Streptococcus equi subsp. Zooepidemicus, application thereof and method for producing hyaluronic acid

Abstract

The invention discloses streptococcus equi subsp. Zooepidemicus, application of the streptococcus equi subsp. Zooepidemicus and a method for producing hyaluronic acid, and belongs to the technical field of microorganisms. The streptococcus equi subsp. Zooepidemicus disclosed by the invention is streptococcus equi subsp. Zooepidemicus SYMY-010, the strain is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms on December 26, 2022, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) NO.26282. The streptococcus equi subsp. Zooepidemicus SYMY-010 disclosed by the invention can be used for preparing the streptococcus equi subsp. Zooepidemicus SYMY-010. The streptococcus equi subsp. Zooepidemicus SYMY-010 is inoculated into a fermentation culture medium for fermentation to produce the hyaluronic acid, the yield of the hyaluronic acid is up to about 15g / L, the average molecular weight of the obtained hyaluronic acid is 0.47 * 10 < 6 > Da, and meanwhile, the hyaluronic acid has good hereditary stability and better acid resistance.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly relates to a Streptococcus equi subsp. zooepidemicus, its application, and a method for producing hyaluronic acid. Background Art

[0002] The information disclosed in the background art of the present invention is only intended to enhance the overall understanding of the present invention, and is not necessarily regarded as an admission or an indication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Hyaluronic acid, also known as hyaluronan or hyaluronic acid, is a linear polysaccharide formed by D-glucuronic acid and N-acetylglucosamine through β-1,3 glycosidic bonds to form disaccharide units, and then repeatedly linked through β-1,4 glycosidic bonds. Due to its excellent moisture retention and viscoelastic properties, as well as its non-immunogenicity and non-toxicity, hyaluronic acid is widely used in the daily chemical industry. In addition, hyaluronic acid is widely distributed in the extracellular matrix of soft connective tissues. As a polysaccharide naturally contained in the human body, it has good biocompatibility and biodegradability, and is therefore also widely used in the field of clinical medicine.

[0004] The production methods of hyaluronic acid mainly include two types: animal tissue extraction method and microbial fermentation method. The former has high production costs and is prone to immune reactions due to factors such as limited sources and low yields. Therefore, at present, the production of hyaluronic acid is mainly carried out by fermentation. Currently, the main production strains of hyaluronic acid in industry include Streptococcus zooepidemicus, Streptococcus equi, etc. The hyaluronic acid obtained by fermentation can be divided into 4 types according to molecular weight: high molecular weight hyaluronic acid (HMWHA), medium molecular weight hyaluronic acid (MMWHA), low molecular weight hyaluronic acid (LMWHA), and hyaluronic acid oligosaccharide (o-HA). Among them, the average molecular weight of HMWHA is generally greater than 1×10 6 Da; MMWHA is 0.5×10 6 Da~1×10 6 Da; LMWHA is 0.01×10 6 Da~0.5×10 6 Da, and o-HA is less than 0.01×10 6 Da. Among them, low molecular weight hyaluronic acid (LMWHA) has functions such as strong permeability, anti-inflammatory and antioxidant functions, and promoting repair.

[0005] Currently, when the Streptococcus equi subsp. zooepidemicus disclosed in the prior art is used to produce hyaluronic acid, the yield of hyaluronic acid is often lower than 10 g / L, which restricts the production of hyaluronic acid and its application in the daily chemical industry and the medical field. Therefore, it is necessary to develop a Streptococcus equi subsp. zooepidemicus with a higher yield of hyaluronic acid. Summary of the Invention

[0006] In view of this, the present invention provides a Streptococcus equi subsp. zooepidemicus and its application and a method for producing hyaluronic acid. Using the Streptococcus equi subsp. zooepidemicus of the present invention to produce hyaluronic acid, the yield of hyaluronic acid in a 250L fermenter can reach about 15 g / L, and it still maintains a high yield of hyaluronic acid at pH 5, showing good acid resistance.

[0007] In the first aspect, the present invention provides a Streptococcus equi subsp. zooepidemicus, which is Streptococcus equi subsp. zooepidemicus ( Streptococcus equi subsp. zooepidemicus ) SYMY-010. This strain was deposited with the China General Microbiological Culture Collection Center on December 26, 2022, and the deposit number is CGMCC NO. 26282.

