Liquid culture medium and fermentation method of mycoplasma synoviae

Abstract

The invention relates to the technical field of microbial fermentation, and particularly provides a liquid culture medium and a fermentation method of mycoplasma synoviae. The liquid culture medium is composed of a basic culture medium containing 1-20 g / L of pplo broth and 0.2-3 g / L of poloxamer. According to the application, the poloxamer is added into the culture medium of the mycoplasma synoviae for the first time, so that the culture density of the mycoplasma synoviae is improved, and the poloxamer is accidentally found to improve the immunogenicity of MS (Murashige and Skoog). In addition, when the liquid culture medium is used for fermenting the mycoplasma synoviae, the fermentation time is shortened to 12-14 hours.

Description

Technical Field

[0001] This application relates to the field of microbial fermentation technology, specifically providing a liquid culture medium and fermentation method for Mycoplasma synoviae. Background Technology

[0002] Mycoplasma synoviae infection is an infectious disease of chickens and turkeys caused by Mycoplasma synoviae (MS). Also known as avian infectious synovitis, it is characterized by joint swelling, inflammation of the synovial bursa and tendons, and swelling of parenchymal organs. Because the disease progresses slowly and has a long course, eradication is very difficult once an infection occurs in a flock. Therefore, it can spread for a long time, leading to problems such as low feed utilization, stunted growth, increased culling rates, and decreased egg production. When this pathogen is present in a flock, mixed infections are likely to occur, exacerbating the disease, increasing mortality, and causing serious economic losses.

[0003] MS cells are typically pear-shaped or oval, about 0.2 μm in diameter, facultatively anaerobic, and lack a cell wall. There are currently two methods for preventing and controlling Mycoplasma synoviae infection in chickens: antibiotic use or immunization. MS is sensitive to many antibiotics, but resistance easily develops. Resistance leads to severe mycoplasma infection and insufficient expected treatment effect, contributing to the transformation into a chronic disease. Inactivated vaccines are the ideal prevention and control method.

[0004] High-density MS culture is a crucial step in the manufacturing of inactivated vaccines. However, large-scale high-density MS culture presents certain challenges. Static culture results in low viable cell counts and long fermentation times. While mechanically stirred and aerated fermenters can increase culture density and shorten fermentation time, the shear forces generated by the lack of a cell wall in MS cells can damage the cells, leading to low viable cell counts.

[0005] In view of the above, this application is hereby submitted. Summary of the Invention

[0006] One of the objectives of this application is to provide a liquid culture medium and fermentation method for Mycoplasma synoviae in chickens, so as to improve the culture density and immunogenicity of Mycoplasma synoviae in chickens.

[0007] To achieve the above objectives, this application adopts the following technical solution:

[0008] Application of poloxamer in increasing the culture density or immunogenicity of Mycoplasma synoviae in chickens.

[0009] Furthermore, the poloxamer is poloxamer 188.

[0010] A liquid culture medium for Mycoplasma synoviae, wherein the liquid culture medium is composed of a basic culture medium containing 1-20 g / L pplo broth and 0.2-3 g / L poloxamer, wherein the basic culture medium is CM2 medium or modified Frey medium.

[0011] Further, the poloxamer content is 1 g / L; preferably, the poloxamer is poloxamer 188;

[0012] Preferably, the pplo broth content is 1-10 g / L, more preferably 5 g / L;

[0013] Preferably, the pH of the liquid culture medium is 7.8 to 8.0.

[0014] A solid culture medium for Mycoplasma synoviae, wherein the solid culture medium is the above-mentioned liquid culture medium containing 1.5% agar powder or agarose.

[0015] A fermentation method for Mycoplasma synoviae, wherein Mycoplasma synoviae is cultured in the above-mentioned liquid culture medium.

[0016] Furthermore, the activated chicken synoviocyte mycoplasma was inoculated into the liquid culture medium for culture. When the dissolved oxygen (DO) dropped below 10%, the rotation speed and aeration were increased to make the DO greater than 10%. The culture was harvested when the pH reached 6.6-6.7, i.e., after 12-14 hours of culture.

