by seed to obtain seed liquid, and then inoculated into the fermentation medium for fermentation. The specific steps are as follows: (1) inoculating Streptococcus equi subspecies zooepidemicus KM-32 strain into a slant culture medium, activating the slant culture, and obtaining activated slant cells; (2) inoculating the activated slant cells in step (1) into a seed culture medium, and performing seed culture to obtain a seed solution; (3) The seed solution obtained in step (2) is transplanted into a fermentation medium, and fermented in a shake flask or in a fermenter to obtain a fermentation liquid containing hyaluronic acid.

14. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 13, characterized in that: The composition of the slant culture medium in step (1) is: 1.5-4 g / L glucose, 3-7 g / L beef extract, 5-8 g / L peptone, 1-4 g / L yeast extract powder, 1.5-5.0 g / L K2HPO4, 1.0-2.0 g / L MgSO4, 15-25 g / L agar, the solvent is tap water, and the pH is 7.0-7.

2.

15. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 13, characterized in that: The composition of the slant culture medium in step (1) is: glucose 1.7-2.3 g / L, beef extract 4-6 g / L, peptone 4-6 g / L, yeast extract powder 1.5-2.5 g / L, K2HPO4 2-3 g / L, MgSO4 1.2-1.8 g / L, agar 18-22 g / L, the solvent is tap water, and the pH is 6.9-7.

1.

16. The use of Streptococcus equi subsp. zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 13, characterized in that: The slant activation culture is carried out at 35-38°C.

17. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 13, characterized in that: The slant activation culture is carried out at 36.5-37.5°C.

18. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 13, characterized in that: The slant activation culture time is 20 to 40 h.

19. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 13, characterized in that: The slant activation culture time is 24 to 36 hours.

20. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 13, characterized in that: The composition of the seed culture medium in step (2) is: 2-6 g / L glucose, 3-7 g / L beef extract, 3-8 g / L peptone, 1-4 g / L yeast extract powder, 1.5-5.0 g / L K2HPO4, 1.0-2.0 g / L MgSO4, the solvent is tap water, and the pH is 7.0-7.

2.

21. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 13, characterized in that: The composition of the seed culture medium in step (2) is: 3.5-4.5 g / L glucose, 4-6 g / L beef extract, 4-6 g / L peptone, 1.5-2.5 g / L yeast extract powder, 2-3 g / L K2HPO4, 1.2-1.8 g / L MgSO4, the solvent is tap water, and the pH is 6.9-7.

1.

22. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 13, characterized in that: The seed culture conditions are culturing at 35-37° C. and 150-300 r / min shaking conditions for 20-26 h.

23. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 13, characterized in that: The seed culture conditions are 36.5-37.5° C. and 200-250 r / min for 23-25 ​​h.

24. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 13, characterized in that: In the step (3), the seed solution obtained in the step (2) is transplanted into the fermentation medium at an inoculation amount of 2% to 5% by volume.

25. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 13, characterized in that: In the step (3), the shaking speed of the fermentation culture in the shake flask is 150-300 r / min.

26. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 25, characterized in that: In the step (3), the shaking speed of the fermentation culture in the shake flask is 200-300 r / min.

27. The use of Streptococcus equi subspecies zooepidemicus KM-32 strain in fermentation production of hyaluronic acid according to claim 13, characterized in that: The fermentation conditions in the fermenter in step (3) are: controlling the temperature to 37±0.5°C, the pH to 7.0±0.2, the ventilation volume to 0.5-1.0 v / v·min, the stirring speed to 400-650 r / min, and the dissolved oxygen saturation to be greater than 60%.

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