Anti-bacteriophage streptococcus equi subsp. Zooepidemicus

Abstract

The invention belongs to the technical field of microorganisms, a streptococcus zooepidemicus mutant strain CGMCC (China General Microbiological Culture Collection Center) No.26991 is screened out, the streptococcus zooepidemicus mutant strain is obtained through screening by adopting a phage secondary infection method, resistance experiments and genetic stability experiments prove that the screened strain is an anti-phage strain, the resistance can be stably inherited, and the strain can be used as a high-yield streptococcus zooepidemicus mutant strain. The strain not only has good resistance to bacteriophages, but also has excellent high-yield hyaluronic acid characteristics, and can be used as an industrial fermentation strain of hyaluronic acid.

Description

[0001] This application claims priority to the Chinese patent application No. 202410909453.X, filed on July 8, 2024, and entitled "A strain of Streptococcus equisubsp. zooepidemicus resistant to bacteriophage", the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application belongs to the field of microbial technology, and specifically relates to a strain of Streptococcus equisubsp. zooepidemicus resistant to bacteriophage. BACKGROUND

[0003] Bacteriophage infection has always been the most common infection problem in microbial fermentation process, which can bring catastrophic losses to fermentation enterprises, and there is no good solution so far. For many years, fermentation factories have tried to use various methods to prevent bacteriophage contamination, the main means are as follows: ensuring the cleanliness of the environment, selecting an environment with low abundance of experimental strains, and sterilizing all fermentation-related equipment and pipelines; establishing a daily inspection system for bacteriophage to detect bacteriophage infection in time and avoid large-scale outbreaks. This increases the additional production cost and additional production link of the preparation of fermentation products.

[0004] Isolating spontaneous mutant bacteriophage-resistant strains from original wild-type strains is a simple and natural method, and since it does not involve genetic manipulation, the use of these strains in industrial environments is not subject to regulatory restrictions. At present, using bacteriophage-resistant strains for fermentation is a good strategy to prevent bacteriophage contamination. Hyaluranic acid (HA) is a linear mucopolysaccharide with excellent moisturizing, lubricating, viscoelastic and biocompatibility, which is composed of UDP-N-acetylglucosamine (UDP-GlcNAc) and D-glucuronic acid (UDP-GlcUA) as a double sugar unit repeated in series, and is widely used in the fields of medicine, plastic surgery, cosmetics, etc. At present, HA is mainly obtained by fermentation of Streptococcus equisubsp. zooepidemicus, which has HA synthesis-related genes and can efficiently synthesize HA, but there is no report on bacteriophage-resistant Streptococcus equisubsp. zooepidemicus strains in the prior art. SUMMARY

[0005] The purpose of the present application is to provide a strain of Streptococcus equisubsp. zooepidemicus resistant to bacteriophage, which is deposited in the China General Microbiological Culture Collection Center with the accession number of CGMCC No. 26991, and the resistance is stably inherited. The strain not only has good resistance to bacteriophage, but also has excellent high-yield hyaluronic acid characteristics, and can be used as a hyaluronic acid industrial fermentation strain.

[0006] On the one hand, this application provides a strain of Streptococcus equisubsp. zooepidemicus, which is resistant to bacteriophages and is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26991.

[0007] On the other hand, this application also provides a microbial preparation containing the aforementioned Streptococcus equi subsp. veterinaria.

[0008] On the other hand, this application also provides the use of Streptococcus equi subsp. veterinaria or its microbial preparations in the production of hyaluronic acid or its salts.

[0009] On the other hand, this application provides a method for fermenting to produce hyaluronic acid or its salts, wherein hyaluronic acid or its salts are produced by fermenting with the above-described Streptococcus equi subsp. vesicerae or its microbial preparation.

[0010] Furthermore, the cultivation method includes: inoculating the above-mentioned Streptococcus equi subsp. veterinaria into a fermentation medium for fermentation culture.

[0011] Furthermore, the fermentation temperature is 30℃-37℃, specifically, the fermentation temperature can be 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃ and any value between them.

[0012] Furthermore, the culture time is 8h-20h, specifically, the culture time can be 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h and any value between them.

[0013] Furthermore, the rotational speed is 200-300 rpm, specifically 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 rpm and any speed in between.