[0008] The Streptococcus equi subsp. zooepidemicus SYMY-010 provided by the present invention was screened from a ditch near Binbei Farm in Binzhou, Shandong. It belongs to Streptococcus equi subsp. zooepidemicus through morphological and molecular biological identification.

[0009] In the second aspect, the present invention provides the application of the above Streptococcus equi subsp. zooepidemicus, and the application is the application in the production of hyaluronic acid.

[0010] In the third aspect, the present invention provides a method for producing hyaluronic acid, and the method includes: Inoculating the above Streptococcus equi subsp. zooepidemicus SYMY-010 onto a slant medium for slant activation culture, and then inoculating the activated bacteria into a seed medium for scale-up culture to obtain a seed liquid; inoculating the seed liquid into a fermentation medium for fermentation culture, and purifying to obtain hyaluronic acid.

[0011] Preferably, the temperature of the fermentation culture is 35-38°C, the time of the fermentation culture is 18-24h; the inoculation amount of inoculating the seed liquid into the fermentation medium is 2-5%.

[0012] Preferably, the composition of the fermentation medium is: glucose 22-28 g / L, peptone 10-18 g / L, yeast extract powder 8-12 g / L, dipotassium hydrogen phosphate trihydrate 2-6 g / L, magnesium sulfate heptahydrate 1-5 g / L, the solvent is water, and the pH is 5.0-7.0; adding a glucose solution during the fermentation process to make the total sugar content in the medium 45-55 g / L.

[0013] Preferably, the composition of the slant medium is: glucose 3-7 g / L, peptone 6-9 g / L, yeast extract powder 3-7 g / L, dipotassium hydrogen phosphate trihydrate 2-3 g / L, magnesium sulfate heptahydrate 0.5-1.5 g / L, agar 18-22 g / L, the solvent is water, and the pH is 6.8-7.2.

[0014] Preferably, the composition of the seed medium is as follows: glucose 3 - 7 g / L, peptone 6 - 9 g / L, yeast extract powder 3 - 7 g / L, dipotassium hydrogen phosphate trihydrate 2 - 3 g / L, magnesium sulfate heptahydrate 0.5 - 1.5 g / L, the solvent is water, and the pH is 6.8 - 7.2.

[0015] Preferably, the temperature for slant activation culture is 34 - 38 °C, and the time for slant activation culture is 20 - 30 h.

[0016] Preferably, the temperature for enlarged culture is 34 - 38 °C, and the time for enlarged culture is 40 - 50 h. More preferably, the specific steps for enlarged culture are as follows: inoculate the activated bacteria into a test tube containing the seed medium at an inoculation amount of 1 - 4%, and culture at 180 - 250 rpm and 34 - 38 °C for 20 - 25 h; then transfer to a shake flask containing the seed medium at an inoculation amount of 1 - 4%, and culture at 180 - 250 rpm and 34 - 38 °C for 20 - 25 h to obtain a seed solution.

[0017] Preferably, the purification step is as follows: add an aqueous solution of sodium dodecyl sulfate to the fermentation broth, treat and then centrifuge, collect the supernatant, resuspend with absolute ethanol, then centrifuge to collect the precipitate, resuspend with a sodium chloride solution, add cetylpyridinium chloride, stir and let stand, then centrifuge to obtain a complex precipitate, resuspend with a sodium chloride solution, add absolute ethanol and let stand, centrifuge to collect the precipitate, and freeze-dry to obtain hyaluronic acid.

[0018] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) Aiming at the problems of low yield and high cost in the existing production route of fermenting hyaluronic acid by Streptococcus equi subsp. zooepidemicus, through a large number of screenings, a strain of Streptococcus equi subsp. zooepidemicus SYMY - 010 was obtained by screening in a ditch near Binbei Farm in Binzhou, Shandong. It was inoculated into a fermentation medium for fermenting hyaluronic acid, and the yield of hyaluronic acid was as high as about 15 g / L, significantly higher than the reported strains of Streptococcus equi subsp. zooepidemicus producing hyaluronic acid; the average molecular weight of the obtained hyaluronic acid was 0.47×10 6 Da, the fermentation time was shortened to within 24 h, and Streptococcus equi subsp. zooepidemicus SYMY - 010 had good genetic stability.

