CN103555641B - A kind of mycoplasma hyopneumoniae culture medium and preparation method thereof - Google Patents

Abstract

A culture medium for mycoplasma hyopneumoniae and a preparation method thereof belong to the technical field of veterinary biological products. Each 1000ml of phosphate buffer contains 20-50g of PPLO, 2-10g of yeast powder, 3-15g of glucose, 0.5-4ml of 1% phenol red, 2-10g of amino acid combination, 15-30g of compound Chinese medicine polysaccharide and 50-100ml of pig serum . The mycoplasma hyopneumoniae culture medium of the present invention can improve the tolerance of mycoplasma hyopneumoniae and reduce the apoptosis rate of mycoplasma hyopneumoniae when culturing mycoplasma hyopneumoniae; The concentration of the cultured bacteria reaches 10 ^10-11 /ml, which is more than 5-10 times higher than that of ordinary medium; the Mycoplasma hyopneumoniae culture medium of the present invention can inhibit the growth of other bacteria at the same time when cultivating Mycoplasma hyopneumoniae, and reduce the growth rate of Mycoplasma hyopneumoniae. Cultured contamination risk.

Description

A kind of mycoplasma hyopneumoniae culture medium and preparation method thereof

technical field

The invention belongs to the technical field of veterinary biological products, and in particular relates to a culture medium for mycoplasma hyopneumoniae and a preparation method thereof.

Background technique

Mycoplasma swine pneumonia is a very common respiratory disease in pig farms. It is prevalent all over the world. It is commonly known as swine asthma. It is caused by Mycoplasma hyopneumoniae (Mhp). The pathogen mainly resides in the trachea, bronchi and bronchioles in pigs On the cilia of the pig's respiratory tract, it severely damages the physical defense mechanism of the pig's respiratory tract - the defense mucosa, which increases the susceptibility of the pig herd to other respiratory pathogens, such as swine influenza virus, porcine reproductive and respiratory syndrome virus, type Ⅱ porcine circovirus, bronchial Bordetella septica, Actinobacillus pleuropneumoniae and Haemophilus parasuis, etc., often cause complex infections of multiple pathogens, causing porcine respiratory syndrome (PRDC) or other diseases, Mycoplasma pneumoniae can reduce pig growth rate by 12.7% And reduce the feed remuneration by 13.8%, seriously endangering the development of the pig industry.

Mycoplasma hyopneumoniae is a facultative anaerobic bacterium with extremely demanding growth requirements and difficult isolation. It has higher nutritional requirements than ordinary mycoplasma. Although Mycoplasma hyopneumoniae can use the nutrients in the culture medium to synthesize the substances needed for its own growth and reproduction, due to the small molecular weight of its genome, the amount of information it carries is not much, and its biosynthetic ability is limited. Therefore, it needs to ingest cholesterol, amino acids, Fatty acids, nucleic acid precursors and vitamins and other substances, which make the composition of the culture medium of mycoplasma more complicated, and some special nutrients must be added.

Because Mycoplasma hyopneumoniae has higher nutritional requirements than ordinary Mycoplasma, the composition of the medium is more complex, the culture time is long, and the culture concentration is low, which greatly affects people's further research on it, and also hinders the development and production of Mycoplasma hyopneumoniae vaccine. . At present, many scholars have designed a variety of culture media for its growth. Although they have their own characteristics, they have not solved the problem of difficult cultivation in general. Therefore, further research should be done on the cultivation of Mycoplasma hyopneumoniae.

Polysaccharides are one of the active ingredients of traditional Chinese medicine. A large number of studies have shown that polysaccharides and sugar complexes are not only used as energy resources and constituent materials in organisms, but more importantly, they exist in all cell membrane structures and participate in various activities of cells in life phenomena. . Polysaccharides are an important part of all living organisms and have the ability to scavenge free radicals, increase the activity of antioxidant enzymes and inhibit lipid peroxidation. In addition, traditional Chinese medicine polysaccharides have special protective effects on lower organisms. Chinese medicine polysaccharides are rich in hydroxyl groups, which can combine with low-level living organisms to build a multi-dimensional network space hydrogen bond structure, form a protective film on the surface of living organisms, and realize the protection of living organisms.

Based on the above technical background, the present invention proposes a mycoplasma culture medium, specifically a culture medium especially suitable for mycoplasma hyopneumoniae, which increases the concentration of mycoplasma hyopneumoniae bacterial liquid.

