Serum for fermentation of haemophilus parasuis as well as preparation method and application of serum

Abstract

The invention discloses serum for fermentation of haemophilus parasuis as well as a preparation method and application thereof, and belongs to the technical field of animal vaccine production. The customized serum comprises the following components: newborn calf serum, sodium glutamate, a plant extract, NAD (Nicotinamide Adenine Dinucleotide), MgSO4, glycerol and glycine. All components of the customized serum do not contain porcine exogenous viruses, and the customized serum is matched with a TSB culture medium for use, so that the fermentation time of haemophilus parasuis is shortened, and the antigen density is improved. According to the method disclosed by the invention, the safety is ensured, the problems that the traditional haemophilus parasuis fermentation only uses TSB, NAD and serum, the time is long, and the antigen density is low are solved, and all components needing to be additionally added are added into the customized serum, so that the culture medium preparation process before fermentation is simplified.

Description

Technical Field

[0001] The invention belongs to the technical field of animal vaccine production, and in particular relates to serum for fermentation of Haemophilus parasuis and a preparation method and application thereof. Background Art

[0002] Haemophilus parasuis is a Gram-negative bacterium, a short rod with more than 15 serotypes, of which serotypes 4, 5, and 13 are the most common. The bacterium requires NAD for growth and grows on blood culture medium and chocolate culture medium. Its colonies are small and transparent, and there is no hemolysis on blood culture medium. It grows well around Staphylococci and forms satellites. It is reported that the actual incidence of Haemophilus parasuis is high, more than 10 times the actual number of confirmed cases.

[0003] Haemophilus parasuis disease, also known as polyfibrinous serositis and arthritis, or Glaser's disease, is caused by the bacterium Haemophilus parasuis. Haemophilus parasuis is common in the environment and is also found in healthy pigs. The consequences of a first infection with this bacterium can be quite serious.

[0004] Vaccination can effectively control Haemophilus parasuis disease, and antigen concentration is an important factor affecting inactivated vaccines. At present, the fermentation of Haemophilus parasuis mostly uses TSB medium supplemented with NAD and newborn calf serum, which not only takes a long time to ferment, but also requires improvement in antigen density. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, the present invention provides a serum for fermentation of Haemophilus parasuis with short fermentation time and high antigen density, as well as a preparation method and application thereof.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is: a serum for fermentation of Haemophilus parasuis, the raw materials comprising by weight: 120-160 parts of newborn calf serum, 20-30 parts of sodium glutamate, 700-800 parts of plant extract, 0.1-1 part of NAD, 0.1-1 part of MgSO4, 50-100 parts of glycerol, and 1-10 parts of glycine.

[0007] The weight composition of the raw materials of the present invention achieves the improvement of fermentation efficiency and antigen density by optimizing the formula, while reducing the cost and being able to meet the needs of large-scale production.

[0008] Preferably, the preparation method of the plant extract is: wash and cut apples into pieces, add 180g~220g of apple pieces into 800mL of purified water, add 7~13g of sodium chloride, maintain the pressure at 112℃~120℃ for 25min~35min, filter with gauze after cooling, and then filter through 0.45μm filter and 0.22μm filter respectively to obtain the filtrate which is the plant extract.

[0009] This method can effectively destroy the cell structure and release more bioactive ingredients by washing and cutting apples and subjecting them to high temperature and high pressure treatment under specific conditions, thereby improving the extraction efficiency. During the high temperature and high pressure treatment process, the oxidation and decomposition of the active ingredients can be reduced, maintaining the stability of the active ingredients in the extract.

[0010] A method for preparing the serum for fermentation of Haemophilus parasuis is as follows: raw materials are taken according to a ratio, dissolved completely, mixed evenly, and filtered through a 0.11 μm filter for sterilization.

[0011] An application of the serum for fermentation of Haemophilus parasuis, wherein a TSB medium added with the serum is used for high-density fermentation of Haemophilus parasuis.

[0012] Preferably, the preparation method of the TSB medium is: prepare the TSB medium and sterilize it under high pressure, after the temperature of the TSB medium drops to room temperature, add the customized serum at an addition amount of 20% (V / V) and mix well for use.

[0013] Preferably, the specific fermentation steps are: 1) Take a freeze-dried strain of Haemophilus parasuis, reconstitute it and streak it on a TSA plate containing 0.01% (M / V) NAD and 5% (V / V) newborn calf serum, incubate it at 37°C for about 36 hours, pick a typical colony and inoculate it in 5 mL of TSB medium with 20% (V / V) of the customized serum added, incubate it at 37°C for 12 hours as the primary strain; 2) Inoculate the primary seed at a 5% (V / V) inoculation volume into TSB medium containing 20% ​​(V / V) of the customized serum and culture at 37°C in a shaking incubator for 6-8 hours as the secondary seed; 3) The secondary seed was inoculated into TSB medium containing 20% ​​(V / V) of the customized serum at a 5% (V / V) inoculation rate and fermented in a tank. The pH was adjusted to 7.2-7.4 with ammonia water. The DO was controlled at 20%-40% by adjusting the speed and air intake. The fermentation was continued for 5-7 hours. OD 600 Fermentation ends when it reaches its peak.