[0014] In a preferred embodiment, a method for producing hyaluronic acid or its salt by fermentation includes the following steps:

[0015] Step 1, Seed Culture Stage: Inoculate Streptococcus equi subsp. veterinaria with seed culture medium at 30℃-37℃ and 200-300 rpm until OD (Organic Depth) is reached. 600 = 0.1-0.5, to obtain seed liquid; more preferably, the temperature is 37℃ and the speed is 200rpm;

[0016] Step 2, Fermentation stage: Inoculate the above seed liquid into the fermentation medium at an inoculation rate of 2-8%, and culture at a temperature of 30℃-37℃ and a speed of 200-300 rpm for 8-18 hours; more preferably, the inoculation rate of the seed liquid is 6%, and the culture is carried out at a temperature of 37℃ and a speed of 200 rpm for 10-18 hours.

[0017] In a preferred embodiment, a method for producing hyaluronic acid or its salt by fermentation includes the following steps:

[0018] Step 1, Seed Culture Stage: Inoculate Streptococcus equi subsp. veterinaria with seed culture medium at 30℃-37℃ and 200-300 rpm until OD (Organic Depth) is reached. 600 =0.1-0.5 to obtain seed liquid; preferably, the temperature is 37℃ and the speed is 200rpm;

[0019] Step 2, First Fermentation Stage: Inoculate the above seed liquid into the fermentation medium at an inoculation rate of 2-8%, and culture for 8-18 hours at a temperature of 30℃-37℃ and a speed of 400-800 r / min; preferably, the inoculation rate of the seed liquid is 6%, and the culture is carried out for 12 hours at a temperature of 36.5℃, a speed of 400 rpm, and a dissolved oxygen level of 3 vvm.

[0020] Step 3, Second fermentation stage: Continue fermentation for 8-18 hours at a temperature of 30℃-37℃ and a speed of 400-800 rpm; preferably, culture for 12 hours at a temperature of 36.5℃, a speed of 800 rpm, and a dissolved oxygen level of 3 vvm.

[0021] Preferably, the pH is adjusted to 7 during the fermentation process.

[0022] In a preferred embodiment, a sodium hydroxide solution is used to control the pH.

[0023] Preferably, the seed culture medium formula is: 5-10 g / L glucose, 8-15 g / L peptone, 0.5-1.5 g / L potassium dihydrogen phosphate, 0.5-2 g / L magnesium sulfate heptahydrate, and 3-15 g / L yeast extract. If solid culture medium is required, 1.5-2% agar is added. After the culture medium is prepared, the sterilization temperature is 115°C and the sterilization time is 20 minutes.

[0024] The glucose concentration in the seed culture medium formulation can be any value among 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, and 10 g / L.

[0025] The peptone concentration in the seed culture medium formulation can be any value among 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, and 15 g / L.

[0026] The concentration of potassium dihydrogen phosphate in the seed culture medium formulation can be any value among 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, and 1.5 g / L.

[0027] The concentration of magnesium sulfate heptahydrate in the seed culture medium formulation can be any value among 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, and 2 g / L.

[0028] The concentration of yeast powder in the seed culture medium formulation can be any value among 3g / L, 4g / L, 5g / L, 6g / L, 7g / L, 8g / L, 9g / L, 10g / L, 11g / L, 12g / L, 13g / L, 14g / L, and 15g / L.

[0029] If it is a solid, agar (concentration) of 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, or 2 g / L can be added.

[0030] Preferably, the fermentation medium is formulated as follows: peptone 12-20 g / L, yeast powder 10-20 g / L, dipotassium hydrogen phosphate 1-3 g / L, magnesium sulfate 1-2 g / L, glucose 60-80 g / L, and sodium glutamate 5-15 g / L; the medium is sterilized at 121°C for 30 minutes after preparation.

[0031] The peptone concentration in the fermentation medium formulation can be any value among 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, and 20 g / L.

[0032] The yeast concentration in the fermentation medium formulation can be any value among 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, and 20 g / L.

[0033] The concentration of dipotassium hydrogen phosphate in the fermentation medium formulation can be any value among 1 g / L, 2 g / L, and 3 g / L.

[0034] The magnesium sulfate concentration in the fermentation medium formulation can be any value among 1 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, and 2 g / L.

