Poultry pasteurella multocida culture medium, preparation method and application

Abstract

The invention discloses a culture medium for poultry pasteurella multocida. The culture medium is prepared from the following components: 24g / L to 25g / L of tryptone, 18g / L to 20g / L of dipotassium phosphate heptahydrate, 7g / L to 8g / L of sodium chloride, 0.25 g / L of anhydrous magnesium sulfate, 1.36 g / L of monopotassium phosphate and 10g / L of glucose. The prepared culture medium for the poultry pasteurella multocida is applied to enrichment culture of the poultry pasteurella multocida, the culture medium suitable for the poultry pasteurella multocida can be obtained by optimizing the components and the component proportion of the culture medium, the culture medium is applied to the enrichment culture of the poultry pasteurella multocida, the obvious enrichment effect is achieved, and the culture medium is suitable for the poultry pasteurella multocida. The cost of the culture medium is obviously lower than that of a conventional culture medium or culture scheme.

Description

Technical Field

[0001] The present invention relates to the technical field of biological culture media, and in particular, to a culture medium for avian Pasteurella multocida, a preparation method thereof, and an application thereof. Background Art

[0002] Avian Pasteurellosis, also known as avian cholera, is a contagious disease mainly characterized by hemorrhagic septicemia, with main pathological changes including punctate hemorrhage of the epicardium, pinpoint necrosis of the liver, and hemorrhage of various organs and mucosae. Poultry are all susceptible to Pasteurella multocida (Pm), among which chickens and turkeys are the most susceptible, followed by quails, pigeons, ducks, and geese. Chickens and turkeys will die acutely after being infected with Pm, and the mortality rate can reach 100%. Ducks and geese have stronger tolerance to Pm compared with chickens. This disease is prevalent in many countries around the world, showing sporadic prevalence, and is one of the common diseases of poultry. This disease is mainly transmitted by contact. Healthy poultry may be infected when they come into contact with the carcasses, excreta, secretions of infected poultry, or contaminated water, litter, feed, soil, etc. in the infected poultry house. This disease can occur throughout the year, but it is more prevalent in the warm and humid spring, the summer and autumn seasons with alternating hot and cold, and the typhoon season. This disease has a high incidence rate and a high fatality rate, and is widely distributed, seriously affecting the healthy development of the poultry industry.

[0003] Pm has relatively high nutritional requirements. The current enrichment culture media for Pasteurella mainly include modified Martin broth or nutrient broth, etc. Modified Martin broth has a high cost, a complex preparation process, and requires the addition of auxiliary preparations such as serum or lysed blood, resulting in exogenous contamination; nutrient broth has a simple preparation process and a low cost, but the enrichment effect is not ideal, and exogenous contamination is caused due to the need to add auxiliary agents such as serum or lysed blood. Therefore, there is currently a lack of a culture medium for culturing avian Pasteurella multocida that has a low cost, a good enrichment effect, a simple preparation process, and does not require the introduction of auxiliary agents such as serum or lysed blood. Therefore, when culturing avian Pasteurella multocida, there are high requirements for both the nutrition of the culture medium and the growth environment. Currently, it is mainly carried out by flat flask solid culture and Erlenmeyer flask liquid culture; even if some production enterprises use fermenters to culture avian Pasteurella multocida, it is only a simple magnification of the traditional culture process for Pasteurella, and there are still problems such as high culture medium cost, low bacterial concentration, and high production cost. Summary of the Invention

[0004] Based on this, in order to solve one of the above problems, the present invention provides a culture medium for avian Pasteurella multocida, a preparation method thereof, and an application thereof. The specific technical solutions are as follows:

[0005] A medium for avian Pasteurella multocida, the medium consists of the following components: 24 g / L - 25 g / L of tryptone, 18 g / L - 20 g / L of dipotassium hydrogen phosphate heptahydrate, 7 g / L - 8 g / L of sodium chloride, 0.25 g / L of anhydrous magnesium sulfate, 1.36 g / L of potassium dihydrogen phosphate, and 10 g / L of glucose.

[0006] Further, the medium includes the following components: 24.24 g / L of tryptone, 18.2 g / L of dipotassium hydrogen phosphate heptahydrate, 7.68 g / L of sodium chloride, 0.25 g / L of anhydrous magnesium sulfate, 1.36 g / L of potassium dihydrogen phosphate, and 10 g / L of glucose.