[0014] Preferably, the Haemophilus parasuis is serotype 4.

[0015] Compared with the prior art, the present invention has the following advantages: the present invention provides a customized serum for high-density fermentation of Haemophilus parasuis and its application, which, while ensuring safety, breaks through the problems of traditional fermentation of Haemophilus parasuis using only TSB, NAD, and serum, which takes a long time and has low antigen density, and all ingredients that need to be added are added to the customized serum, simplifying the culture medium preparation process before fermentation. DETAILED DESCRIPTION

[0016] The present invention uses tank fermentation of Haemophilus parasuis type 4 to compare TSB+serum of the present invention with traditional TSB+NAD+newborn calf serum, which shows that the customized serum of the present invention can shorten the fermentation time of Haemophilus parasuis and increase the fermentation antigen density. Example 1

[0017] Preparation of customized serum: Take 150 parts of newborn calf serum, 25 parts of sodium glutamate, 775 parts of plant extract, 0.5 parts of NAD, 0.1 parts of MgSO4, 75 parts of glycerol, and 5 parts of glycine, dissolve them completely and mix them evenly, and filter them through a 0.11μm filter for sterilization.

[0018] Preparation of culture medium: Prepare TSB culture medium according to the instructions and sterilize by high pressure. After the temperature of TSB culture medium drops to room temperature, add the customized serum at a rate of 20% (V / V) and mix well.

[0019] Fermentation process: 1) Take a freeze-dried strain of Haemophilus parasuis type 4, reconstitute it and streak it on a TSA plate containing 0.01% (M / V) NAD and 5% (V / V) newborn calf serum, incubate it at 37°C for about 36 hours, pick a typical colony and inoculate it in 5 mL of TSB medium with 20% (V / V) added serum, and incubate it at 37°C in a shaking incubator for 12 hours as the primary strain; 2) Inoculate the primary seed at a 5% (V / V) inoculation volume into TSB medium containing 20% ​​(V / V) of the customized serum and culture at 37°C in a shaking incubator for 6 hours as the secondary seed; 3) The secondary strain was inoculated into TSB medium containing 20% ​​(V / V) of the customized serum at a 5% (V / V) inoculation rate and fermented in a tank. The pH was adjusted to 7.2-7.4 with ammonia water. The DO was controlled at 20%-40% by adjusting the speed and air intake. The fermentation was continued for 5 hours. After the bacterial solution was diluted 10 times, the OD 600 When the concentration was 0.626, the fermentation was terminated and the live bacteria were counted. Example 2

[0020] Preparation of customized serum: Take 120 parts of newborn calf serum, 25 parts of sodium glutamate, 805 parts of plant extract, 0.5 parts of NAD, 0.1 parts of MgSO4, 75 parts of glycerol, and 5 parts of glycine, dissolve them completely and mix them evenly, and filter them through a 0.11μm filter for sterilization.

[0021] Preparation of culture medium: Prepare TSB culture medium according to the instructions and sterilize by high pressure. After the temperature of TSB culture medium drops to room temperature, add the customized serum at a rate of 20% (V / V) and mix well.

[0022] Fermentation process: 1) Take a freeze-dried strain of Haemophilus parasuis type 4, reconstitute it and streak it on a TSA plate containing 0.01% (M / V) NAD and 5% (V / V) newborn calf serum, incubate it at 37°C for about 36 hours, pick a typical colony and inoculate it in 5 mL of TSB medium with 20% (V / V) added serum, and incubate it at 37°C in a shaking incubator for 12 hours as the primary strain; 2) Inoculate the primary seed at a 5% (V / V) inoculation volume into TSB medium containing 20% ​​(V / V) of the customized serum and culture at 37°C in a shaking incubator for 6 hours as the secondary seed; 3) The secondary strain was inoculated into TSB medium containing 20% ​​(V / V) of the customized serum at a 5% (V / V) inoculation rate and fermented in a tank. The pH was adjusted to 7.2-7.4 with ammonia water. The DO was controlled at 20%-40% by adjusting the speed and air intake. The fermentation was continued for 7 hours. After the bacterial solution was diluted 10 times, the OD 600 When the concentration was 0.418, the fermentation was terminated and the live bacteria were counted. Example 3

[0023] Preparation of customized serum: Take 150 parts of newborn calf serum, 25 parts of sodium glutamate, 800 parts of plant extract, 0.5 parts of NAD, 0.1 parts of MgSO4, 50 parts of glycerol, and 5 parts of glycine, dissolve them completely and mix them evenly, and filter them through a 0.11μm filter for sterilization.