[0035] The glucose concentration in the fermentation medium formulation can be any value among 60 g / L, 61 g / L, 62 g / L, 63 g / L, 64 g / L, 65 g / L, 66 g / L, 67 g / L, 68 g / L, 69 g / L, 70 g / L, 71 g / L, 72 g / L, 73 g / L, 74 g / L, 75 g / L, 76 g / L, 77 g / L, 78 g / L, 79 g / L, and 80 g / L.

[0036] The sodium glutamate concentration in the fermentation medium formulation can be any value among 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, and 15 g / L.

[0037] The solvent for the above-mentioned culture medium can be water.

[0038] The yield of hyaluronic acid or its salt obtained by fermentation using the method described in this application can reach more than 12 g / L.

[0039] On the other hand, this application also provides hyaluronic acid or its salt prepared by fermentation using the above-described method.

[0040] On the other hand, this application also provides a composition comprising the above-described hyaluronic acid or a salt thereof.

[0041] On the other hand, this application also provides the application of the above-mentioned Streptococcus equi subsp. veterinaria, or the above-mentioned microbial preparations, or the above-mentioned hyaluronic acid or its salts, or the above-mentioned compositions in the chemical, pharmaceutical and / or cosmetic fields.

[0042] The beneficial effects of this invention are as follows: This invention obtains a high-hyaluronic acid-producing mutant strain of Streptococcus equi subsp. zooepidemicus, which was screened using a phage secondary infection method. After resistance and genetic stability experiments, it was proven that the screened strain is a phage-resistant strain, and the resistance can be stably inherited. It not only has good resistance to phages, but also has excellent high-hyaluronic acid production characteristics, and can be used as a hyaluronic acid industrial fermentation strain.

[0043] Biological Preservation Information

[0044] Streptococcus equi subsp. zooepidemicus was deposited on March 31, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 26991. Attached Figure Description

[0045] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0046] Figure 1 This is a diagram of colony morphology;

[0047] Figure 2 This is the HA standard curve. Detailed Implementation

[0048] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0049] Unless otherwise specified in the examples, the conditions shall be performed according to the standard conditions or the conditions recommended by the manufacturer.

[0050] Unless otherwise specified, in the following embodiments, reagents or instruments whose manufacturers are not indicated are all conventional products that can be purchased commercially.

[0051] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0052] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0053] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in the fields of microbiology, biochemistry, analytical chemistry, cell culture, and related areas.

[0054] Phage mixture (as a screening pressure, derived from the fermentation broth of phages contaminated during the hyaluronic acid production process).

[0055] The seed culture medium formula is: 5 g / L glucose, 10 g / L peptone, 1 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, and 5 g / L yeast extract. If solid culture medium is required, add 2% agar. After the culture medium is prepared, sterilize it at 115℃ for 20 minutes.

[0056] The fermentation medium formula is as follows: 15 g / L peptone, 10 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 1.5 g / L magnesium sulfate, 70 g / L glucose, and 5 g / L sodium glutamate. After the medium is prepared, it is sterilized at 121℃ for 30 minutes.

[0057] Example 1: Selection of antiphage strains

[0058] A phage-resistant mutant strain of *Streptococcus equi* subsp. *porcineus* was obtained using a secondary infection method. The specific process is as follows:

[0059] Streptococcus equi subsp. veterinaria was inoculated into seed culture medium at a volume ratio of 2% and cultured at 37°C until OD500. 600 ≈0.5, inoculate 500μL of phage mixture, and incubate at 37℃ until clear. The OD of the clear culture medium is... 600 It is 0.143.

[0060] The clarified culture medium was incubated for another 12 hours. Then, a small amount of culture medium was streaked onto seed agar using an inoculation loop, and the plates were incubated at 37°C for another 12 hours. After incubation, colonies formed on the seed agar. A single, uniform colony was picked up with an inoculation loop and streaked onto seed agar. The resulting plates were incubated at 37°C as described above. Through resistance testing, a phage-resistant and stably heritable mutant strain of *Streptococcus equi* subsp. *porphyria* was successfully screened. After the strain grew well, colony morphology was observed and recorded (see...). Figure 1 ).according to Figure 1 It can be seen that the colony morphology of this strain is round, the colony surface is smooth and moist, and the edges are neat.