Contents of the invention

In view of the problems existing in the prior art, the purpose of the present invention is to design and provide a technical scheme for a Mycoplasma hyopneumoniae culture medium and a preparation method thereof. By cultivating Mycoplasma hyopneumoniae through the culture medium, the concentration of Mycoplasma hyopneumoniae bacterium can be increased, and the growth rate of Mycoplasma hyopneumoniae can be improved. Mycoplasma antigen production.

Described a kind of Mycoplasma hyopneumoniae culture medium is characterized in that every 1000ml phosphate buffer contains the following weight ratio components:

PPLO 20～50g

Yeast powder 2～10g

Glucose 3～15g

1% phenol red 0.5～4ml

Amino acid combination 2～10g

Compound traditional Chinese medicine polysaccharide 15～30g

Pig serum 50~100ml;

The amino acid combination contains L-arginine, glycine, tyrosine, L-cysteine and serine;

The compound traditional Chinese medicine polysaccharide is extracted from the following traditional Chinese medicine compositions: Coptis chinensis, Ginkgo biloba, Achyranthes bidentata, Codonopsis pilosula, Poria cocos, wolfberry, licorice and salvia miltiorrhiza.

Described a kind of Mycoplasma hyopneumoniae culture medium is characterized in that every 1000ml phosphate buffer contains the following weight ratio components:

PPLO 30～40g

Yeast powder 4～8g

Glucose 5～10g

1% phenol red 1～3ml

Amino acid combination 4～8g

Compound traditional Chinese medicine polysaccharide 20～25g

Pig serum 60-80ml.

The Mycoplasma hyopneumoniae culture medium is characterized in that the amino acid combination contains 10-30% of L-arginine, 5-15% of glycine, 10-30% of tyrosine, and 20% of L-cysteine. ~40% and serine 10~30%.

The Mycoplasma hyopneumoniae culture medium is characterized in that the compound Chinese medicine polysaccharide is extracted from the following Chinese medicine compositions: 5-15 parts of Coptidis Rhizome, 10-30 parts of Ginkgo biloba, 10-20 parts of Achyranthes bidentata, Codonopsis pilosula 10-30 parts, Poria cocos 5-50 parts, wolfberry 10-30 parts, licorice 10-20 parts and salvia miltiorrhiza 5-15 parts.

Described a kind of Mycoplasma hyopneumoniae culture medium is characterized in that described compound Chinese medicine polysaccharide is made through the following steps:

a. Weigh each raw material of traditional Chinese medicine, chop it up, wash it, soak it in cold water overnight, then add purified water 15 times the weight of the raw material, take a water bath to 90°C, and keep it at 90°C, and boil for 2 hours. Stir once every 10 minutes;

b. Discard the dregs of the traditional Chinese medicine in a, collect the medicinal liquid, and centrifuge at 10,000 rpm for 10 minutes after cooling at room temperature, discard the precipitate, and collect the supernatant;

c. The supernatant in b is subjected to alcohol precipitation, and the precipitation is crude Chinese medicine polysaccharide;

d. Wash the crude traditional Chinese medicine polysaccharide in c with sterilized purified water. After washing, dissolve it with 20 times the amount of sterilized purified water at 42°C. After dissolving, add activated carbon at a ratio of 0.5% to absorb overnight at 4°C. After adsorption, centrifuge at 10,000 rpm After 10 minutes, discard the precipitate and collect the supernatant;

e. After the supernatant in d is filtered and sterilized by a 0.22um filter membrane, it is concentrated 10 times to obtain a Chinese medicine polysaccharide concentrate;

f. Vacuum freeze-drying the concentrated Chinese medicine polysaccharide in e to obtain the compound Chinese medicine polysaccharide.

The preparation method of described a kind of Mycoplasma hyopneumoniae culture medium is characterized in that comprising the following processing steps:

1) Prepare phosphate buffer solution;

2) 5-15 parts of Coptis chinensis, 10-30 parts of Ginkgo biloba, 10-20 parts of Achyranthes bidentata, 10-30 parts of Codonopsis pilosula, 5-50 parts of Poria cocos, 10-30 parts of wolfberry, 10-20 parts of licorice and 5 parts of salvia miltiorrhiza ~15 parts were extracted to obtain compound Chinese medicine polysaccharides;

3) Weigh 20-50g of PPLO, 2-10g of yeast powder, 3-15g of glucose, 2-10g of amino acid combination, 15-30g of compound Chinese medicine polysaccharide, dissolve in the 1000mL phosphate buffer solution prepared in step 1), add 1% Mix 0.5-4ml of phenol red thoroughly, adjust the pH to 7.4-7.8 with acid or alkali, pass through a 0.22μm filter membrane under aseptic conditions, filter and sterilize, store at 4°C for later use, add healthy pig serum 50-70℃ before use 100ml to obtain the Mycoplasma hyopneumoniae culture medium.