[0017] Further, the fermentation method includes the following steps: inoculating MS freeze-dried bacteria into the liquid culture medium and incubating at 37°C until the pH reaches 6.8–6.9, as primary seed; inoculating 10% of the primary seed into the liquid culture medium and incubating at 37°C until the pH reaches 6.8–6.9, as secondary seed; inoculating the secondary seed into the liquid culture medium in a large-volume fermenter, and when the dissolved oxygen (DO) drops below 10%, increasing the rotation speed and aeration to make the DO greater than 10%, and harvesting when the pH reaches 6.6–6.7, i.e., culturing for 12–14 hours;

[0018] Preferably, the large-capacity fermenter is a 15L or 150L fermenter, wherein the stirring speed of the 15L fermenter is 50-150 rpm and the aeration rate is 1-2 L / min; the stirring speed of the 150L fermenter is 30-50 rpm and the aeration rate is 5-12 L / min.

[0019] Preferably, the inoculum size in the fermenter is 2% to 10%, more preferably 5%.

[0020] The chicken synoviae mycoplasma obtained by the above fermentation method.

[0021] The above-mentioned application of Mycoplasma synoviae in inactivated Mycoplasma synoviae vaccine.

[0022] Compared with the prior art, the technical effects of this application are as follows:

[0023] This application is the first to add poloxamer to the culture medium of Mycoplasma synoviae, which not only increases the culture density of Mycoplasma synoviae, but also achieves a viable cell density of 1×10⁻⁶. 10 The concentration of CFU / ml was found to be above a certain level, and poloxamer was unexpectedly found to enhance the immunogenicity of MS. Animal immunization results using the inactivated vaccine prepared from *Mycoplasma synoviae* produced in this application showed an IHA titer of 7.9 log2 and a lesion reduction rate of 82%, providing effective protection for chickens. Furthermore, fermentation using the liquid culture medium of this application shortened the fermentation time to 12-14 hours. Detailed Implementation

[0024] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions described in this application will be further described in detail below with reference to specific embodiments.

[0025] The term "poloxam" refers to a polyoxyethylene-polyoxypropylene ether block copolymer, a novel type of high-molecular-weight nonionic surfactant used as an emulsifier and stabilizer in industrial production. As a nonionic surfactant, poloxamer prevents cells from attaching to air bubbles and thus protects cells from damage when the bubbles burst. It also reduces vibration during bubble bursting. Some reports also indicate that poloxamer can enter cells and affect their metabolism. Currently, there are no reports of poloxamer being used for mycoplasma culture. This application uses poloxamer 188 for mycoplasma culture to reduce shear stress damage to mycoplasma during fermentation.

[0026] The term "CCU" stands for Color Change Unit Count, which quantifies mycoplasma by observing color changes caused by its metabolism in liquid culture media. It is a commonly used method for mycoplasma quantification. It typically involves 10-fold serial dilutions to 10-10. 12 As described above, this process requires high stability in experimental operations and has a relatively large systematic error in result interpretation. The test results can also be affected by inconsistent color changes in the culture medium. In large-scale production, unstable viable cell counts can negatively impact product quality.

[0027] The term "CFU" stands for Colony Forming Unit Count, which provides a more accurate count of viable cells by measuring the number of colonies formed on a solid culture medium. The CFU dilution gradient is much lower than that of CCU (10⁻⁶). 6(Approximately 10-20 cm) This reduces errors in the process, ensuring consistency and reproducibility of results. Based on visible colonies, it provides more accurate quantitative results, significantly reducing batch-to-batch variability, which is beneficial for product quality control and evaluation. Secondly, the CFU method can also help assess the activity of the cultured cells in a batch. Compared to the CCU method, the CFU method has advantages such as higher accuracy, intuitiveness, and ease of standardization.

[0028] The term "CM2 medium" has the following formula: NaCl 5 g / L, KCl 0.4 g / L, MgSO4 0.2 g / L, Na2HPO4 1.6 g / L, KH2PO4 0.1 g / L, hydrolyzed milk protein 2 g / L, bovine heart extract 14.8 g / L, yeast extract powder 4 g / L, glucose 4 g / L, porcine serum 120 ml / L, phenol red 0.1 g / L, and 80,000 units of penicillin.

