CN111349592B - Spore production culture medium and preparation method of bacterial spores - Google Patents

Abstract

The invention relates to a preparation method of bacterial spores. The method comprises the steps of culturing bacteria by using an activation culture medium and a seed culture medium, inoculating the bacteria to a spore production culture medium, carrying out shake culture at the temperature of 35-37 ℃ and the speed of 150-. By exploring the formula of a spore production culture medium, culture conditions and nutrient treatment, the invention effectively solves the problems of long time, complicated steps and incapability of ensuring the spore quality in the conventional spore preparation process. Compared with the method known in the field, the spore preparation method can obtain a large amount of high-purity spores in a short time, and does not influence the basic characteristics of the morphology, germination rate and heat resistance of the spores. Has important application value for developing various biological agents based on spore forms, reducing production cost, increasing production benefit and ensuring product quality.

Description

A kind of sporulation medium and preparation method of bacterial spores

technical field

The invention belongs to the technical field of spore preparation, and in particular relates to a preparation method of bacterial spores.

Background technique

Spores are some bacteria in the late stage of growth and development, due to the lack of nutrients in the environment or the excessive accumulation of harmful metabolites, so that they form a round or oval shape, thick wall, extremely low water content, and extremely stress resistance in the cell. Strong dormant body. Spores are one of the most resistant organisms in the entire biological world, such as heat resistance, chemical resistance, radiation resistance and so on. The study of bacterial spores has important theoretical and practical significance, as follows:

1. The spores of different bacteria have different characteristics. The presence or absence, shape, size and location of spores are the basis or important reference for bacterial classification and identification.

2. Spores have strong heat resistance. Heat treatment of the bacteria-containing suspension to kill all vegetative cells can screen out the bacterial species that form spores, which is beneficial to improve the screening efficiency of spore-producing species.

3. Spores have strong resistance to adverse environments such as low temperature and dryness, and can maintain their vitality for decades.

4. Many spore-forming bacteria are potent pathogens. For example, Bacillus anthracis, Clostridium botulinum, Clostridium tetani, etc., whether it can kill some representative bacteria spores is the main basis for measuring and formulating various disinfection and sterilization standards.

5. Some spore-forming bacteria can produce useful products concomitantly. For example, Bacillus thuringiensis can produce biconical crystalline inclusions, called paraspore crystals, which are protein toxins with a high degree of specificity while producing spores. Monosexual, can kill the larvae of some insects (especially Lepidoptera), but is completely non-toxic to other animals and plants. Therefore, they make an ideal biopesticide, which can be produced without isolating the protein, but simply by cultivating a large number of bacteria, harvesting and drying them as they form spores and crystals, and make Powder can.

6. Due to the unique production mode of spores, their formation and development are strictly genetically regulated, making spores ideal materials for studying morphogenesis and genetic control.

The development of a rapid spore preparation method is not only of great theoretical significance for the study of spore formation mechanism and spore resistance mechanism, but also for the development of various biological preparations based on spore form, such as BT insecticides, fungicides, etc., to reduce experimental and production costs It has important application value to protect crops and human health.

The problem with the reported spore preparation method is that it takes a long time. Generally, the cells are cultured in DSM medium, and the transfer amount is 1:10 (v:v), which requires 37 °C treatment for 48 hours. Fully mature spores need 90h-100h, and often require complicated and tedious operations, such as 4 °C cold water starvation overnight treatment, repeated 2-3 times, and differential centrifugation, etc., to increase the time in the experiment or enterprise production process and economic costs.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a spore-forming medium and a method for preparing bacterial spores for the deficiencies of the above-mentioned prior art. Through the exploration of medium formula and bacterial culture conditions, the method solves the problems of long spore production time, cumbersome operation steps and unguaranteed spore quality prepared by the existing method, improves production efficiency, and reduces the number of experiments and experiments. Enterprise production costs, promote scientific and economic development. The time required to prepare spores by this method is short, about 54 hours, the spore yield is as high as about 95%, and the spore purity is not less than 98%.

