Streptococcus equi subspecies zooepidemicus, its application and method for producing hyaluronic acid

Abstract

The present invention discloses a Streptococcus equi subspecies zooepidemicus and its application and a method for producing hyaluronic acid, belonging to the field of microbial technology. The Streptococcus equi subspecies zooepidemicus of the present invention is Streptococcus equi subspecies zooepidemicus ( Streptococcus equi subsp. zooepidemic ) SYMY-010, which was deposited in the General Microbiology Center of the China National Committee for the Collection of Microorganisms on December 26, 2022, with the deposit number CGMCC NO. 26282. In the present invention, Streptococcus equi subsp. zooepidemicus SYMY-010 was inoculated into a fermentation medium for fermentation to produce hyaluronic acid. The hyaluronic acid yield was as high as about 15 g / L, and the average molecular weight of the obtained hyaluronic acid was 0.47×10 6 Da, and has both good genetic stability and good acid resistance.

Description

Technical Field

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

[0002] The information disclosed in the background of the invention is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Hyaluronic acid, also known as glassy acid or hyaluronic acid, is a linear polysaccharide composed of D-glucuronic acid and N-acetylglucosamine linked via β-1,3-glycosidic bonds to form disaccharide units, which are then repeatedly linked via β-1,4-glycosidic bonds. Due to its excellent moisturizing and water-retaining properties, viscoelastic properties, and lack of immunogenicity and toxicity, hyaluronic acid is widely used in the daily chemical industry. Furthermore, hyaluronic acid is widely distributed in the extracellular matrix of soft connective tissue. As a polysaccharide naturally present in the human body, it possesses excellent biocompatibility and biodegradability, leading to its widespread use in clinical medicine.

[0004] The production methods of hyaluronic acid mainly include animal tissue extraction and microbial fermentation. The former has high production costs and is prone to immune reactions due to factors such as limited sources and low yields. Therefore, the current production of hyaluronic acid is mainly carried out by fermentation. At present, the main strains used to produce hyaluronic acid in industry include Streptococcus zooepidemicus and Streptococcus equi. Hyaluronic acid obtained by fermentation can be divided into four 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 oligosaccharides (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,o-HA is less than 0.01×10 6 Among them, low molecular weight hyaluronic acid (LMWHA) has strong permeability, anti-inflammatory and antioxidant functions, and promotes repair.

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

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

[0007] In a first aspect, the present invention provides a Streptococcus equi subspecies zooepidemicus, wherein the Streptococcus equi subspecies zooepidemicus is Streptococcus equi subspecies zooepidemicus ( Streptococcus equi subsp. zooepidemicus )SYMY-010, the strain was deposited in the General Microbiology Center of China Culture Collection Administration on December 26, 2022, with the deposit number CGMCCNO. 26282.

[0008] The Streptococcus equi subspecies zooepidemicus SYMY-010 provided by the present invention was obtained by screening in a ditch near the Binbei breeding farm in Binzhou, Shandong Province, and was identified as the Streptococcus equi subspecies zooepidemicus through morphological and molecular biological identification.

[0009] In a second aspect, the present invention provides an application of the aforementioned Streptococcus equi subspecies Zooepidemicus, wherein the application is an application in the production of hyaluronic acid.

[0010] In a third aspect, the present invention provides a method for producing hyaluronic acid, comprising:

[0011] The Streptococcus equi subsp. zooepidemicus SYMY-010 is inoculated into a slant culture medium for slant activation culture, and then the activated bacteria are inoculated into a seed culture medium for expansion culture to obtain a seed liquid; the seed liquid is inoculated into a fermentation culture medium for fermentation culture, and hyaluronic acid is obtained through purification.

[0012] Preferably, the fermentation culture temperature is 35-38° C., and the fermentation culture time is 18-24 h; and the inoculation amount of the seed liquid inoculated into the fermentation medium is 2-5%.

[0013] Preferably, the fermentation medium is composed of: 22-28 g / L glucose, 10-18 g / L peptone, 8-12 g / L yeast extract powder, 2-6 g / L dipotassium hydrogen phosphate trihydrate, 1-5 g / L magnesium sulfate heptahydrate, water as the solvent, and a pH of 5.0-7.0; glucose solution is added during the fermentation process to make the total sugar content in the culture medium 45-55 g / L.

[0014] Preferably, the composition of the slant culture 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.

[0015] Preferably, the seed culture medium is composed of: 3-7 g / L glucose, 6-9 g / L peptone, 3-7 g / L yeast extract powder, 2-3 g / L dipotassium hydrogen phosphate trihydrate, 0.5-1.5 g / L magnesium sulfate heptahydrate, the solvent is water, and the pH is 6.8-7.2.