[0029] The term "modified Frey medium" has the following formula: NaCl 5 g / L, KCl 0.4 g / L, MgSO4 0.2 g / L, Na2HPO4 1.6 g / L, KH2PO4 0.1 g / L, yeast extract 5 g / L, hydrolyzed milk protein 5 g / L, glucose 10 g / L, coenzyme I 0.1 g / L, cysteine ​​0.1 g / L, arginine 0.4 g / L, 100,000 units of penicillin, phenol red 0.01 g / L, and 10% porcine serum.

[0030] The term "pplo broth" refers to a recipe for... Peptone 1g / L, peptone 10g / L, beef heart extract 4g / L, sodium chloride 5g / L.

[0031] MS (Mycoplasma synoviae) is typically cultured in mechanically stirred and aerated fermenters to increase culture density and shorten fermentation time. However, the shear forces generated can damage MS cell membranes, resulting in low viable cell counts. This application involves adding poloxamer to the culture medium to reduce shear damage to MS, and unexpectedly, it was discovered that poloxamer can enhance the immunogenicity of MS. Therefore, this application provides the application of poloxamer in increasing the culture density or immunogenicity of Mycoplasma synoviae in chickens.

[0032] In one implementation, poloxamer is poloxamer 188.

[0033] This application provides a liquid culture medium for Mycoplasma synoviae, which consists of a basic culture medium containing 1-20 g / L pplo broth and 0.2-3 g / L poloxamer, wherein the basic culture medium is CM2 medium or modified Frey medium.

[0034] The content of pplo broth may be, but is not limited to, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 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, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, or 20 g / L; the content of poloxamer may be, but is not limited to, 0.2 g / L, 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, or 3 g / L.

[0035] In some implementations, poloxamer is poloxamer 188.

[0036] Based on practical application needs, 1.5% agar powder or agarose can be added to the above liquid culture medium to make a solid culture medium for MS culture.

[0037] In some implementations, the pH of the liquid culture medium is adjusted to 7.8–8.0 for MS culture.

[0038] A fermentation method for Mycoplasma synoviae:

[0039] Step 1: Resuscitate MS lyophilized bacteria using the liquid culture medium of this application, inoculate the lyophilized bacteria into the liquid culture medium, and incubate at 37°C until the pH reaches 6.8-6.9, as the primary seed culture.

[0040] Step 2: Inoculate the primary seeds into liquid culture medium at a 10% inoculum and incubate at 37°C until the pH reaches 6.8-6.9 to obtain the secondary seeds.

[0041] Step 3: Inoculate the secondary seed (activated MS) into the liquid culture medium of this application in a large-volume fermenter. When the DO drops below 10%, increase the rotation speed and aeration to make the DO greater than 10%. Harvest when the pH reaches 6.6-6.7, i.e., after 12-14 hours of cultivation.

[0042] In one embodiment, the large-capacity fermenter is a 15L or 150L fermenter, wherein the stirring speed of the 15L fermenter is 50-150 rpm and the aeration rate is 1-2 L / min; the stirring speed of the 150L fermenter is 30-50 rpm and the aeration rate is 5-12 L / min.

[0043] In a preferred embodiment, the inoculum size of the fermenter is 2%-10%, and preferably, it is 5%.

[0044] As one embodiment of this application, the viable cell count of the samples after fermentation is performed using the CCU and CFU counting methods. By comparing the results of the CCU and CFU viable cell counting methods of MS, a more reasonable CFU counting method is selected to provide a reliable number of viable cells for evaluating the quality of the process and improving the accuracy of seedling preparation.

[0045] The present application will be further described below with reference to specific embodiments, and the advantages and features of the present application will become clearer with the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present application without departing from the spirit and scope of the present application, but such modifications and substitutions all fall within the protection scope of the present application.

[0046] Unless otherwise specified, the experimental methods described in this application are conventional methods; the biological materials described are all commercially available unless otherwise specified. This application uses the HN1 strain as an example to describe specific embodiments, and the strains involved include, but are not limited to, the HN1 strain. The basic culture medium is a modified Frey culture medium, including, but not limited to, modified Frey.