The method for preparing a culture medium for Mycoplasma hyopneumoniae is characterized in that the compound Chinese medicine polysaccharide in the step 2) is prepared through the following steps:

a. Weigh each raw material of traditional Chinese medicine, chop it up, wash it, soak it in cold water overnight, then add purified water 15 times the weight of the raw material, take a water bath to 90°C, and keep it at 90°C, and boil for 2 hours. Stir once every 10 minutes;

b. Discard the dregs of the traditional Chinese medicine in a, collect the medicinal liquid, and centrifuge at 10,000 rpm for 10 minutes after cooling at room temperature, discard the precipitate, and collect the supernatant;

c. The supernatant in b is subjected to alcohol precipitation, and the precipitation is crude Chinese medicine polysaccharide;

d. Wash the crude traditional Chinese medicine polysaccharide in c with sterilized purified water. After washing, dissolve it with 20 times the amount of sterilized purified water at 42°C. After dissolving, add activated carbon at a ratio of 0.5% to absorb overnight at 4°C. After adsorption, centrifuge at 10,000 rpm After 10 minutes, discard the precipitate and collect the supernatant;

e. After the supernatant in d is filtered and sterilized by a 0.22um filter membrane, it is concentrated 10 times to obtain a Chinese medicine polysaccharide concentrate;

f. Vacuum freeze-drying the concentrated Chinese medicine polysaccharide in e to obtain the compound Chinese medicine polysaccharide.

The method for preparing a culture medium for Mycoplasma hyopneumoniae is characterized in that the amino acid combination described in step 3) contains 10-30% of L-arginine, 5-15% of glycine, and 10-15% of tyrosine. 30%, L-cysteine 20-40% and serine 10-30%.

The preparation method of a Mycoplasma hyopneumoniae culture medium is characterized in that in the step 3), PPLO 30-40g, yeast powder 7-8g, glucose 5-10g, amino acid combination 4-8g, compound Chinese medicine polysaccharide 20 ~ 25g, dissolved in 1000mL phosphate buffer solution prepared in step 1), add 1% phenol red 1 ~ 3ml and mix well, adjust the pH to 7.4 ~ 7.8 with acid or alkali, pass through 0.22μm under sterile conditions filter membrane, filter and sterilize, store at 4°C for later use, add 60-80ml of healthy pig serum before use.

In the present invention, PPLO refers to the purchase of PPLO medium by Senbeijia (Nanjing) Biotechnology Co., Ltd.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The Mycoplasma hyopneumoniae culture medium prepared by the present invention can improve the tolerance of Mycoplasma hyopneumoniae and reduce the apoptosis rate of Mycoplasma hyopneumoniae when culturing Mycoplasma hyopneumoniae.

(2) The mycoplasma hyopneumoniae culture medium prepared by the present invention can increase the number of cultured bacteria of mycoplasma hyopneumoniae, and the concentration of the cultured bacteria liquid reaches 10 ^10-11 /ml (ccu counting method), which is more than 5-10 times higher than that of ordinary culture medium .

(3) The culture medium for Mycoplasma hyopneumoniae prepared by the present invention can inhibit the growth of other bacteria at the same time when culturing Mycoplasma hyopneumoniae, and reduce the contamination risk of culturing Mycoplasma hyopneumoniae.

Detailed ways

In order to make the present invention easier to understand, the present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

Example 1

A kind of mycoplasma hyopneumoniae culture medium, this culture medium contains the following components in weight ratio in every 1000ml phosphate buffer:

PPLO 20g

Yeast powder 6g

Glucose 8g

1% phenol red 4ml

Amino acid combination 4g

Compound Traditional Chinese Medicine Polysaccharide 15g

Pig Serum 50ml

A preparation method for mycoplasma hyopneumoniae culture medium, the steps are:

(1) Preparation of phosphate buffer solution: Accurately weigh 8g of sodium chloride, 0.2g of potassium chloride, 1.15g of disodium hydrogen phosphate, 0.2g of potassium dihydrogen phosphate, 0.1g of calcium chloride, and magnesium chloride containing 6 crystal water Dissolve 0.1g in 1000ml double distilled water, pass through a 0.22μm filter membrane under aseptic conditions, filter and sterilize for later use.