[0016] Preferably, the temperature of the slant activation culture is 34-38° C., and the time of the slant activation culture is 20-30 h.

[0017] Preferably, the temperature of the expanded culture is 34-38°C, and the time of the expanded culture is 40-50 hours. More preferably, the specific steps of the expanded culture are: inoculating the activated bacteria into a test tube containing a seed culture medium at an inoculum size of 1-4%, and culturing at 180-250 rpm and 34-38°C for 20-25 hours; then transferring the inoculum size of 1-4% to a shake flask containing a seed culture medium, and culturing at 180-250 rpm and 34-38°C for 20-25 hours to obtain a seed solution.

[0018] Preferably, the purification step comprises: adding an aqueous solution of sodium lauryl sulfate to the fermentation broth, treating the broth, and centrifuging the broth, collecting the supernatant, resuspending the broth with anhydrous ethanol, and then centrifuging the precipitate, resuspending the broth with a sodium chloride solution, adding cetylpyridinium chloride, stirring and standing the broth, and then centrifuging the broth to obtain a complex precipitate, resuspending the broth with a sodium chloride solution, adding anhydrous ethanol, and standing the broth, centrifuging the precipitate, and freeze-drying the broth to obtain hyaluronic acid.

[0019] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0020] (1) The present invention addresses the problems of low yield and high cost in the existing hyaluronic acid production route produced by fermentation of Streptococcus equi subsp. Zooepidemicus. Through extensive screening, a strain of Streptococcus equi subsp. Zooepidemicus SYMY-010 was obtained from a ditch near Binbei Farm in Binzhou, Shandong Province. The strain was inoculated into a fermentation medium for fermentation to produce hyaluronic acid. The hyaluronic acid yield was as high as about 15 g / L, which was significantly higher than the existing reported hyaluronic acid-producing Streptococcus equi subsp. Zooepidemicus strains. The average molecular weight of the obtained hyaluronic acid was 0.47×10 6 Da, the fermentation time was shortened to less than 24 h, and Streptococcus equi subspecies zooepidemicus SYMY-010 had good genetic stability.

[0021] (2) The method of the present invention has a high yield in preparing hyaluronic acid products. Moreover, when the pH drops to 5, the yield of hyaluronic acid can still be maintained at above 14 g / L, and has good acid resistance. The method is low in cost and simple in preparation process. It can improve industrial efficiency in the low and medium molecular weight hyaluronic acid market, is suitable for industrial production, and has broad application prospects. DETAILED DESCRIPTION

[0022] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0023] The technical solution of the present invention is further described below with reference to specific examples. Unless otherwise specified, the present invention has no particular limitation on the sources of the reagents used in the following examples, and commercially available products known to those skilled in the art can be used.

[0024] In the following examples, the culture medium specifically used is as follows:

[0025] (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 5 g / L, potassium dihydrogen 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.

[0026] (2) The composition of the seed culture medium is as follows: glucose 5 g / L, peptone 7.5 g / L, yeast extract 5 g / L, potassium dihydrogen phosphate trihydrate 2.5 g / L, magnesium sulfate heptahydrate 1 g / L, the solvent is water, and the pH is 7.0.

[0027] (3) The composition of the fermentation medium is as follows: glucose 25 g / L, peptone 15 g / L, yeast extract 10 g / L, potassium dihydrogen phosphate trihydrate 5 g / L, magnesium sulfate heptahydrate 2 g / L, the solvent is water, and the pH is 7.0.

[0028] Example 1

[0029] This example provides screening of strains.

[0030] (1) Samples collected from the natural environment were diluted 10 times with sterile water, and 0.5 mL of the sample was spread on a solid culture medium and cultured at 37°C for 24 h. Strains with colony morphology similar to that of Streptococcus equi subsp. zooepidemicus were selected and picked out with a sterile toothpick. The strains were inoculated into a 24-well deep-well plate containing fermentation medium, with each well containing 2 mL of liquid. After incubation at 37°C and 250 rpm for 20 h, the hyaluronic acid content in the fermentation liquid was detected.