[0019] (2) The method for preparing hyaluronic acid product of the present invention has a high yield, and when the pH drops to 5, the yield of hyaluronic acid can still be maintained above 14 g / L, having good acid resistance; low cost, simple preparation process, can improve the industrial efficiency in the market of medium and low molecular weight hyaluronic acid, is suitable for industrial production, and has broad application prospects. Detailed implementation manners

[0020] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention pertains.

[0021] The technical solution of the present invention will be further elaborated below in conjunction with specific embodiments. Unless otherwise specified, the present invention has no special restrictions on the sources of the reagents used in the following embodiments, and commercially available products well-known to those skilled in the art can be used.

[0022] In the following embodiments, the specific culture media used are as follows: (1) The composition of the slant culture medium or solid culture medium is as follows: glucose 5 g / L, peptone 7.5 g / L, yeast extract powder 5 g / L, dipotassium hydrogen phosphate trihydrate 2.5 g / L, magnesium sulfate heptahydrate 1 g / L, agar 20 g / L, the solvent is water, and the pH is 7.0.

[0023] (2) The composition of the seed culture medium is as follows: glucose 5 g / L, peptone 7.5 g / L, yeast extract powder 5 g / L, dipotassium hydrogen phosphate trihydrate 2.5 g / L, magnesium sulfate heptahydrate 1 g / L, the solvent is water, and the pH is 7.0.

[0024] (3) The composition of the fermentation culture medium is as follows: glucose 25 g / L, peptone 15 g / L, yeast extract powder 10 g / L, dipotassium hydrogen phosphate trihydrate 5 g / L, magnesium sulfate heptahydrate 2 g / L, the solvent is water, and the pH is 7.0.

[0025] Example 1 This example provides the screening of strains.

[0026] (1) Dilute the samples collected from the natural environment 10 times with sterile water, take 0.5 mL of the samples and spread them on the solid culture medium, culture at 37 °C for 24 h, select the strains with colony morphology similar to Streptococcus equi subsp. zooepidemicus, pick them out with a sterile toothpick, and inoculate them into a 24-well deep-well plate containing the fermentation culture medium. The liquid loading volume in each well is 2 mL, and after culturing at 37 °C and a rotation speed of 250 rpm for 20 h, detect the content of hyaluronic acid in the fermentation broth.

[0027] (2) Detection method for hyaluronic acid (GA) content: After centrifuging the fermentation broth in step (1), transfer the supernatant to a new 24-well plate. Add 2 mL of absolute ethanol to each well, shake well, and let it stand for 30 min. After centrifuging again, discard the supernatant. Add 2 mL of deionized water to each well of the precipitate to resuspend the precipitate, and take out a part of the solution and transfer it to a new well plate, dilute it with water to 1 mL to prepare the sample to be tested. Place the well plate in an ice-water bath. Slowly add 2 mL of precooled sodium tetraborate sulfuric acid solution with a concentration of 19.08 g / L, shake well, put it in boiling water and boil for about 12 min, and then cool it in an ice-water bath. Then add 0.1 mL of carbazole ethanol solution with a concentration of 1.25 g / L to each well, shake well, put it in boiling water and boil for about 12 min, and then cool it in an ice-water bath. After the reaction, hyaluronic acid reacts to form glucuronic acid, and glucuronic acid has an absorption wavelength at 530 nm. Calculate the content of hyaluronic acid according to the content of glucuronic acid, as shown in formula ①. Take part of the sample in the well plate and measure the absorbance at 530 nm. At the same time, set up a control group and a standard product.

[0028] Glucuronic acid (g / L) = 2.07 × n × GA (g / L) Formula ①.

[0029] In formula ①, n is the sample dilution factor; 2.07 is obtained by dividing the relative molecular mass 401.3 of the repeating disaccharide unit of hyaluronic acid by the relative molecular mass 194.1 of GA under theoretical conditions.