[0024] Preparation of culture medium: Prepare TSB culture medium according to the instructions and sterilize by high pressure. After the temperature of TSB culture medium drops to room temperature, add the customized serum at a rate of 20% (V / V) and mix well.

[0025] Fermentation process: 1) Take a freeze-dried strain of Haemophilus parasuis type 4, reconstitute it and streak it on a TSA plate containing 0.01% (M / V) NAD and 5% (V / V) newborn calf serum, incubate it at 37°C for about 36 hours, pick a typical colony and inoculate it in 5 mL of TSB medium with 20% (V / V) added serum, and incubate it at 37°C in a shaking incubator for 12 hours as the primary strain; 2) Inoculate the primary seed at a 5% (V / V) inoculation volume into TSB medium containing 20% ​​(V / V) of the customized serum and culture at 37°C in a shaking incubator for 6 hours as the secondary seed; 3) The secondary strain was inoculated into TSB medium containing 20% ​​(V / V) of the customized serum at a 5% (V / V) inoculation rate and fermented in a tank. The pH was adjusted to 7.2-7.4 with ammonia water. The DO was controlled at 20%-40% by adjusting the speed and air intake. The fermentation was continued for 6 hours. After the bacterial solution was diluted 10 times, the OD 600 When the concentration was 0.545, the fermentation was terminated and the live bacteria were counted.

[0026] Comparative Example 1 Culture medium preparation: Prepare TSB culture medium according to the instructions and sterilize by high pressure. After the temperature of TSB culture medium drops to room temperature, add NAD at 0.01% (M / V) and newborn calf serum at 3% (V / V) and mix well.

[0027] Fermentation process: 1) Take a freeze-dried strain of Haemophilus parasuis type 4, reconstitute it and streak it on a TSA plate containing 0.01% (M / V) NAD and 5% (V / V) newborn calf serum, and incubate it at 37°C for about 36 hours. Pick a typical colony and inoculate it in 5 mL of TSB medium containing 0.01% (M / V) NAD and 3% (V / V) newborn calf serum, and incubate it at 37°C in a shaking incubator for 16 hours as the primary strain; 2) Inoculate the primary seed at a 5% (V / V) inoculum into TSB medium containing 0.01% (M / V) NAD and 3% (V / V) newborn calf serum, and culture at 37°C in a shaking incubator for 10 h as the secondary seed; 3) The secondary strain was inoculated at a 5% (V / V) inoculum into TSB medium containing 0.01% (M / V) NAD and 3% (V / V) newborn calf serum for fermentation. The pH was adjusted to 7.2-7.4 with ammonia water. The DO was controlled at 20%-40% by adjusting the speed and air intake. The fermentation was continued for 8 hours. After the bacterial solution was diluted 10 times, the OD 600 When the concentration was 0.235, the fermentation was terminated and the live bacteria were counted.

[0028] Comparative Example 2 Culture medium preparation: Prepare TSB culture medium according to the instructions and sterilize by high pressure. After the temperature of TSB culture medium drops to room temperature, add NAD at 0.01% (M / V) and newborn calf serum at 3% (V / V) and mix well.

[0029] Fermentation process: 1) Take a freeze-dried strain of Haemophilus parasuis type 4, reconstitute it and streak it on a TSA plate containing 0.01% (M / V) NAD and 5% (V / V) newborn calf serum, and incubate it at 37°C for about 36 hours. Pick a typical colony and inoculate it in 5 mL of TSB medium containing 0.01% (M / V) NAD and 3% (V / V) newborn calf serum, and incubate it at 37°C in a shaking incubator for 16 hours as the primary strain; 2) Inoculate the primary seed at a 5% (V / V) inoculum into TSB medium containing 0.01% (M / V) NAD and 3% (V / V) newborn calf serum, and culture at 37°C in a shaking incubator for 10 h as the secondary seed; 3) The secondary strain was inoculated at a 5% (V / V) inoculum into TSB medium containing 0.01% (M / V) NAD and 3% (V / V) newborn calf serum for fermentation. The pH was adjusted to 7.2-7.4 with ammonia water. The DO was controlled at 20%-40% by adjusting the speed and air intake. The fermentation was continued for 9 hours. After the bacterial solution was diluted 10 times, the OD 600 When the concentration was 0.242, the fermentation was terminated and the live bacteria were counted.