In order to realize above-mentioned technical effect, the technical scheme adopted in the present invention is:

A sporulation medium used in the preparation of bacterial spores, the sporulation medium is composed of: yeast powder 0.3-0.5 g added per 1000 mL of distilled water, peptone 0.3-0.5 g, glucose 0.8-1 g, K ₂ HPO ₄ 5-7 g, KH ₂ PO ₄ 4-5 g, MgSO ₄ 7H ₂ O 0.01-0.1 g, NaCl 0.05-0.2 g, CaCl ₂ 0.01-0.05 g, FeSO ₄ 0.0005-0.001 g, pH adjusted is 7.0-7.5.

Preferably, the sporulation medium is composed of: yeast powder 0.3g added per 1000 mL of distilled water, peptone 0.4g, glucose 0.85g, K ₂ HPO ₄ 5.6g, KH ₂ PO ₄ 4.5g, MgSO ₄ · 7H ₂ O 0.08g, NaCl 0.15g, CaCl ₂ 0.04g, FeSO ₄ 0.0005g, pH was adjusted to 7.2.

A method for preparing bacterial spores using the described sporulation medium, comprising the steps of:

(1) Bacterial activation: streak the bacteria on the activation medium;

(2) Bacterial culture: pick a single colony and inoculate it into the seed medium, cultivate to logarithmic phase to obtain seed bacterial liquid, and the bacterial concentration is 2×10 ⁷ -6×10 ⁸ CFU/mL;

(3), spore production: seed bacterial liquid is inoculated in the described sporulation medium to continue to cultivate;

(4), lysing vegetative bodies: adding triton x-100 to the spore-forming medium to lyse vegetative bodies without spores;

(5), spore collection and preservation.

Preferably, in the step (1), the activation medium is composed of: 4-5 g of yeast powder, 8-10 g of peptone, 8-10 g of NaCl, 18-20 g of agar powder per 1000 mL of distilled water g, pH 7.0-7.5.

Preferably, in the step (1), the activation medium consists of: adding 5 g of yeast powder, 8.5 g of peptone, 9 g of NaCl, 18.5 g of agar powder, and pH 7.5 per 1000 mL of distilled water.

Preferably, in the step (1), the culturing temperature is 28-30 °C, and the culturing time is 12-16 h.

Preferably, the seed medium in the step (2) is composed of: adding 4-5 g yeast powder, 8-10 g peptone, 8-10 g NaCl, pH 7.0-7.5 per 1000 mL of distilled water.

Preferably, the culturing conditions in the step (2) are 28-30 °C, 150-200 rpm, and the culturing time is 10-12 h.

Preferably, in the step (3), the culturing conditions are 35-37 °C, 150-200 rpm, and the culturing time is 20-24 h.

Preferably, the vegetative lysis conditions in the step (4) are 37 °C, 150-200 rpm, and the lysis time is 40-60 min.

LB medium culture control in the prior art: Purification of spores on step gradients of Renografin, in Molecular Biological Methods for Bacillus , pp.391-450, 1990

The prior art spore preparation steps are cumbersome and time-consuming, including 16 hours of strain activation, 3 hours of strain expansion, 20 hours of cold treatment, and 48 hours of heat treatment. The total time required for spore preparation is about 90 hours. The specific time and operation steps are as follows: (1) Strain activation, take out glycerol from -80°C refrigerator to preserve the strain, streak on LB medium, and cultivate at 37°C for 16 h. (2) Pick a single colony and inoculate it in 25 mL of DSM medium, incubate at 150 rpm at 37 °C for 2-3 h, until the OD value is between 0.45-0.60. (3) Inoculate in 250 mL DSM medium at a ratio of 1:10, shake the bacteria at 37 °C and 150 rpm for 48 h. (4) Centrifuge at 10,000 x g for 10 min, and discard the supernatant suspension. (5) Suspend in sterile water at 4 °C, centrifuge at 10,000 x g for 10 min, repeat once, and place in a refrigerator at 4 °C overnight. (6) Repeat steps 4 and 5. (7) Add 50% Renografin reagent, centrifuge at 10000×g for 30 min, and then add sterile water at 4 °C and centrifuge at 7000×g for 10 min. (8) Repeat step 7 3 times. (9) Suspended in sterile water and stored at 4 °C.