[0030] From more than 100 samples, more than 2,000 strains were screened in total. Finally, a strain of Streptococcus equi subsp. zooepidemicus with high activity was screened from the ditch near Binbei Farm in Binzhou, Shandong. This strain was named Streptococcus equi subsp. zooepidemicus ( Streptococcus equi subsp. zooepidemicus ) SYMY-010, which was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on December 26, 2022, with the deposit number CGMCC NO. 26282.

[0031] Example 2 This example provides a method for producing hyaluronic acid using Streptococcus equi subsp. zooepidemicus SYMY-010 screened in Example 1.

[0032] (1)Slant activation culture: Inoculate the Streptococcus equi subsp. zooepidemicus SYMY-010 strain on a slant medium and culture it at 37 °C for 24 h to obtain the activated slant bacteria.

[0033] (2)Seed activation culture: Inoculate the Streptococcus equi subsp. zooepidemicus SYMY-010 obtained in step (1) into a test tube containing seed medium at an inoculation amount of 2%, culture at 250 rpm and 37°C for 24 h; then transfer it to a flask containing seed medium at an inoculation amount of 2% and culture at 200 rpm and 37°C for 24 h to obtain a seed solution.

[0034] (3)Fermentation culture: Inoculate the seed solution obtained in step (2) into the fermentation medium. The fermentation is carried out in a 250 L fermenter with a liquid volume of 150 L, an inoculation amount of 3%, a set fermentation temperature of 37°C, a rotation speed of 500 rpm, control the fermentation pH = 7.0, control the dissolved oxygen at about 35%, and add a glucose solution during the fermentation process with an addition amount of 25 g / L. Ferment for 20 h. When the residual sugar in the fermentation broth is lower than 1 g / L, the fermentation can be ended. After the fermentation ends, the content of hyaluronic acid is detected to be 15 g / L.

[0035] (4)Purification and extraction of hyaluronic acid: Add an aqueous solution of 0.6 wt% sodium dodecyl sulfate (SDS) to the fermentation broth at the end of fermentation in step (3) and treat for 30 min. After the treatment, centrifuge the fermentation broth to remove the precipitate and bacteria. Add anhydrous ethanol twice the volume of the solution to the supernatant of the obtained fermentation broth for resuspension, stir and treat for 8 h. Centrifuge the resuspended fermentation broth again to collect the precipitate. Resuspend the precipitate with a 5 g / L NaCl solution, add cetylpyridinium chloride with a final concentration of 1.5 wt% to the solution, stir and let stand for 3 h. After centrifuging the solution, a complex precipitate is obtained. Resuspend the complex precipitate with a 10 g / L NaCl solution, then add anhydrous ethanol twice the volume of the solution, let stand for 0.5 h, centrifuge to collect the precipitate, and then obtain hyaluronic acid through freeze-drying. After detection, the average molecular weight of the obtained hyaluronic acid is 0.47×10 6 Da.

[0036] Example 3 This example provides a genetic stability test of the Streptococcus equi subsp. zooepidemicus SYMY-010 screened in Example 1.

[0037] The Streptococcus equi subsp. zooepidemicus SYMY-010 was streaked on a solid medium to obtain the first-generation strain. According to this method, the first-generation strain was continuously streaked to obtain the second-generation strain, and so on to obtain the third, fourth, fifth, sixth, seventh, and eighth-generation strains. Each generation of the strain was activated and inoculated into a fermentation medium, and the fermentation was carried out in a 5 L fermenter. The inoculation amount was 3%, the fermentation temperature was set at 37 °C, the rotation speed was 500 rpm, the fermentation pH was controlled at 7.0, the dissolved oxygen was controlled at about 35%, and a glucose solution was added during the fermentation process, with the addition amount being 25 g / L. The fermentation was carried out for 20 h, and the fermentation could be ended when the residual sugar in the fermentation broth was lower than 1 g / L. After the fermentation was completed, the content of hyaluronic acid was detected, and the detection results are shown in Table 1.

[0038] Table 1 The yields of hyaluronic acid of strains with different generations

[0039] As can be seen from Table 1, with the increase in the number of passages, the yield of hyaluronic acid was relatively stable, indicating that the Streptococcus equi subsp. zooepidemicus SYMY-010 had good genetic stability.

