Actinobacillus pleuropneumoniae culture medium and application thereof

Abstract

The invention provides an actinobacillus pleuropneumoniae culture medium and application thereof, and relates to the technical field of fermentation culture, the actinobacillus pleuropneumoniae culture medium comprises 6-10 g / L of tryptone, 2-4 g / L of yeast extract powder, 20-60 ml / L of bovine serum, 8-12 mg / L of coenzyme, 1-4 g / L of ammonium sulfate, 0.2-1 g / L of magnesium sulfate, 2-6 g / L of sodium chloride and 2-6 g / L of dipotassium phosphate, the culture medium is suitable for high-density fermentation culture of serotype 1, serotype 2, serotype 4, serotype 5, serotype 7 and serotype 15 which are mainly epidemic to actinobacillus pleuropneumoniae; the growth rhythm of each serotype strain is basically consistent when the serotype strains are fermented and cultured to a logarithmic phase and a plateau phase; the technical problems that in the prior art, the culture effect difference of a culture medium on actinobacillus pleuropneumoniae is large, the enrichment effect is poor, and high-density fermentation is difficult to achieve are solved.

Description

Technical Field

[0001] This invention relates to the field of fermentation culture technology, and in particular to a culture medium for Actinobacillus pleuropneumoniae and its application. Background Technology

[0002] Actinobacillus pleuropneumoniae (APP) is the pathogen that causes porcine infection pleuropneumonia, a highly contagious respiratory disease in pigs that is characterized by outbreaks and is primarily fibrinous, hemorrhagic, and necrotic pneumonia.

[0003] Actinobacillus contagious pleuropneumoniae has at least 18 serotypes, and there is no effective cross-protection between different serotypes. Since 2000, porcine contagious pleuropneumoniae has been prevalent throughout China, with serotypes 1, 2, 4, 5, and 7 being the most common. In recent years, serotype 15 has gradually become more prevalent nationwide, with a high infection rate. Currently, most culture media for Actinobacillus contagious pleuropneumoniae are BHI basal medium, but this medium shows differences in the timing of entry into the logarithmic, plateau, and death phases for strains of the above serotypes, and the viable count in the plateau phase is mostly around 1.0 × 10⁻⁶. 10 With a CFU / ml level below a certain threshold, it is difficult to achieve high-density fermentation.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide a universal Actinobacillus pleuropneumoniae culture medium suitable for the main circulating serotypes of Actinobacillus pleuropneumoniae, in order to solve the technical problems of large differences in the culture effect of existing culture media on Actinobacillus pleuropneumoniae, poor enrichment effect and difficulty in achieving high-density fermentation.

[0006] The second objective of this invention is to provide a method for preparing Actinobacillus pleuropneumoniae culture medium.

[0007] A third objective of this invention is to provide the application of the above-mentioned culture medium in the fermentation culture of Actinobacillus pleuropneumoniae.

[0008] The fourth objective of this invention is to provide a fermentation culture method for Actinobacillus pleuropneumoniae.

[0009] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a culture medium for Actinobacillus pleuropneumoniae, the culture medium comprising 6-10 g / L tryptone, 2-4 g / L yeast extract, 20-60 ml / L bovine serum, 8-12 mg / L coenzyme, 1-4 g / L ammonium sulfate, 0.2-1 g / L magnesium sulfate, 2-6 g / L sodium chloride and 2-6 g / L dipotassium hydrogen phosphate.

[0010] Furthermore, the culture medium comprises 8 g / L tryptone, 3 g / L yeast extract, 50 ml / L bovine serum, 10 mg / L coenzyme, 2 g / L ammonium sulfate, 0.5 g / L magnesium sulfate, 5 g / L sodium chloride, and 5 g / L dipotassium hydrogen phosphate.

[0011] Furthermore, the pH of the culture medium is 7 to 7.2.

[0012] Secondly, the present invention provides a method for preparing Actinobacillus pleuropneumoniae culture medium, comprising dissolving each component in water according to the above-mentioned culture medium formula to obtain a fermentation culture medium.

[0013] Thirdly, the present invention provides the application of the above-mentioned culture medium or the culture medium prepared by the above-mentioned preparation method in the fermentation culture of Actinobacillus pleuropneumoniae.