[0007] In addition, the present invention also provides a preparation method for a medium of avian Pasteurella multocida, the preparation method includes the following steps:

[0008] Based on a total volume of 1 L of the medium, add 24 g - 25 g of tryptone, 18 g - 20 g of dipotassium hydrogen phosphate heptahydrate, 7 g - 8 g of sodium chloride, 0.25 g of anhydrous magnesium sulfate, and 1.36 g of potassium dihydrogen phosphate to water until the total volume reaches 980 mL, stir and heat to dissolve, after cooling to room temperature by the first cooling treatment, adjust the pH to 7.2 - 8.2, then sterilize at 100 KPa - 125 KPa and 110 °C - 125 °C for 10 min - 20 min, and then add 20 mL of 50% glucose after cooling to room temperature by the second cooling treatment.

[0009] The present invention also provides an application of a medium for avian Pasteurella multocida, the application is the application of the medium in the enrichment culture of avian Pasteurella multocida.

[0010] Further, the enrichment culture is: Scratch and resuscitate avian Pasteurella multocida, pick a single colony and inoculate it into 8 mL of Martin broth containing 3% calf serum, and shake culture at 37 °C at a speed of 180 r / min for 12 h as the primary seed solution;

[0011] Inoculate the primary seed solution into the medium for shake culture, the culture temperature is 35 °C - 38 °C, the shake speed is 150 r / min - 200 r / min, and the aeration culture time is 12 - 14 h.

[0012] In the above solution, by optimizing the components and the component ratio of the medium, a medium suitable for avian Pasteurella multocida can be obtained and applied to the enrichment culture of avian Pasteurella multocida, which has a significant enrichment effect, and the cost of the medium is significantly lower than that of conventional media or culture schemes. Description of the Drawings

[0013] Figure 1Schematic diagram of the effects of different culture media;

[0014] Figure 2 Schematic diagram of the response surface and contour lines of the culture medium to the interaction of different components. Detailed implementation manners

[0015] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with its embodiments. It should be understood that the specific implementation manners described herein are only used to explain the present invention and do not limit the protection scope of the present invention.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention in this specification are only for the purpose of describing specific implementation manners and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0017] The implementation scheme of the present invention will be described in detail below in conjunction with specific embodiments.

[0018] Example 1:

[0019] A preparation method of a culture medium for avian Pasteurella multocida, comprising the following steps:

[0020] Based on a total volume of 1 L of the culture medium, 24.24 g of tryptone, 18.2 g of dipotassium hydrogen phosphate heptahydrate, 7.68 g of sodium chloride, 0.25 g of anhydrous magnesium sulfate, and 1.36 g of potassium dihydrogen phosphate are added to water to a total volume of 980 mL, stirred and heated to dissolve. After being cooled to room temperature by the first cooling treatment, the pH is adjusted to 7.6, and then sterilized at 100 KPa and 121 °C for 15 min. After being cooled to room temperature by the second cooling treatment, 20 mL of 50% glucose is added;

[0021] Method for enrichment culture: The enrichment culture is as follows: Scratch and resuscitate avian Pasteurella multocida, pick a single colony into 8 mL of Martin broth containing 3% calf serum, and shake and culture at 37 °C and a rotation speed of 180 r / min for 12 h as the primary seed solution;

[0022] The primary seed solution is inoculated into the culture medium for shaking culture. The culture temperature is 37 °C, the rotation speed of the shaker is 180 r / min, the aeration culture time is 16 h, and 1 mL of the bacterial solution is taken every 1 h to measure the OD value and perform viable cell counting.

[0023] To verify that the components and their ratios of the present invention have a significant effect on increasing the number of bacteria, the following effect verification was carried out: Using Martin broth medium, nutrient broth + 1% serum medium, and basal medium as control groups, the results are shown in Table 1. Among them, the basal medium is: potassium dihydrogen phosphate 1.36 g / L, dipotassium hydrogen phosphate 10 g / L, sodium chloride 1.19 g / L, anhydrous magnesium sulfate 0.25 g / L, tryptone 10 g / L, and glucose 10 g / L.

[0024] Table 1: Effects of different media

[0025]

[0026] From the data in Table 1 and Figure 1 analysis shows that Figure 1 As shown in the schematic diagram of the effects of different media, based on the basal medium formula, the present application optimizes the components and their ratios of the medium and applies it to the enrichment culture of avian Pasteurella multocida. It can not only obtain a significant enrichment culture effect, but also significantly reduce the preparation components, with the advantage of being easier to obtain.

[0027] In addition, to verify the effect of the components and their ratios of the present application on the enrichment of avian Pasteurella multocida, using the same method as in Example 1, through different component ratios, that is, the steepest ascent test, the OD value was measured and recorded as OD600, as shown in Table 2 below.