Compared with the prior art, the present invention has the following advantages:

Nutrient stress is an external environmental signal for bacteria to produce spores. Bacteria generally produce spores when nutrients are insufficient. For example, when bacteria are depleted of nutrients in the late growth stage or severely lack of nutrients in the growth environment, a large number of spores are produced to survive adverse environmental conditions. Because the sporulation medium in the present invention contains the basic nutrient elements necessary for bacterial growth, but the carbon and nitrogen sources are seriously insufficient, only about one-tenth of the content in a normal bacterial medium such as LB, in which bacteria grow Due to the inability to obtain sufficient nutrients, this environmental condition induces the expression of genes related to bacterial spore formation, resulting in the production of a large number of spores. triton x-100 is a non-ionic surfactant that can dissolve lipids to increase the permeability of cell membranes, help bacteria and sporangia in vivo to lyse, and greatly shorten the process of spore release. According to this principle, time-consuming and cost-intensive processes such as cold treatment and heat treatment are avoided, the spore preparation steps of the present invention are simple, and after the bacterial strain is activated and the seed bacterial liquid is obtained, it only needs to be inoculated in a spore-forming medium to continue culturing for 20-24 h, then add triton x-100 to lyse the vegetative bodies that did not form spores.

The spore preparation time of the present invention is short, the strain is activated for 12-16 hours, the strain is cultured for 10-12 hours, the spore is produced for 20-24 hours, the vegetative body is split for 40-60 minutes, the spore is collected for 8-10 minutes, and the total time for spore preparation A total of 43-53 hours are required.

Description of drawings

Fig. 1 is the microscopic observation result of spore morphology;

Figure 2 shows the test results of heat resistance of spores produced by different strains.

Detailed ways

Example 1

Bacillus cereus (ATCC 14579) was streaked on the activated medium, and the incubation temperature was 30 °C for 12 h. The activation medium is composed of: yeast powder 5 g, peptone 10 g, NaCl 10 g, agar powder 20 g, distilled water 1000 mL, pH 7.2.

A single colony was picked and inoculated into the seed medium, and cultured at 30 °C and 150 rpm for 10 h to obtain a seed bacterial liquid at a stable stage with a bacterial concentration of 5×10 ⁷ CFU/mL. The seed medium is composed of: 5 g of yeast powder, 10 g of peptone, 10 g of NaCl, 1000 mL of distilled water, pH 7.2.

The seed bacterial liquid was inoculated into the spore-forming medium at a ratio of 1:50 (v/v) and continued to cultivate to obtain a large number of spores. The culturing conditions were 35°C, 200 rpm, and 24 h, and the sporulation medium consisted of: 0.5 g of yeast powder, 0.5 g of peptone, 1 g of glucose, 5 g of K ₂ HPO ₄ , and 5 g of KH ₂ PO ₄ 5 g, MgSO ₄ ·7H ₂ O 0.01 g, NaCl 0.05 g, CaCl ₂ 0.01 g, FeSO ₄ 0.001 g, distilled water 1000 mL, pH 7.2.

Triton x-100 was added to a final concentration of 0.02% (v:v) to lyse vegetative bodies in which no spores were formed. The vegetative lysis conditions were 37 °C, 200 rpm, and the lysis time was 40 min.

Spore collection, drying and preservation. The collection conditions are: centrifuging the spore-forming medium after vegetative lysis at 7000 rpm for 10 min, and discarding the supernatant. Described storage condition is 4 ℃ of preservations.

Observation of spore morphology and calculation of yield and purity: 25 minutes after adding 0.02% triton x-100 to lyse the vegetative body, samples were taken for spore staining, smears were made and dried and fixed. 0.5% safranin solution was counterstained for 2 min, and the tablets were dried and observed with an oil microscope. The spores were green and the bacteria were red. The spores that were not released were red rod-shaped bacteria containing green spores. In the figure, green spores are in granular shape, and bacterial bodies are in rod shape. Spore staining and microscopic observation are carried out with the bacteria cultured in LB in the prior art as a control. As can be seen, Figures 1A-B show that there is no difference in the morphology of the spores formed by this method and conventional culture in LB medium. It shows that this method does not affect the basic morphology of Bacillus cereus (ATCC 14579) spores.

The spores were sampled after 24 h of culture in the sporulation medium, and 10 visual fields were counted for the oil microscope observation to calculate the average value. Spore yield=(M1+M3)/(M1+M2+M3) ×100%

where M1 is the number of spores, M2 is the number of bacteria, and M3 is the number of spores that have been formed but not completely released.