[0061] Example 2: Phage Resistance Detection and Genetic Stability Experiment

[0062] The resistance and genetic stability of single colonies of the strain obtained in Example 1 were tested, and the specific methods are as follows:

[0063] (1) First transfer: Inoculate a single colony of the above strain into the seed culture medium at a volume ratio of 2% and culture at 37°C until OD. 600 ≈0.5, inoculate with phage mixture, the inoculation volume of phage mixture is 500μL, and then incubate at 37℃ for 12h to obtain culture medium;

[0064] (2) Second transfer: The culture medium obtained in step (1) was inoculated into a new seed culture medium at a volume ratio of 2%, and cultured at 37°C until OD. 600 ≈0.5, inoculate with phage mixture, the inoculation volume of phage mixture is 500μL, incubate at 37℃ for 12h to obtain culture medium.

[0065] Repeat the above process until the strain has been continuously transferred ten times in the seed culture medium.

[0066] Using a culture medium without phage mixture as a control group, the strain was continuously subcultured ten times in seed culture medium according to the above method.

[0067] The growth of the above-mentioned strains in the presence of phage mixtures was compared using the plate colony counting method. Under the condition of adding phage mixtures, the OD of the culture medium obtained after the tenth subculturing of the above-mentioned strains in seed medium was [not specified]. 600 The value was 1.74, and the viable count increased from the initial 10. 8 cfu / mL increased to 10 10 The cfu / mL indicates that the above strain can grow in the presence of bacteriophages and is not lysed by them.

[0068] The growth of the above strains in the absence of phage mixture was compared using the plate colony counting method. No phage mixture was added to the culture medium. The culture medium obtained after the tenth subculturing of the above strains in seed medium had an OD value of [missing value]. 600 The value was 1.77, and the viable count increased from the initial 10. 8 CFU / mL increased to 1.15 × 10⁻⁶ 10 cfu / mL.

[0069] The above resistance and genetic stability experiments proved that the strains were phage resistant and that the resistance could be stably inherited.

[0070] The genome of the above-mentioned phage-resistant mutant strain was sequenced using whole-genome sequencing. The whole-genome sequencing was commissioned to BGI Genomics Co., Ltd. in Shenzhen. The 16S rRNA sequence of the above-mentioned strain was compared with data in the NCBI NT database to determine the strain's classification information. The identification results showed that the above-mentioned strain is *Streptococcus equi subsp. zooepidemicus*, and the 16S rRNA sequence of the strain has approximately 100% similarity to that of *Streptococcus equi subsp. zooepidemicus*. Its 16S rRNA sequence is shown in SEQ ID NO:1 and was deposited on March 31, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 26991.

[0071]

[0072] Example 3: Fermentation yield of mutant strain HA

[0073] (1) Select a single colony of the phage-resistant mutant strain CGMCC No. 26991 and inoculate it into seed culture medium. After incubation at 37℃ and 200 rpm for 12 h, transfer 1 mL of fresh bacterial culture to 200 mL of seed culture medium and incubate until OD500. 600 =0.2 (approximately 3.5-6 hours). The entire seed culture medium was inoculated into 3.5 L of fermentation medium. Fermentation was carried out at 400 rpm, 36.5℃, and dissolved oxygen 3 vvm for 12 hours. The fermentation speed was then adjusted to 800 rpm, and fermentation continued for another 12 hours to obtain the fermentation broth. During fermentation, a 40% sodium hydroxide solution was used to maintain the pH at 7.

[0074] (2) Detection of hyaluronic acid fermentation broth yield

[0075] The HA is broken down into disaccharides using enzymatic hydrolysis. The specific method is as follows:

[0076] Standard curve preparation: Accurately weigh 0.2g of HA powder and accurately pipette 10mL of purified water. Dissolve at 26℃ and 200rpm. After shaking well, accurately pipette 10mL of the HA solution and add water to bring the volume to 100mL. Pipe 0.0, 0.4, 0.8, 1.2, 1.6, and 2mL of the HA solution into test tubes respectively, add water to each to bring the volume to 2mL, add 8mL of 5mM PBS buffer, and then add 3mL of HA solution and 100uL of HA enzyme to each test tube. Mix well. Incubate at 42℃ for 1h. After enzymatic digestion, boil for 2min and measure the absorbance at 232nm. Figure 2 As shown, the HA standard curve is plotted with absorbance on the vertical axis and HA content on the horizontal axis.