(2) Preparation of compound traditional Chinese medicine polysaccharides

15 parts of Coptis chinensis, 30 parts of Ginkgo biloba, 10 parts of Achyranthes bidentata, 10 parts of Codonopsis pilosula, 20 parts of Poria cocos, 10 parts of wolfberry, 10 parts of licorice and 15 parts of salvia miltiorrhiza were weighed, chopped, washed and soaked in cold water Overnight, then add purified water 15 times the weight of the raw material, take a water bath to 90°C, and keep it at 90°C, cook for 2 hours, and stir every 10 minutes during the cooking process;

b. Discard the dregs of the traditional Chinese medicine in a, collect the medicinal liquid, and centrifuge at 10,000 rpm for 10 minutes after cooling at room temperature, discard the precipitate, and collect the supernatant;

c. The supernatant in b is subjected to alcohol precipitation, and the precipitation is crude Chinese medicine polysaccharide;

d. Wash the crude traditional Chinese medicine polysaccharide in c with sterilized purified water. After washing, dissolve it with 20 times the amount of sterilized purified water at 42°C. After dissolving, add activated carbon at a ratio of 0.5% to absorb overnight at 4°C. After adsorption, centrifuge at 10,000 rpm After 10 minutes, discard the precipitate and collect the supernatant;

e. After the supernatant in d is filtered and sterilized by a 0.22um filter membrane, it is concentrated 10 times to obtain a Chinese medicine polysaccharide concentrate;

f. Vacuum freeze-drying the concentrated Chinese medicine polysaccharide in e to obtain the compound Chinese medicine polysaccharide.

(3) Preparation of Mycoplasma hyopneumoniae medium: Weigh 20g of PPLO, 6g of yeast powder, 8g of glucose, 4ml of 1% phenol red, 4g of amino acid combination (0.8g of L-arginine, 0.4g of glycine, tyrosine 1.0g, L-cysteine 1.2g and serine 0.6g), compound Chinese medicine polysaccharide 15g, dissolve in the phosphate buffer solution prepared in step (1), add 1% phenol red 4mL and mix well, add acid or alkali Adjust the pH to 7.4-7.8, pass through a 0.22 μm filter membrane under aseptic conditions, filter and sterilize, store at 4°C for later use, and add 50 ml of healthy pig serum before use.

Example 2

A kind of mycoplasma hyopneumoniae culture medium, this culture medium contains the following components in weight ratio in every 1000ml phosphate buffer:

PPLO 35g

Yeast powder 2g

Glucose 15g

1% phenol red 0.5ml

Amino acid combination 10g

Compound Traditional Chinese Medicine Polysaccharide 30g

Pig serum 80ml.

A preparation method of a mycoplasma hyopneumoniae culture medium, the steps of which are the same as in Example 1. The compound traditional Chinese medicine polysaccharide is extracted from 10 parts of Coptis chinensis, 20 parts of Ginkgo biloba, 15 parts of Achyranthes bidentata, 20 parts of Codonopsis pilosula, 50 parts of Poria cocos, 20 parts of wolfberry, 15 parts of licorice and 5 parts of salvia miltiorrhiza.

Example 3

A kind of mycoplasma hyopneumoniae culture medium, this culture medium contains the following components in weight ratio in every 1000ml phosphate buffer:

PPLO 50g

Yeast powder 10g

Glucose 3g

1% phenol red 2.5ml

Amino acid combination 2g

Compound Traditional Chinese Medicine Polysaccharide 25g

Pig serum 100ml.

A preparation method of a mycoplasma hyopneumoniae culture medium, the steps of which are the same as in Example 1. The compound traditional Chinese medicine polysaccharide is extracted from 5 parts of Coptis chinensis, 10 parts of Ginkgo biloba, 20 parts of Achyranthes bidentata, 30 parts of Codonopsis pilosula, 5 parts of Poria cocos, 30 parts of wolfberry, 20 parts of licorice and 10 parts of salvia miltiorrhiza.

Comparative example 1 culture medium prepared by the present invention and conventional culture medium comparison test

1 Mhp strains and culture conditions

The Mycoplasma hyopneumoniae J strain purchased from ATCC was respectively inoculated with the Mycoplasma hyopneumoniae culture medium of Example 1 and the two kinds of Mycoplasma hyopneumoniae culture medium of the prior art, and was inoculated with Mycoplasma hyopneumoniae in each culture medium at a ratio of 5% (V/V). culture medium at 37°C, when the color of the medium turns yellow and the pH drops to 6.4-6.8, take out the culture aseptically.