[0047] Example 1: Preparation and sterilization of culture medium

[0048] Liquid culture medium formulation: NaCl 5g / L, KCl 0.4g / L, MgSO4 0.2g / L, Na2HPO4 1.6g / L, KH2PO4 0.1g / L, yeast extract 5g / L, hydrolyzed milk protein 5g / L, glucose 10g / L, PPL0 broth 5g / L, coenzyme I 0.1g / L, cysteine ​​0.1g / L, arginine 0.4g / L, 100,000 units of penicillin, phenol red 0.01g / L, and 10% porcine serum. Yeast extract, hydrolyzed milk protein, and PPL0 broth were sterilized in a fermenter at 121℃ for 30 min. Penicillin, coenzyme I, cysteine, and arginine were sterilized by filtration. Porcine serum was inactivated at 56℃ for 30 min. The remaining components were sterilized at 116℃ for 30 min. After sterilization, all components were mixed, and the pH was adjusted to 7.8–8.0 with NaOH.

[0049] Adding 1.5% agar powder or agarose to the above culture medium creates a solid culture medium, which is used for the detection of CFU viable counts.

[0050] Example 2: 15L Fermentation Tank Process

[0051] Resuscitate MS HN1 lyophilized bacteria in liquid medium. Inoculate the lyophilized bacteria into the liquid medium and incubate at 37°C until the pH reaches 6.8-6.9. This serves as the primary seed culture. Inoculate 10% of the primary seed culture into the liquid medium and incubate at 37°C until the pH reaches 6.8-6.9. This serves as the secondary seed culture. Adjust the fermentation temperature to 37°C, pH to 7.8-8.0, agitator speed to 50 rpm, and deep aeration at a flow rate of 1-2 L / min. Inoculate the secondary seed culture at a 5% inoculation rate into the liquid medium in a 15L fermenter. When the dissolved oxygen (DO) drops below 10%, increase the agitator speed and aeration to bring the DO above 10%. Incubate until the pH reaches 6.6-6.7 before harvesting.

[0052] Take sterile test tubes and add 4.5 ml of liquid culture medium to each tube. Add 0.5 ml of the culture to be tested to the liquid culture medium. Place the test tubes on a shaker to mix thoroughly. Take 0.5 ml of the diluted solution from the mixed test tube and add it to 4.5 ml of liquid culture medium. Perform serial dilutions 10-fold to the appropriate dilution following the above steps. CFU viable cell count method: Add 0.1 ml of the final diluted bacterial solution to a solid culture medium, invert it, and incubate at 37℃ in a CO2 incubator for 7 days. Count the colonies; each plate should have 30-200 colonies. CCU viable cell count method: Stopper the diluted test tubes and incubate at 37℃ for 7 days. Observe and record the color change of the culture medium daily. Perform three replicates for both counting methods.

[0053] The results showed that the viable bacterial counts of CCU and CFU were 1×10⁻⁶. 10 CCU / ml, 2.3×10 9 CFU / ml. CFU can more accurately characterize antigen content and provide more accurate vaccine preparation requirements, so the CFU counting method is used for viable cell counting.

[0054] Example 3: 15L fermentation process with added poloxamer 188

[0055] The difference from Example 2 was the addition of 1 g / L poloxamer 188 to the liquid culture medium. The results showed that the CFU viable count with the addition of poloxamer 188 was 1.0 × 10⁻⁶. 10 The CFU / ml count was 4.3 times higher than that of Example 2 without the addition of poloxamer 188.

[0056] Example 4: MS Inactivation and Vaccine Preparation

[0057] Formaldehyde solution with a final concentration of 0.3% was added to the MS bacterial culture, and the culture was inactivated at 37°C for 24 hours. The formaldehyde-inactivated MS antigen was then adjusted with PBS to ensure that the viable bacterial count of MS HN1 strain in the antigen solution was 6.4 × 10⁻⁶ before inactivation. 9CFU / ml, antigen solution: Tween-80 = 96:4, mix well to obtain the aqueous phase. Take 94 parts white oil and 6 parts Span-80, sterilize at 121℃ for 30 minutes, shake until clear, and set aside; this is the oil phase. First, add the oil phase to a beaker, and while stirring in an emulsifier at 11000 rpm, slowly pour in the aqueous phase, then stir at 14000 rpm for 5 minutes. After stirring, dispense the vaccine.