[0014] Furthermore, the Actinobacillus pleuropneumoniae includes at least one of Actinobacillus pleuropneumoniae type 1, Actinobacillus pleuropneumoniae type 2, Actinobacillus pleuropneumoniae type 4, Actinobacillus pleuropneumoniae type 5, and Actinobacillus pleuropneumoniae type 7.

[0015] Fourthly, the present invention provides a fermentation culture method for Actinobacillus pleuropneumoniae, wherein Actinobacillus pleuropneumoniae is inoculated into the culture medium described above or the culture medium prepared by the above preparation method for culture.

[0016] Furthermore, this also includes adding an antifoaming agent to the culture medium; Preferably, the amount of defoamer used is 0.3~0.5 g / L, and more preferably 0.4 g / L.

[0017] Furthermore, the culture temperature is 36~38℃, preferably 37℃; Preferably, the stirring speed of the culture system is 100~400 r / min; Preferably, the aeration rate of the culture system is 0.3~0.5 vvm; Preferably, the dissolved oxygen content of the culture system is 5% to 10%; Preferably, the residual sugar content in the culture system is controlled to be 1~3 g / L; Preferably, the pH of the culture system is controlled at 7.0~7.2.

[0018] The Actinobacillus pleuropneumoniae culture medium provided by this invention is suitable for high-density fermentation culture of the main prevalent serotypes 1, 2, 4, 5, 7, and 15 of Actinobacillus pleuropneumoniae, and has a wider range of applications. When culturing strains of each serotype using the culture medium of this invention, the viable count of each strain is not less than 1.0 × 10⁻⁶ after 6 hours of culture. 10 CFU / mL, among which APP5-336 strain had the highest viable count after culture, reaching 1.57 × 10⁻⁶ after 6 hours of culture. 10 The CFU / mL concentration is 2-3 times that of existing technologies. All serotype strains can rapidly enter the logarithmic growth phase 2 hours after inoculation, and enter the plateau phase after 4 hours. The plateau phase lasts for about 2-4 hours before entering the decline phase. The growth rhythm of each serotype strain in the fermentation culture to the logarithmic and plateau phases is basically consistent, which solves the technical problems of large differences in the culture effect of the culture medium on Actinobacillus pleuropneumoniae and poor enrichment effect in existing technologies, making it difficult to achieve high-density fermentation. Attached Figure Description To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 The growth curves of different serotype strains cultured in culture medium 1 provided in Example 2 of the present invention; Figure 2 The growth curves of different serotype strains cultured in culture medium 2 provided in Example 2 of the present invention; Figure 3 The growth curves of different serotype strains cultured in culture medium 3 provided in Example 2 of the present invention; Figure 4 The growth curves of different serotype strains cultured in culture medium 4 provided in Example 2 of the present invention; Figure 5 The growth curves of different serotype strains cultured in culture medium 5 provided in Example 2 of this invention; Figure 6 Growth curves of different serotype strains cultured in culture medium 8 provided in Example 2 of the present invention; Figure 7 The growth curves of different serotype strains cultured in culture medium 9 provided in Example 2 of the present invention; Figure 8 The growth curves of different serotype strains cultured in culture medium 10 provided in Example 2 of the present invention are shown. Figure 9The growth curves of different serotype strains cultured in culture medium 11 provided in Example 2 of the present invention; Figure 10 The growth curves of different serotype strains cultured in culture medium 12 provided in Example 2 of the present invention; Figure 11 The growth curves of different serotype strains cultured in culture medium 13 provided in Example 2 of the present invention; Figure 12 The growth curves of different serotype strains cultured in culture medium 14 provided in Example 2 of the present invention; Figure 13 The growth curves of different serotype strains cultured in culture medium 15 provided in Example 2 of the present invention; Figure 14 The growth curves of different serotype strains cultured in culture medium 16 provided in Example 2 of the present invention are shown. Detailed Implementation

[0020] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0021] Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

[0022] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] One aspect of this invention provides a culture medium for Actinobacillus pleuropneumoniae, the culture medium comprising 6-10 g / L tryptone, 2-4 g / L yeast extract, 20-60 ml / L bovine serum, 8-12 mg / L coenzyme, 1-4 g / L ammonium sulfate, 0.2-1 g / L magnesium sulfate, 2-6 g / L sodium chloride, and 2-6 g / L dipotassium hydrogen phosphate.