2 Viable count (CCU method)

Take 14 sterile test tubes for each strain culture, each tube is filled with 4.5ml of culture medium containing Mycoplasma hyopneumoniae, add 0.5ml of well-grown culture to the first tube, after mixing evenly, draw 0.5ml into the second tube, and so on Perform a 10-fold serial dilution to the last tube, and set the Mycoplasma hyopneumoniae culture medium without bacterial solution as a negative control. Test tubes were run in triplicate. After serial dilution, place the test tube in a constant temperature incubator at 37°C for static culture, observe once a day, mainly observe the color change and turbidity of the medium, the continuous observation time is 14 days, the dilution of the small tube with the last color change is the culture The CCU titer of the substance.

3 results

3.1 See Table 1 for the CCU measurement results of three growth experiments in which the Z strain of Mycoplasma hyopneumoniae was inoculated with three kinds of media.

Table 1 The CCU measurement results of three growth experiments of Mycoplasma hyopneumoniae Z strain inoculated with three kinds of media

The mycoplasma hyopneumoniae culture medium prepared by the invention can increase the number of mycoplasma hyopneumoniae cultured bacteria, and the concentration of the cultured bacteria liquid reaches 10 ^10-11 /ml (ccu counting method), which is more than 5-10 times higher than that of ordinary culture medium.

The Mycoplasma hyopneumoniae culture medium in Examples 2 and 3 is applied in the same way as in Comparative Example 1, and it can finally achieve the same technical effect as that of Comparative Example 1.

Claims (2)

1. a preparation method for mycoplasma hyopneumoniae substratum, is characterized in that comprising following processing step: 1) Prepare phosphate buffer solution; 2) 5-15 parts of Coptis chinensis, 10-30 parts of Ginkgo biloba, 10-20 parts of Achyranthes bidentata, 10-30 parts of Codonopsis pilosula, 5-50 parts of Poria cocos, 10-30 parts of wolfberry, 10-20 parts of licorice and 5 parts of salvia miltiorrhiza ~15 parts were extracted to obtain compound Chinese medicine polysaccharides; 3) Weigh 20-50g of PPLO, 2-10g of yeast powder, 3-15g of glucose, 2-10g of amino acid combination, 15-30g of compound Chinese medicine polysaccharide, dissolve in the 1000mL phosphate buffer solution prepared in step 1), add 1% Mix 0.5-4ml of phenol red thoroughly, adjust the pH to 7.4-7.8 with acid or alkali, pass through a 0.22μm filter membrane under aseptic conditions, filter and sterilize, store at 4°C for later use, add healthy pig serum 50-70℃ before use 100ml, to obtain Mycoplasma hyopneumoniae culture medium; Said step 2) Chinese compound traditional Chinese medicine polysaccharide is prepared through the following steps: a. Weigh each raw material of traditional Chinese medicine, chop it up, wash it, soak it in cold water overnight, then add purified water 15 times the weight of the raw material, take a water bath to 90°C, and keep it at 90°C, and boil for 2 hours. Stir once every 10 minutes; b. Discard the dregs of the traditional Chinese medicine in a, collect the medicinal liquid, and centrifuge at 10,000 rpm for 10 minutes after cooling at room temperature, discard the precipitate, and collect the supernatant; c. The supernatant in b is subjected to alcohol precipitation, and the precipitation is crude Chinese medicine polysaccharide; d. Wash the crude traditional Chinese medicine polysaccharide in c with sterilized purified water. After washing, dissolve it with 20 times the amount of sterilized purified water at 42°C. After dissolving, add activated carbon at a ratio of 0.5% to absorb overnight at 4°C. After adsorption, centrifuge at 10,000 rpm After 10 minutes, discard the precipitate and collect the supernatant; e. After the supernatant in d is filtered and sterilized by a 0.22um filter membrane, it is concentrated 10 times to obtain a Chinese medicine polysaccharide concentrate; f. Carrying out vacuum freeze-drying of the concentrated Chinese medicine polysaccharide in e to obtain the compound Chinese medicine polysaccharide; The amino acid combination in step 3) contains 10-30% of L-arginine, 5-15% of glycine, 10-30% of tyrosine, 20-40% of L-cysteine and 10-30% of serine . 2. The preparation method of a Mycoplasma hyopneumoniae culture medium as claimed in claim 1, characterized in that in said step 3), PPLO 30-40g, yeast powder 7-8g, glucose 5-10g, amino acid combination 4 ～8g, compound Chinese medicine polysaccharide 20～25g, dissolve in 1000mL phosphate buffer solution prepared in step 1), add 1% phenol red 1～3ml and mix well, adjust the pH to 7.4～7.8 with acid or alkali, without Pass through a 0.22μm filter membrane under bacterial conditions, filter and sterilize, and store at 4°C for later use. Add 60-80ml of healthy pig serum before use.