[0030] Comparative Example 3 Culture medium preparation: Prepare TSB culture medium according to the instructions and sterilize by high pressure. After the temperature of TSB culture medium drops to room temperature, add NAD at 0.01% (M / V) and newborn calf serum at 3% (V / V) and mix well.

[0031] Fermentation process: 1) Take a freeze-dried strain of Haemophilus parasuis type 4, reconstitute it and streak it on a TSA plate containing 0.01% (M / V) NAD and 5% (V / V) newborn calf serum, and incubate it at 37°C for about 36 hours. Pick a typical colony and inoculate it in 5 mL of TSB medium containing 0.01% (M / V) NAD and 3% (V / V) newborn calf serum, and incubate it at 37°C in a shaking incubator for 16 hours as the primary strain; 2) Inoculate the primary seed at a 5% (V / V) inoculum into TSB medium containing 0.01% (M / V) NAD and 3% (V / V) newborn calf serum, and culture at 37°C in a shaking incubator for 10-12 hours as the secondary seed; 3) The secondary strain was inoculated at a 5% (V / V) inoculum into TSB medium containing 0.01% (V / V) NAD and 3% (V / V) newborn calf serum for fermentation. The pH was adjusted to 7.2-7.4 with ammonia water. The DO was controlled at 20%-40% by adjusting the speed and air intake. The fermentation was continued for 10 hours. After the bacterial solution was diluted 10 times, the OD 600 When the concentration was 0.247, the fermentation was terminated and the live bacteria were counted.

[0032] Fermentation results Fermentation time, OD after 10-fold dilution 600 The results of live bacteria count are shown in Table 1.

[0033] Table 1 Results of 6 fermentations of Haemophilus parasuis

[0034] As can be seen from Table 1, the results of the three embodiments are better than those of the comparative example. The fermentation time of the embodiments is short and the number of viable bacteria is high. Compared with the three embodiments, the fermentation time of embodiment 1 is shorter and the number of viable bacteria is higher, which can reach 90×10 8 Therefore, the customized serum of the present invention is of great significance for the fermentation of Haemophilus parasuis.

[0035] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A serum for fermentation of Haemophilus parasuis, characterized in that: The raw materials include, by weight: 120-160 parts of newborn calf serum, 20-30 parts of sodium glutamate, 700-800 parts of plant extract, 0.1-1 part of NAD, 0.1-1 part of MgSO4, 50-100 parts of glycerol, and 1-10 parts of glycine.

2. The serum for fermentation of Haemophilus parasuis according to claim 1, characterized in that: The preparation method of the plant extract is as follows: wash and cut apples into pieces, add 180g-220g of apple pieces into 800mL of purified water, add 7-13g of sodium chloride, maintain the pressure at 112°C-120°C for 25min-35min, filter with gauze after cooling, and then filter through a 0.45μm filter and a 0.22μm filter respectively to obtain a filtrate, which is the plant extract.

3. A method for preparing serum for fermentation of Haemophilus parasuis according to claim 1 or 2, characterized in that: Take the raw materials according to the ratio, dissolve them completely, mix them evenly, and filter them through a 0.11μm filter to sterilize them.

4. An application of serum for fermentation of Haemophilus parasuis, characterized in that: The TSB medium added with the serum according to claim 1 or 2 is used for high-density fermentation of Haemophilus parasuis.

5. The use of serum for fermentation of Haemophilus parasuis according to claim 4, characterized in that: The preparation method of the TSB culture medium is as follows: prepare the TSB culture medium and sterilize it under high pressure, and after the temperature of the TSB culture medium drops to room temperature, add the customized serum at a 20% (V / V) addition amount and mix well for use.

6. The use of serum for fermentation of Haemophilus parasuis according to claim 5, characterized in that: The specific fermentation steps are: 1) Take a freeze-dried strain of Haemophilus parasuis, reconstitute it and then streak it on a TSA plate containing 0.01% (M / V) NAD and 5% (V / V) newborn calf serum, incubate it at 37°C for about 36 hours, pick a typical colony and inoculate it in 5 mL of TSB medium to which the serum is added at a rate of 20% (V / V), and incubate it at 37°C in a shaking incubator for 12 hours as the primary strain; 2) Inoculate the primary seed at a 5% (V / V) inoculation volume into TSB medium containing 20% ​​(V / V) of the serum, and culture at 37°C in a shaking incubator for 6-8 hours as the secondary seed; 3) The secondary seed was inoculated into TSB medium containing 20% ​​(V / V) of the serum at a 5% (V / V) inoculation rate and fermented in a tank. The pH was adjusted to 7.2-7.4 with ammonia water. The DO was controlled at 20%-40% by adjusting the speed and air intake. The fermentation was continued for 5-7 hours. OD 600 Fermentation ends when it reaches its peak.

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