[0031] (2) Detection method of hyaluronic acid (GA) content: Step (1) After the fermentation liquid is centrifuged, the supernatant is transferred to a new 24-well plate, 2 mL of anhydrous ethanol is added to each well, shaken and then allowed to stand for 30 minutes. After centrifugation again, the supernatant is discarded, 2 mL of deionized water is added to each well of the precipitate to resuspend the precipitate, and part of the solution is transferred to a new well plate, diluted with water to 1 mL to prepare the sample to be tested. The well plate is placed in an ice water bath. Slowly add 2 mL of pre-cooled sodium tetraborate sulfuric acid solution with a concentration of 19.08 g / L, shake and mix, put it in boiling water and boil for about 12 minutes, and then cool it in an ice water bath. Then add 0.1 mL of 1.25 g / L carbazole ethanol solution to each well, shake and mix, put it in boiling water and boil for about 12 minutes, and then cool it in an ice water bath. After the reaction is completed, hyaluronic acid reacts to generate glucuronic acid, which has an absorption wavelength at 530 nm. The content of hyaluronic acid is calculated based on the content of glucuronic acid, as shown in formula ①. Take some samples from the well plate and measure the absorbance at 530 nm, and set up a control group and standard at the same time.

[0032] Glucuronic acid (g / L) = 2.07 × n × GA (g / L) Formula ①.

[0033] In formula ①, n is the sample dilution multiple; 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.

[0034] More than 2,000 strains were screened from more than 100 samples, and a highly viable strain of Streptococcus equi subsp. zooepidemicus was finally found from a ditch near Binbei Farm in Binzhou, Shandong Province. The strain was named Streptococcus equi subsp. zooepidemicus ( Streptococcus equi subsp. zooepidemicus )SYMY-010 was deposited in the General Microbiology Center of China Culture Collection Administration on December 26, 2022, with the deposit number CGMCC NO. 26282.

[0035] Example 2

[0036] This example provides a method for producing hyaluronic acid using the Streptococcus equi subspecies zooepidemicus SYMY-010 screened in Example 1.

[0037] (1) Slant activation culture: Inoculate the Streptococcus equi subsp. zooepidemicus SYMY-010 strain into the slant culture medium and culture at 37°C for 24 h to obtain activated slant bacteria.

[0038] (2) Seed activation culture: The Streptococcus equi subsp. zooepidemicus SYMY-010 obtained in step (1) was inoculated into a test tube containing a seed culture medium at a rate of 2%, and cultured at 250 rpm and 37°C for 24 h; then, the inoculation rate was transferred to a shake flask containing a seed culture medium at a rate of 2%, and cultured at 200 rpm and 37°C for 24 h to obtain a seed solution.

[0039] (3) Fermentation culture: The seed liquid obtained in step (2) was inoculated into the fermentation medium, and the fermentation was carried out in a 250 L fermentor with a liquid volume of 150 L and an inoculum volume of 3%. The fermentation temperature was set at 37°C and the rotation speed was 500 rpm. The fermentation pH was controlled to 7.0, the dissolved oxygen was controlled to about 35%, and glucose solution was added during the fermentation process at a supplementary amount of 25 g / L. The fermentation was continued for 20 h. When the residual sugar in the fermentation liquid was less than 1 g / L, the fermentation was terminated. After the fermentation was completed, the hyaluronic acid content was detected to be 15 g / L.

[0040] (4) Purification and extraction of hyaluronic acid: 0.6 wt% sodium dodecyl sulfate (SDS) aqueous solution was added to the fermentation broth after fermentation in step (3) and treated for 30 min. After treatment, the fermentation broth was centrifuged to remove the precipitate and bacteria. Anhydrous ethanol twice the volume of the solution was added to the supernatant of the fermentation broth to resuspend it. The mixture was stirred and treated for 8 h. The resuspended fermentation broth was centrifuged again to collect the precipitate. The precipitate was resuspended with 5 g / L NaCl solution. Hexadecylpyridinium chloride with a final concentration of 1.5 wt% was added to the solution. The mixture was stirred and allowed to stand for 3 h. The solution was centrifuged to obtain a complex precipitate. The complex precipitate was resuspended with 10 g / L NaCl solution. Anhydrous ethanol twice the volume of the solution was added and allowed to stand for 0.5 h. The precipitate was collected by centrifugation and then freeze-dried to obtain hyaluronic acid. The average molecular weight of the obtained hyaluronic acid was 0.47×10 6 Da.

[0041] Example 3

[0042] This example provides a genetic stability test of Streptococcus equi subsp. zooepidemicus SYMY-010 screened in Example 1.