[0077] The specific method for detecting the HA content in the fermentation broth is as follows:

[0078] Accurately pipette 2 mL of the fermentation broth from step (1), add 15 mL of anhydrous ethanol for precipitation, centrifuge at 13000 rpm for 5 min, discard the supernatant, add 10 mL of purified water, dissolve at 26℃ and 200 rpm to obtain a 5× dilution. Then pipette 1 mL of the 2× dilution and add 19 mL of 5 mM PBS buffer to obtain a 100× dilution. Take 3 mL of HA 10× dilution and 100 μL of HA enzyme, mix well; treat at 42℃ for 1 h; after enzymatic digestion, boil for 2 min. Two parallel groups were set up.

[0079] Absorbance measurement: The absorbance was measured at 232 nm, and the obtained absorbance was used to calculate the HA content in the fermentation broth by substituting it into the standard curve formula y = 11.502x - 0.0192. The calculated results are shown in Table 1. The HA fermentation yield of the phage-resistant mutant strain reached 12-13 g / L in a 5L fermenter.

[0080] Table 1. HA production of the mutant strain

[0081]

[0082]

[0083] This patent is the first to report a phage-resistant mutant strain of Streptococcus equi subsp. vesicans, CGMCC No. 26991. Compared with the wild strain, this strain has good phage resistance characteristics and a high HA yield.

[0084] Comparative Example 1: Detection and genetic stability of phage resistance to Streptococcus equi subsp. 39920 (ATCC39920)

[0085] Resistance and genetic stability experiments were performed on single colonies of Streptococcus equi strain ATCC39920 (purchased from Ningbo Mingzhou Technology Co., Ltd.). The specific methods are as follows:

[0086] (1) First transfer: Inoculate a single colony of the above strain into the seed culture medium at a volume ratio of 2% and culture at 37°C until OD. 600 ≈0.5, inoculate with phage mixture, the inoculation volume of phage mixture is 500μL, and then incubate at 37℃ for 12h to obtain culture medium;

[0087] (2) Second transfer: The culture medium obtained in step (1) was inoculated into a new seed culture medium at a volume ratio of 2%, and cultured at 37°C until OD. 600 ≈0.5, inoculate with phage mixture, the inoculation volume of phage mixture is 500μL, incubate at 37℃ for 12h to obtain culture medium.

[0088] Repeat the above process until the strain has been continuously transferred ten times in the seed culture medium.

[0089] Using a culture medium without phage mixture as a control group, the strain was continuously subcultured ten times in seed culture medium according to the above method.

[0090] Using a culture medium without phage mixture as a control group, the strain was continuously subcultured ten times in seed culture medium according to the above method.

[0091] The growth of the ATCC39920 strain in the presence of phage mixture was compared using the plate colony counting method. Under the phage mixture condition, the culture medium obtained after the tenth subculture of the above strain in seed medium showed the following OD values: 600 The value was 0.057, and the viable cell count increased from the initial 5 x 10⁻⁶. 7 The decrease in cfu / mL to 0 cfu / mL indicates that the above strain could not grow in the presence of bacteriophage and was lysed by bacteriophage.

[0092] The growth of the ATCC39920 strain was compared using the plate count method under conditions without phage mixture. The culture medium did not contain phage mixture; the culture medium obtained after the tenth subculture of the above strain in seed medium had an OD of [missing value]. 600 The value was 0.874, and the viable count increased from the initial 5 x 10⁻⁴. 7 CFU / mL increased to 5.5 x 10⁻⁶ 9 cfu / mL.

[0093] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A phage-resistant Streptococcus equi subsp. The genera *zooepidemicus* is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26991.

2. A microbial preparation containing the Streptococcus equi subsp. veterinaria as described in claim 1.

3. The use of Streptococcus equi subsp. veterinaria as described in claim 1 or the microbial preparation as described in claim 2 in the production of hyaluronic acid or its salts.

4. A method for producing hyaluronic acid or its salt by fermentation, characterized in that, Hyaluronic acid or its salts can be produced by fermentation using Streptococcus equi subsp. veterinaria as described in claim 1 or the microbial preparation as described in claim 2.

5. A hyaluronic acid or a salt thereof obtained by fermentation as described in claim 4.

6. A composition, characterized in that, The composition comprises the hyaluronic acid or a salt thereof as described in claim 5.

7. The use of Streptococcus equi subsp. veterinaria as described in claim 1, or the microbial preparation as described in claim 2, or the hyaluronic acid or its salt as described in claim 5, or the composition as described in claim 6, in the chemical, pharmaceutical and / or cosmetic fields.

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