[0024] The concentration of tryptone can be, but is not limited to, 6 g / L, 6.3 g / L, 6.5 g / L, 6.8 g / L, 7 g / L, 7.3 g / L, 7.5 g / L, 7.8 g / L, 8 g / L, 8.3 g / L, 8.5 g / L, 8.8 g / L, 9 g / L, 9.3 g / L, 9.5 g / L, 9.8 g / L, or 10 g / L, or any value between 6 and 10 g / L, preferably 8 g / L.

[0025] The concentration of the yeast extract can be, but is not limited to, 2 g / L, 2.3 g / L, 2.5 g / L, 2.8 g / L, 3 g / L, 3.3 g / L, 3.5 g / L, 3.8 g / L or 4 g / L, or any value between 2 and 4 g / L, preferably 3 g / L.

[0026] The concentration of the bovine serum can be, but is not limited to, 20 ml / L, 25 ml / L, 30 ml / L, 35 ml / L, 40 ml / L, 45 ml / L, 50 ml / L, 55 ml / L or 60 ml / L, or any value between 20 and 60 ml / L, preferably 50 ml / L.

[0027] The concentration of the coenzyme can be, but is not limited to, 8 mg / L, 8.5 mg / L, 9 mg / L, 9.5 mg / L, 10 mg / L, 10.5 mg / L, 11 mg / L, 11.5 mg / L or 12 mg / L, or any value between 8 and 12 mg / L, preferably 10 mg / L.

[0028] The concentration of ammonium sulfate can be, but is not limited to, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L or 4 g / L, or any value between 1 and 4 g / L, preferably 2 g / L.

[0029] The concentration of magnesium sulfate can be, but is not limited to, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L or 1 g / L, or any value between 0.2 and 1 g / L, preferably 0.5 g / L.

[0030] The concentration of sodium chloride can be, but is not limited to, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L or 6 g / L, or any value between 2 and 6 g / L, preferably 5 g / L.

[0031] The concentration of the dipotassium hydrogen phosphate can be, but is not limited to, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L or 6 g / L, or any value between 2 and 6 g / L, preferably 5 g / L.

[0032] In some specific embodiments, the pH of the culture medium is 7 to 7.2. Specifically, ammonia can be used to adjust the pH of the culture medium.

[0033] According to another aspect of the present invention, a method for preparing a culture medium for Actinobacillus pleuropneumoniae is also provided, comprising dissolving each component in water according to the above-mentioned culture medium formulation to obtain a fermentation culture medium.

[0034] This culture medium ensures that the logarithmic and plateau phases of fermentation are essentially consistent across different serotypes, enabling rapid and standardized fermentation culture processes that meet the needs of whole-cell inactivated vaccines for disease control. According to another aspect of the invention, the application of the above-described culture medium or the culture medium prepared by the above-described method in the fermentation culture of Actinobacillus pleuropneumoniae is also provided.

[0035] In some specific embodiments, the Actinobacillus pleuropneumoniae includes at least one of Actinobacillus pleuropneumoniae type 1, Actinobacillus pleuropneumoniae type 2, Actinobacillus pleuropneumoniae type 4, Actinobacillus pleuropneumoniae type 5, and Actinobacillus pleuropneumoniae type 7.

[0036] According to another aspect of the present invention, a fermentation culture method for Actinobacillus pleuropneumoniae is also provided, wherein Actinobacillus pleuropneumoniae is inoculated into the culture medium described above or the culture medium prepared by the above preparation method for culture.

[0037] Using the above-mentioned culture medium, the number of viable bacteria in each serotype can be no less than 10 billion CFU / ml, realizing high-density fermentation culture of Actinobacillus pleuropneumoniae strains of serotypes 1, 2, 4, 5, 7 and 15.