[0043] Streptococcus equi subsp. Zooepidemicus SYMY-010 was streaked onto solid culture medium to obtain the first-generation strain. Following this method, the first-generation strain was further streaked to obtain the second-generation strain, and so on for the third, fourth, fifth, sixth, seventh, and eighth generations. Each generation was activated and inoculated into the fermentation medium. Fermentation was performed in a 5 L fermentor. The inoculum size was 3%, the fermentation temperature was set at 37°C, the rotation speed was 500 rpm, the pH was controlled at 7.0, and the dissolved oxygen was maintained at approximately 35%. During fermentation, glucose solution was added at a rate of 25 g / L. Fermentation was terminated for 20 h, and the residual sugar content in the fermentation broth was less than 1 g / L. Hyaluronic acid content was measured after fermentation. The results are shown in Table 1.

[0044] Table 1 Hyaluronic acid production of different generations of bacterial strains

[0045]

[0046] As can be seen from Table 1, with the increase in the number of passages, the yield of hyaluronic acid is relatively stable, indicating that Streptococcus equi subspecies zooepidemicus SYMY-010 has good genetic stability.

[0047] Example 4

[0048] This example provides verification of the acid resistance of Streptococcus equi subsp. zooepidemicus SYMY-010 screened in Example 1.

[0049] The hyaluronic acid production process in this example was the same as in Example 2. The fermentation process was carried out in a 5 L fermentor. The pH of the fermentation medium was controlled between 5 and 7. The hyaluronic acid yield was measured after the fermentation was completed. The test results are shown in Table 2.

[0050] Table 2 Acid resistance test results of strains

[0051]

[0052] According to the test results, the hyaluronic acid production of the Streptococcus equi subspecies Zooepidemicus SYMY-010 strain showed a downward trend with the decrease in the pH of the fermentation medium. However, when the pH dropped to 5, the hyaluronic acid production could be maintained at 14.4 g / L, indicating that the Streptococcus equi subspecies Zooepidemicus SYMY-010 strain has good acid resistance.

[0053] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for producing hyaluronic acid, characterized in that: The method comprises: Streptococcus equi subsp. zooepidemicus ( Streptococcus equi subsp. zooepidemicus SYMY-010 is inoculated into a slant culture medium for slant activation culture, and then the activated bacteria are inoculated into a seed culture medium for expansion culture to obtain a seed solution; the seed solution is inoculated into a fermentation culture medium for fermentation culture, and hyaluronic acid is obtained after purification; the fermentation culture medium is composed of: 22-28 g / L glucose, 10-18 g / L peptone, 8-12 g / L yeast extract powder, 2-6 g / L dipotassium hydrogen phosphate trihydrate, 1-5 g / L magnesium sulfate heptahydrate, water as the solvent, and a pH of 5.0-7.0; glucose solution is added during the fermentation process to adjust the total sugar content of the culture medium to 45-55 g / L; the fermentation culture temperature is 35-38° C., and the fermentation culture time is 18-24 hours; The Streptococcus equi subsp. zooepidemicus ( Streptococcus equi subsp. zooepidemicus )SYMY-010 was deposited in the General Microbiology Center of China Culture Collection Administration on December 26, 2022, with the deposit number CGMCC NO. 26282.

2. The method according to claim 1, wherein The seed liquid is inoculated into the fermentation medium at an inoculation rate of 2-5%.

3. The method according to claim 1, wherein The slant culture medium is composed of: 3-7 g / L glucose, 6-9 g / L peptone, 3-7 g / L yeast extract powder, 2-3 g / L dipotassium hydrogen phosphate trihydrate, 0.5-1.5 g / L magnesium sulfate heptahydrate, 18-22 g / L agar, the solvent is water, and the pH is 6.8-7.

2.

4. The method according to claim 1, wherein The seed culture medium is composed of: 3-7 g / L glucose, 6-9 g / L peptone, 3-7 g / L yeast extract powder, 2-3 g / L dipotassium hydrogen phosphate trihydrate, 0.5-1.5 g / L magnesium sulfate heptahydrate, water as the solvent, and a pH of 6.8-7.

2.

5. The method according to claim 1, wherein The temperature of the slant activation culture is 34-38° C., and the time of the slant activation culture is 20-30 h.

6. The method according to claim 1, wherein The temperature of the expanded culture is 34-38° C., and the time of the expanded culture is 40-50 hours.

7. The method according to claim 6, wherein The specific steps of the expanded culture are: inoculating the activated bacteria into a test tube containing a seed culture medium at an inoculum size of 1-4%, and culturing at 180-250 rpm and 34-38°C for 20-25 hours; then transferring the inoculum size of 1-4% into a shake flask containing a seed culture medium, and culturing at 180-250 rpm and 34-38°C for 20-25 hours to obtain a seed solution.

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