[0028] Table 2: Results of the steepest ascent test

[0029] Group 1 2 3 4 5 6 Tryptone 12 16 20 24 28 30 Dipotassium hydrogen phosphate 12 14 16 18 20 22 Sodium chloride 5 6 7 8 9 10 OD 600 1.505 1.728 1.755 2.005 1.829 1.819

[0030] Taking the OD600 (Y) of the bacterial solution in Table 2 as the response value, a Box-Behnken test was carried out. A: tryptone, B: potassium dihydrogen phosphate heptahydrate, C: sodium chloride were used as independent variables. Using Design-Expert 8.0 software, a Box-Behnken central composite test with 3 factors and 3 levels was designed. The Box-Behnken test design and results are shown in Table 3, and the variance analysis of the regression model is shown in Table 4.

[0031] Table 3: Results of the Box-Behnken test

[0032] Group A: Tryptone B: Potassium dihydrogen phosphate heptahydrate C: Sodium chloride OD600 1 20 18 7 1.738 2 20 16 8 1.682 3 24 18 8 1.848 4 28 20 8 1.672 5 28 16 8 1.785 6 28 18 7 1.768 7 24 20 7 1.815 8 24 18 8 1.823 9 24 16 7 1.731 10 28 18 9 1.701 11 20 20 8 1.747 12 24 16 9 1.774 13 24 18 8 1.838 14 20 18 9 1.667 15 24 18 8 1.802 16 24 20 9 1.751 17 24 18 8 1.82

[0033] Table 4: Variance analysis of the regression model

[0034]

[0035]

[0036] From the combined analysis of Table 3 and Table 4, it can be seen that the P value of the model is less than 0.01, indicating that the model is significant; the P value of the lack-of-fit term is 0.1246, which is greater than 0.05, indicating that the lack-of-fit term is not significant, meaning that the model can be used for optimizing the enrichment medium. From the F value, it can be known that the influence of the three factors on the enrichment medium is potassium hydrogen phosphate (B) > tryptone (A) > sodium chloride (C). The steeper the response surface slope and the elliptical contour lines indicate a significant interaction between the two. The response surface and contour lines of the interaction between various factors on the enrichment medium are shown in Figure 2 . After testing with Design-Expert 8.0 software, the optimized composition ratio of the medium for avian Pasteurella multocida was finally obtained as follows: A: tryptone 24.24 g / L; B: potassium dihydrogen phosphate heptahydrate 18.2 g / L; C: sodium chloride 7.68 g / L. In addition, the optimized components and composition ratio can significantly improve the enrichment effect of avian Pasteurella multocida. The raw materials are easily available, the preparation process is simple, which can not only ensure the enrichment effect but also significantly reduce the preparation cost of the medium.

[0037] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0038] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A culture medium for avian Pasteurella multocida, characterized in that: The culture medium is composed of the following ingredients: 24 g / L-25 g / L tryptone, 18 g / L-20 g / L potassium hydrogen phosphate heptahydrate, 7 g / L-8 g / L sodium chloride, 0.25 g / L anhydrous magnesium sulfate, 1.36 g / L potassium dihydrogen phosphate and 10 g / L glucose.

2. The culture medium according to claim 1, characterized in that The culture medium includes the following components: 24.24 g / L tryptone, 18.2 g / L potassium hydrogen phosphate heptahydrate, 7.68 g / L sodium chloride, 0.25 g / L anhydrous magnesium sulfate, 1.36 g / L potassium dihydrogen phosphate and 10 g / L glucose.

3. A method for preparing a culture medium for avian Pasteurella multocida, characterized in that: The preparation method comprises the following steps: Based on the total volume of the culture medium as 1L, add 24g-25g of tryptone, 18g-20g of dipotassium hydrogen phosphate heptahydrate, 7g-8g of sodium chloride, 0.25g of anhydrous magnesium sulfate and 1.36g of potassium dihydrogen phosphate to water to a total volume of 980mL, stir and heat to dissolve, cool to room temperature through the first cooling treatment, adjust the pH to 7.2-8.2, and then sterilize at 100KPa-125KPa, 110°C-125°C for 10min-20min, and then cool to room temperature through the second cooling treatment, and then add 20mL of 50% glucose.

4. An application of a culture medium for avian Pasteurella multocida, characterized in that: The application is the application of the culture medium described in any one of claims 1 to 2 in the enrichment culture of avian Pasteurella multocida or the application of the culture medium obtained by the preparation method described in claim 3 in the enrichment culture of avian Pasteurella multocida.

5. The use according to claim 4, characterized in that: The bacterial enrichment culture comprises: scarifying and reviving avian Pasteurella multocida, picking a single colony in 8 mL of Martin broth containing 3% calf serum, and culturing it on a shaking table at 37° C. and at a speed of 180 r / min for 12 hours as a primary seed solution; The primary seed solution is inoculated into the culture medium for shaking culture, the culture temperature is 35° C. to 38° C., the shaking speed is 150 r / min to 200 r / min, and the ventilation culture time is 12 to 14 hours.

Images