[0038] To suppress or eliminate fermentation foam, in some specific embodiments, the preparation method further includes the step of adding an antifoaming agent to the culture medium. In some specific embodiments, the dosage of the antifoaming agent is 0.3~0.5 g / L to achieve the effect of suppressing or eliminating fermentation foam without affecting the growth of the strain. Specific antifoaming agents can be at least one of Boyihe, Punio, or BASF.

[0039] The concentration of the defoamer can be, but is not limited to, 0.3 g / L, 0.35 g / L, 0.4 g / L, 0.45 g / L or 0.5 g / L, or any value between 0.3 and 0.5 g / L, preferably 0.4 g / L.

[0040] In some specific embodiments, the culture temperature is 36~38℃, preferably 37℃; in some specific embodiments, the stirring speed of the culture system is 100~400r / min; in some specific embodiments, the aeration rate of the culture system is 0.3~0.5vvm; in some specific embodiments, the dissolved oxygen content of the culture system is 5%~10%.

[0041] In some specific implementations, the residual sugar content in the culture system is controlled to be 1~3 g / L; in some specific implementations, the pH of the culture system is controlled to be 7.0~7.2.

[0042] The present invention will be further illustrated by the following embodiments. Unless otherwise specified, all materials used in the embodiments are commercially available.

[0043] Example 1 Fermentation medium The components and concentrations per liter of the fermentation medium are shown in Tables 1 and 2.

[0044] Table 1

[0045] Table 2

[0046] Prepare culture media 1 to 11 according to the table above: weigh each component according to the formula of the culture medium, add water for injection and stir thoroughly to dissolve, make up to 1000ml, filter with a 0.22μm filter cartridge to sterilize, and prepare fermentation culture medium. Store at 4℃ for later use.

[0047] Culture medium 12 Unlike culture medium 2, 5 g / L of lyophilized bovine serum albumin powder was used instead of bovine serum.

[0048] Culture medium 13 Unlike culture medium 2, casein peptone is used instead of tryptone.

[0049] Culture medium 14 Unlike culture medium 2, soybean peptone was used instead of trypsin.

[0050] Culture medium 15: BHI basal medium (manufacturer: BD, batch number: 1159790) Culture medium 16: TSB (manufacturer: BD, batch number: 3114509) plus FBS basal culture medium (manufacturer: Jinyuankang, batch number: 20230705), with a TSB to FBS ratio of 9:1.

[0051] Example 2 Fermentation culture and growth curve monitoring 1. Microbial strain resuscitation and culture The freeze-dried strains APP1-332, APP2-333, APP4-335, APP5-336, APP7-338, and APP15-346 were rehydrated in a biosafety cabinet, streaked into solid culture medium in four zones, and incubated at 37°C for 20-24 hours for later use.

[0052] 2. Seed liquid preparation Three typical solid culture media were aseptically picked with an inoculation loop to revive single colonies, which were then aseptically inoculated into 1L shake flask culture medium and placed in a 2L shake flask at 37℃ and 200r / min for 12h to harvest the seed culture.

[0053] 3. Reactor fermentation culture The prepared culture medium was inoculated into the fermenter, a certain amount of defoamer was added, and the pH was adjusted to 7.2 using ammonia water. 5% seed culture was then added. The following control parameters were used during the cultivation process: dissolved oxygen 10%, aeration rate 0.5 vvm, stirring speed 300 r / min, pH maintained at 7.0 using ammonia water during cultivation, residual sugar testing started after 1 hour of cultivation, and intermittent feeding was used to control the residual sugar content during fermentation to 1-3 g / L. Culture medium 1 contained 0.3 g / L of Puniol defoamer; culture medium 2 contained 0.3 g / L of Boyi defoamer; culture medium 3 contained 0.35 g / L of Boyi defoamer; culture medium 4 contained 0.4 g / L of Boyi defoamer; culture medium 5 contained 0.4 g / L of Puniol defoamer; culture medium 6 contained 0.45 g / L of Boyi defoamer; culture medium 7 contained 0.5 g / L of Boyi defoamer; and culture medium 8 contained 0.5 g / L of Puniol defoamer. BASF defoamer at a concentration of 0.3 g / L was added to culture medium 9; BASF defoamer at a concentration of 0.4 g / L was added to culture medium 10; BASF defoamer at a concentration of 0.5 g / L was added to culture medium 11; BASF defoamer at a concentration of 0.35 g / L was added to culture medium 12; BASF defoamer at a concentration of 0.35 g / L was added to culture medium 13; BASF defoamer at a concentration of 0.35 g / L was added to culture medium 14; BASF defoamer at a concentration of 0.4 g / L was added to culture medium 15; and BASF defoamer at a concentration of 0.4 g / L was added to culture medium 16.