[0058] Example 5 Immunogenicity Evaluation

[0059] Thirty 6-7 week old SPF chickens were randomly divided into three groups of 10 each. The immunization dose was 0.5 ml. The first group was subcutaneously injected with MS vaccine prepared from the antigen cultured in Example 2, and the second group was subcutaneously injected with MS vaccine prepared from the antigen cultured in Example 3. The third group was not immunized and served as a control. Blood was collected 28 days after immunization to separate serum and detect HI antibody efficacy.

[0060] Twenty-eight days after immunization, chickens were challenged with 1.0 ml of MS HN1 strain intravenously. The challenge dose was 1.0 × 10⁻⁶ ml. 8 CFU. Fourteen days after challenge, lesions on the footpads, tarsal joints, toe joints, wing joints, and keel of chickens were observed and scored, with 3 points for each site, for a total of 9 sites. 0 points indicated no abnormalities; 1 point indicated mild swelling; 2 points indicated moderate swelling; and 3 points indicated severe swelling, for a total of 27 points. The lesion reduction rate in the immunized group was calculated using the formula: Lesion reduction rate = (average score of control group - average score of immunized group) / average score of control group.

[0061] The results are shown in Table 1. The results show that the HI antibody and lesion reduction rate of vaccine 2 are higher than those of vaccine 1, indicating that the addition of poloxamer 188 can improve the immunogenicity of MS.

[0062] Table 1. Effects of poloxamer 188 on the immunogenicity of MS antigen. vaccine Antigen culture process Lesion reduction rate HI antibody titer (log2HI) 1 Example 2 65％ 7.0 2 Example 3 82％ 7.9 3 Blank control / 0

[0063] Example 6: Process Scalability Verification

[0064] Resuscitate MS HN1 lyophilized bacteria in liquid medium. Inoculate the lyophilized bacteria into the liquid medium and incubate at 37°C until the pH reaches 6.8-6.9. This serves as the primary seed culture. Inoculate 10% of the primary seed culture into a 15L seed tank containing liquid medium supplemented with 1g / L poloxamer 188. Maintain a stirring speed of 50 rpm and deep aeration at a flow rate of 1L / min. Incubate at 37°C until the pH reaches 6.8-6.9. This serves as the secondary seed culture. Adjust the temperature of a 150L fermenter to 37°C and the pH to 7.8-8.0. Maintain a stirring speed of 30 rpm and deep aeration at a flow rate of 5-12L / min. Inoculate 5% of the secondary seed culture into the liquid medium of the 150L fermenter, supplemented with 1g / L poloxamer 188. When the dissolved oxygen (DO) drops below 10%, increase the stirring speed and aeration to bring the DO above 10%. Harvest when the pH reaches 6.6-6.7, i.e., after 12-14 hours of incubation.

[0065] The results showed that the viable count of MS HN1 strain cultured in a 150L fermenter was 1.4 × 10⁻⁶. 10 The CFU / ml indicates that the process in this application is easy to scale up.

[0066] Example 7: Verification of Process Universality

[0067] Resuscitate MSSD01 strain of lyophilized bacteria in liquid medium. Inoculate the lyophilized bacteria into the liquid medium and incubate at 37°C until the pH reaches 6.8-6.9. This serves as the primary seed culture. Inoculate 10% of the primary seed culture into a 15L seed tank containing liquid medium with or without 1 g / L poloxamer 188. Maintain a stirring speed of 50 rpm and deep aeration at a flow rate of 1 L / min. Incubate at 37°C until the pH reaches 6.8-6.9. This serves as the secondary seed culture. Adjust the temperature of a 150L fermenter to 37°C and the pH to 7.8-8.0. Maintain a stirring speed of 30 rpm and deep aeration at a flow rate of 5-12 L / min. Inoculate 5% of the secondary seed culture into the liquid medium of the 150L fermenter, with or without 1 g / L poloxamer 188. When the dissolved oxygen (DO) drops below 10%, increase the stirring speed and aeration to bring the DO above 10%. Harvest when the pH reaches 6.6-6.7, i.e., after 12-14 hours of cultivation. In Process 1, poloxamer is added to both the 15L seed tank and the 150L fermentation tank; in Process 2, poloxamer is not added to either the 15L seed tank or the 150L fermentation tank.