[0040] Example 4 This example provides the acid tolerance verification of the Streptococcus equi subsp. zooepidemicus SYMY-010 screened in Example 1.

[0041] The process of producing hyaluronic acid in this example was the same as that in Example 2. The fermentation process was carried out in a 5 L fermenter, and the pH of the fermentation medium was controlled at 5 - 7. After the fermentation was completed, the yield of hyaluronic acid was measured. The detection results are shown in Table 2.

[0042] Table 2 The results of the acid tolerance experiment of the strain

[0043] Judging from the detection results, for the Streptococcus equi subsp. zooepidemicus SYMY-010 strain, as the pH of the fermentation medium decreased, the yield of hyaluronic acid showed a downward trend. However, when the pH dropped to 5, the yield of hyaluronic acid could still be maintained at 14.4 g / L, indicating that the Streptococcus equi subsp. zooepidemicus SYMY-010 had good acid tolerance.

[0044] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A Streptococcus equi subsp. zooepidemicus, characterized in that, The Streptococcus equi subsp. zooepidemicus is Streptococcus equi subsp. zooepidemicus ( Streptococcus equi subsp. zooepidemicus ) SYMY-010, which was deposited in the China General Microbiological Culture Collection Center on December 26, 2022, with the deposit number of CGMCC NO. 26282.

2. Use of Streptococcus equi subsp. zooepidemicus according to claim 1, characterized in that, The application is the application in the production of hyaluronic acid.

3. A method for producing hyaluronic acid, characterized in that, The method includes: Inoculating Streptococcus equi subsp. zooepidemicus SYMY-010 described in claim 1 onto a slant medium for slant activation culture, and then inoculating the activated bacterial cells into a seed medium for enlarged culture to obtain a seed solution; inoculating the seed solution into a fermentation medium for fermentation culture, and purifying to obtain hyaluronic acid.

4. The method according to claim 3, wherein The temperature of the fermentation culture is 35-38 °C, and the time of the fermentation culture is 18-24 h; the inoculation amount of inoculating the seed solution into the fermentation medium is 2-5%.

5. The method according to claim 3, characterized in that, The composition of the fermentation medium is: 22-28 g / L of glucose, 10-18 g / L of peptone, 8-12 g / L of yeast extract powder, 2-6 g / L of dipotassium hydrogen phosphate trihydrate, 1-5 g / L of magnesium sulfate heptahydrate, the solvent is water, and the pH is 5.0-7.0; adding a glucose solution during the fermentation process to make the total sugar content in the medium 45-55 g / L.

6. The method according to claim 3, characterized in that The composition of the slant medium is: 3-7 g / L of glucose, 6-9 g / L of peptone, 3-7 g / L of yeast extract powder, 2-3 g / L of dipotassium hydrogen phosphate trihydrate, 0.5-1.5 g / L of magnesium sulfate heptahydrate, 18-22 g / L of agar, the solvent is water, and the pH is 6.8-7.

2.

7. The method according to claim 3, wherein The composition of the seed medium is: 3-7 g / L of glucose, 6-9 g / L of peptone, 3-7 g / L of yeast extract powder, 2-3 g / L of dipotassium hydrogen phosphate trihydrate, 0.5-1.5 g / L of magnesium sulfate heptahydrate, the solvent is water, and the pH is 6.8-7.

2.

8. The method according to claim 3, wherein The temperature of the slant activation culture is 34-38 °C, and the time of the slant activation culture is 20-30 h.

9. The method according to claim 3, wherein The temperature of the enlarged culture is 34-38 °C, and the time of the enlarged culture is 40-50 h.

10. The method according to claim 9, wherein The specific steps of the enlarged culture are: inoculating the activated bacterial cells into a test tube containing the seed medium at an inoculation amount of 1-4%, culturing at 180-250 rpm and 34-38 °C for 20-25 h; then transferring at an inoculation amount of 1-4% to a shake flask containing the seed medium, and culturing at 180-250 rpm and 34-38 °C for 20-25 h to obtain a seed solution.

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