[0054] 4. Growth curve monitoring: After inoculation, samples were taken every 2 hours to test the viable bacterial count. The monitoring was carried out continuously for 12 hours, and the statistical data was collected to plot the growth curve. Method for viable cell counting: Take 0.1 mL of APP bacterial culture from different fermentation medium groups and harvested at different times, and add it to a 2 mL EP tube containing 0.9 mL of fermentation medium in tube 1. Vortex to mix. Change the pipette tip and transfer 0.1 mL of the mixture from tube 1 to a 2 mL EP tube containing 0.9 mL of fermentation medium in tube 2. Repeat this step until the mixture is diluted to tube 8. Vortex to mix. Transfer 0.1 mL of the mixture from tube 5 to tube 8 and spread it evenly on a solid plate containing NAD, TSA and NBS. Repeat on 3 plates. Incubate at 37℃ for 12-16 hours and count the colonies.

[0055] 5. Results (1) The culture results of culture medium 1 are as follows Figure 1 As shown in Table 3, the results indicated that all six serotype strains rapidly entered the logarithmic growth phase 2 hours after inoculation, reached the plateau phase at 6 hours of culture, and maintained the plateau phase for approximately 2 hours, meaning they began to decline after 8 hours of culture. The growth rhythm of the six serotypes was basically consistent under this culture medium and process; strain APP2-333 had the highest viable count at the plateau phase (8 hours), reaching 1.22 × 10⁻⁶. 10 CFU / mL; APP1-332 and APP7-338 strains had lower viable counts during the plateau phase compared to other serotypes, but still reached 1.11 × 10⁻⁶ CFU / mL. 10 CFU / mL. Therefore, medium 1 is suitable for the fermentation culture of APP.

[0056] Table 3

[0057] (2) The culture results of culture medium 2 are as follows Figure 2 As shown in Table 4, the results indicate that all serotype strains rapidly entered the logarithmic growth phase 2 hours after inoculation, reached the plateau phase at 6 hours, maintained the plateau phase for about 2 hours, and began to decline after 8 hours. It is evident that the growth rhythm of the six serotypes was basically consistent under this culture medium and process; strain APP5-336 had the highest viable count at the plateau phase (8 hours), reaching 1.28 × 10⁻⁶. 10 CFU / mL; APP1-332 strain had a lower viable count during the plateau phase compared to other serotypes, but it still reached 1.06 × 10⁻⁶. 10 CFU / mL.

[0058] Table 4

[0059] (3) The culture results of culture medium 3 are as follows Figure 3As shown in Table 5, all serotype strains rapidly entered the logarithmic growth phase 2 hours after inoculation, reached the plateau phase at 6 hours, maintained the plateau phase for about 2 hours, and began to decline after 8 hours. The growth rhythm of the six serotypes was basically consistent under this culture medium and process; strain APP5-336 had the highest viable count at the plateau phase (6 hours), reaching 1.57 × 10⁻⁶. 10 CFU / mL; APP2-333 strain had a lower viable count during the plateau phase compared to other serotypes, but it still reached 1.16 × 10⁻⁶. 10 CFU / mL.

[0060] Table 5

[0061] (4) The culture results of culture medium 4 are as follows Figure 4 As shown in Table 6, all serotype strains rapidly entered the logarithmic growth phase 2 hours after inoculation, lasted for 4 hours, then entered the plateau phase, which lasted for about 2 hours before beginning the decline phase. The growth rhythm of the six serotypes was basically consistent under this culture medium and process; strain APP5-336 had the highest viable count at the plateau phase (6 hours), reaching 1.32 × 10⁻⁶. 10 CFU / mL; APP1-332 strain had a lower viable count during the plateau phase compared to other serotypes, but it still reached 1.10 × 10⁻⁶ CFU / mL. 10 CFU / mL.