[0068] Table 2 Effect of poloxamer on viable cell count of MSSD01 strain process 15L seed container 150L fermentation tank viable bacteria count (CFU / ml) 1 + + <![CDATA[1.2×10 10 ]]> 2 - - <![CDATA[2.9×10 9 ]]> Note: + indicates that poloxamer 188 is added to the culture medium; - indicates that poloxamer 188 is not added to the culture medium.

[0069] The results showed that the number of viable MSSD01 cells cultured on liquid medium with and without poloxamer 188 was 1.2 × 10⁻⁶. 10 2.9×10 9 The CFU / ml of the process described in this application can also increase the viable count of the MSSD01 strain, indicating that the process described in this application is universally applicable to different strains.

[0070] Unless otherwise defined, all technical and scientific terms used throughout this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. In case of any inconsistency, the meaning as stated in this application or derived from the content described herein shall prevail. Furthermore, the terminology used in this description is for the purpose of describing embodiments of this application only and is not intended to limit this application.

Claims

1. Application of poloxamer in improving the culture density or immunogenicity of Mycoplasma synoviae in chickens.

2. The application according to claim 1, characterized in that, The poloxamer in question is poloxamer 188.

3. A liquid culture medium for Mycoplasma synoviae, characterized in that, The liquid culture medium consists of a basic culture medium containing 1–20 g / L pelo broth and 0.2–3 g / L poloxamer, wherein the basic culture medium is CM2 medium or modified Frey medium.

4. The liquid culture medium according to claim 3, characterized in that, The poloxamer content is 1 g / L; preferably, the poloxamer is poloxamer 188; Preferably, the pplo broth content is 1-10 g / L, more preferably 5 g / L; Preferably, the pH of the liquid culture medium is 7.8 to 8.

0.

5. A solid culture medium for Mycoplasma synoviae, characterized in that, The solid culture medium is the liquid culture medium of claim 3 or 4 containing 1.5% agar powder or agarose.

6. A fermentation method for Mycoplasma synoviae, characterized in that, Chicken synoviae Mycoplasma is cultured using the liquid culture medium described in claim 3 or 4.

7. The fermentation method according to claim 6, characterized in that, The activated chicken synoviocyte mycoplasma was inoculated into a fermenter containing the liquid culture medium and cultured. When the dissolved oxygen (DO) dropped below 10%, the rotation speed and aeration of the fermenter were increased to make the DO greater than 10%. The fermentation was carried out until the pH reached 6.6-6.7, i.e., after 12-14 hours of culture.

8. The fermentation method according to claim 7, characterized in that, The fermentation method includes the following steps: MS freeze-dried bacteria are inoculated into the liquid culture medium and cultured statically at 37°C until the pH reaches 6.8–6.9, serving as primary seed; 10% of the primary seed is inoculated into the liquid culture medium and cultured at 37°C until the pH reaches 6.8–6.9, serving as secondary seed; the secondary seed is inoculated into the liquid culture medium in a large-volume fermenter; when the dissolved oxygen (DO) drops below 10%, the fermenter speed and aeration are increased to make the DO greater than 10%; the fermentation is carried out until the pH reaches 6.6–6.7, at which point the bacteria are harvested, i.e., cultured for 12–14 hours. Preferably, the large-capacity fermenter is a 15L or 150L fermenter, wherein the stirring speed of the 15L fermenter is 50-150 rpm and the aeration rate is 1-2 L / min; the stirring speed of the 150L fermenter is 30-50 rpm and the aeration rate is 5-12 L / min. Preferably, the inoculum size in the fermenter is 2% to 10%, more preferably 5%.

9. Mycoplasma synoviae obtained by the fermentation method according to any one of claims 6 to 8.

10. The use of Mycoplasma synoviae of claim 9 in an inactivated Mycoplasma synoviae vaccine.