[0062] Table 6

[0063] (5) The culture results of culture medium 5 are as follows Figure 5 As shown in Table 7, all different serotype strains rapidly entered the logarithmic growth phase 2 hours after inoculation, lasted for 4 hours, then entered the plateau phase, which lasted for about 2 hours, and began to decline after 8 hours of culture. The growth rhythm of the six serotypes was basically consistent under this culture medium and process; strain APP7-338 had the highest viable count at the plateau phase (8 hours), reaching 1.21 × 10⁻⁶. 10 CFU / mL; APP1-332 and APP15-346 strains had lower viable counts during the plateau phase compared to other serotypes, but still reached 1.08 × 10⁻⁶. 10 CFU / mL.

[0064] Table 7

[0065] (6) After fermentation culture inoculation, the dissolved oxygen level of culture medium 6 and culture medium 7 remained high, the culture solution was relatively clear, and the bacterial concentration was low. Culture medium 6 and culture medium 7 are not suitable for the culture of the above five serotype strains.

[0066] (7) The culture results of culture medium 8 are as follows Figure 6 As shown in Table 8, strain APP2-333 had the highest viable bacterial count after 8 hours of culture, at only 7.8 × 10⁻⁶. 8 The CFU / mL concentration of the strains did not show significant effects in the culture of any serotype, making them unsuitable for APP fermentation culture.

[0067] Table 8

[0068] (8) The culture results of culture medium 9 are as follows Figure 7 As shown in Table 9, the viable counts of each serotype strain at the peak of culture ranged from 7.06 to 8.2 × 10⁻⁶. 9 The CFU / mL concentration and the time and duration of the plateau phase vary among different strains, making this culture medium unsuitable for planning the production of multivalent vaccines.

[0069] Table 9

[0070] (9) The culture results of culture medium 10 are as follows Figure 8 As shown in Table 10, the viable count of each serotype strain at the peak of culture ranged from 5.8 to 7.2 × 10⁻⁶. 9 The CFU / mL concentration and the time and duration of the plateau phase vary among different strains, making this culture medium unsuitable for planning the production of multivalent vaccines.

[0071] Table 10

[0072] (10) The culture results of culture medium 11 are as follows Figure 9 As shown in Table 11, the viable counts of each serotype strain at the peak of culture ranged from 6.6 to 8.3 × 10⁻⁶. 9 The CFU / mL concentration varies, and the time and duration of the plateau phase also differ among strains. This culture medium is not suitable for the concentrated culture and production of multiple serotypes of bacteria in the later stages.

[0073] Table 11

[0074] (11) The culture results of culture medium 12 are as follows Figure 10 As shown in Table 12, the viable count of each serotype strain at the peak of culture was 5.0 × 10⁻⁶. 9 The CFU / mL concentration and the varying times at which different serotypes of the strain enter the plateau phase are detrimental to the production planning of multivalent vaccines.

[0075] Table 12

[0076] (12) The culture results of culture medium 13 are as follows Figure 11 As shown in Table 13, the viable count of each serotype strain at the peak of culture was 7.2 × 10⁻⁶. 9 The CFU / mL concentration and the varying times at which different serotypes of the strain enter the plateau phase are detrimental to the production planning of multivalent vaccines.

[0077] Table 13

[0078] (13) The culture results of culture medium 14 are as follows Figure 12 As shown in Table 14, the viable count of each serotype strain at the highest point of culture was 9.0 × 10⁹ CFU / mL. The plateau phase of each serotype strain was relatively short, which is not conducive to the production planning of multivalent vaccines.

[0079] Table 14

[0080] (14) The culture results of culture medium 15 are as follows Figure 13 As shown in Table 15, different serotype strains entered the plateau phase after 6 hours of culture, and the plateau phase lasted for 2 hours. Except for the APP4 strain, which could reach a maximum viable count of 6.8 × 10⁻⁶ after 6 hours of culture. 9 CFU / mL, with the highest values ​​for other cultures ranging from 4.4 to 5.2 × 10⁻⁶. 9 The concentration of CFU / mL was between [value missing]. When culturing APP15-346 strain, flocculent precipitate was found after inoculation, making normal counting impossible. This culture medium is not suitable for culturing APP15-346 strain and has poor culturing effect on other serotype strains.

[0081] Table 15

[0082] In Table 15, " / " indicates that the count could not be completed.

[0083] (15) The culture results of culture medium 16 are as follows Figure 14 As shown in Table 16, during the plateau phase after 4-8 hours of incubation, the highest viable count for each serotype strain can reach 5-6 × 10⁻⁶. 9 The CFU / mL concentration can basically meet the existing requirements for antigen preparation; however, strain APP4-335 entered a plateau phase after 6 hours of culture, with the highest viable count being 4.6 × 10⁻⁶. 9The CFU / mL count is relatively low, making it unsuitable for high-density fermentation of this strain. The time it takes for different serotypes to enter the plateau phase varies considerably; APP2-333 and APP5-336 enter the plateau phase after 4 hours of culture, while APP1-332 and APP15-346 enter the plateau phase after 8 hours. The duration of the plateau phase also varies among serotypes, which is detrimental to the production planning of multivalent vaccines.

[0084] Table 16

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture medium for Actinobacillus pleuropneumoniae, characterized in that, The culture medium comprises 6-10 g / L tryptone, 2-4 g / L yeast extract, 20-60 ml / L bovine serum, 8-12 mg / L coenzyme, 1-4 g / L ammonium sulfate, 0.2-1 g / L magnesium sulfate, 2-6 g / L sodium chloride, and 2-6 g / L dipotassium hydrogen phosphate.

2. The culture medium according to claim 1, characterized in that, The culture medium comprises 8 g / L tryptone, 3 g / L yeast extract, 50 ml / L bovine serum, 10 mg / L coenzyme, 2 g / L ammonium sulfate, 0.5 g / L magnesium sulfate, 5 g / L sodium chloride, and 5 g / L dipotassium hydrogen phosphate.

3. The culture medium according to claim 1 or 2, characterized in that, The pH of the culture medium is 7 to 7.

2.

4. A method for preparing a culture medium for Actinobacillus pleuropneumoniae, characterized in that, The fermentation medium is prepared by dissolving the components in water according to the culture medium formulation of any one of claims 1 to 3.

5. The application of the culture medium according to any one of claims 1 to 3 or the culture medium prepared by the preparation method according to claim 4 in the fermentation culture of Actinobacillus pleuropneumoniae.

6. The application according to claim 5, characterized in that, The Actinobacillus pleuropneumoniae includes at least one of Actinobacillus pleuropneumoniae type 1, Actinobacillus pleuropneumoniae type 2, Actinobacillus pleuropneumoniae type 4, Actinobacillus pleuropneumoniae type 5, and Actinobacillus pleuropneumoniae type 7.

7. A fermentation culture method for Actinobacillus pleuropneumoniae, characterized in that, Actinobacillus pleuropneumoniae is inoculated into the culture medium according to any one of claims 1 to 3 or the culture medium prepared by the preparation method according to claim 4 and cultured.

8. The cultivation method according to claim 7, characterized in that, This also includes adding an antifoaming agent to the culture medium; Preferably, the amount of defoamer used is 0.3~0.5 g / L, and more preferably 0.4 g / L.

9. The cultivation method according to claim 7, characterized in that, The incubation temperature is 36~38℃, preferably 37℃; Preferably, the stirring speed of the culture system is 100~400 r / min; Preferably, the aeration rate of the culture system is 0.3~0.5 vvm; Preferably, the dissolved oxygen content of the culture system is 5% to 10%; Preferably, the residual sugar content in the culture system is controlled to be 1~3 g / L; Preferably, the pH of the culture system is controlled at 7 to 7.

2.

10. The cultivation method according to any one of claims 7-9, characterized in that, The Actinobacillus pleuropneumoniae includes at least one of Actinobacillus pleuropneumoniae type 1, Actinobacillus pleuropneumoniae type 2, Actinobacillus pleuropneumoniae type 4, Actinobacillus pleuropneumoniae type 5, and Actinobacillus pleuropneumoniae type 7.

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