40 min after adding 0.02% triton x-100 to lyse the vegetative body, samples were taken for spore staining, and 10 visual fields were counted to calculate the average value under oil microscope observation. Spore purity=N1/(N1+N2+N3)×100%

Among them, N1 is the number of spores, N2 is the number of bacteria, and N3 is the number of spores that have been formed but not completely released.

Spore heat resistance test: After adding 0.02% triton x-100 to lyse the vegetative body for 40 minutes, the spores were collected by centrifugation, resuspended in sterile water and diluted to ^10-6 times, and then divided into two equal parts, one directly The count of the coated plate is A; the count of another plate after 80 °C water bath for 10 min is B; the germination rate of spores=(B/A)×100%. Compared with the germination rate of spores obtained by LB culture in the prior art, three parallel samples were used in each group. Figure 2 shows that the germination rate of spores obtained by this method is not significantly different from the control, indicating that this method does not affect the heat resistance of Bacillus cereus (ATCC 14579) spores.

This example effectively solves the problems of long time and cumbersome process for spores prepared by conventional methods, and reduces the cost of experiments and enterprise production by exploring the medium formula and culture conditions. The Bacillus cereus spores prepared by this method The total time required was 47 h, the spore yield was 98%, and the spore purity was 92%. Microscopic examination and heat resistance experiments showed that the prepared spore morphology and heat resistance remained unchanged.

Example 2

Bacillus subtilis B.subtilis BGSC 168 was streaked on the activated medium, and the incubation temperature was 28 °C for 16 h. The activation medium is composed of: yeast powder 4 g, peptone 8 g, NaCl 8 g, agar powder 18 g, distilled water 1000 mL, pH 7.0.

A single colony was picked and inoculated into the seed medium, and cultured at 28 °C and 180 rpm for 12 h to obtain a seed bacterial liquid with a bacterial concentration of 5×10 ⁸ CFU/mL. The seed medium is composed of: 4 g of yeast powder, 8 g of peptone, 8 g of NaCl, 1000 mL of distilled water, pH 7.0.

The seed bacterial solution was inoculated into the sporulation medium at a ratio of 1:50 (v/v) to continue the cultivation, so that the bacterial cells produced a large number of spores. The culturing conditions were 37°C, 150 rpm, and 20 h, and the sporulation medium was composed of: yeast powder 0.4 g, peptone 0.3 g, glucose 0.8 g, K ₂ HPO ₄ 6 g, KH ₂ PO ₄ 4 g, MgSO ₄ ·7H ₂ O 0.05 g, NaCl 0.1 g, CaCl ₂ 0.03 g, FeSO ₄ 0.0008 g, distilled water 1000 mL, pH 7.0.

Triton x-100 was added to a final concentration of 0.02% (v/v) to lyse vegetative bodies in which no spores were formed. The vegetative lysis conditions were 37 °C, 200 rpm, and 50 min for lysis time.

Spore collection and preservation. The collection conditions described were centrifuging the spore-forming medium after lysis of the vegetative body at 8000 rpm for 8 min, and discarding the supernatant. Described storage condition is 4 ℃ of preservations.

Observation of spore morphology and calculation of yield and purity: After adding 0.02% triton x-100 lysate for 30 minutes, samples were taken for spore staining, smears were made and dried and fixed, first stained with 5% malachite green aqueous solution for 5 minutes, and then used 0.5% safranin solution was counterstained for 2 min, and the tablets were dried and observed with an oil microscope. The spores were green and the bacteria were red. The spores that were not released were red rod-shaped bacteria containing green spores. Spore staining and microscopic observation were carried out with LB cultured cells in the prior art as a control. The granular shape is the spore, and the rod shape is the bacterial body. Figures 1C-D show that there is no difference in the morphology of the spores formed under the conventional culture of the LB medium treated by this method and the control in the prior art. It shows that this method does not affect the basic morphology of B. subtilis BGSC168 spores.

After 20 hours of culture in the sporulation medium, samples were taken for spore staining, and 10 visual fields were counted to calculate the average value under oil microscope observation. Spore yield=(M1+M3)/(M1+M2+M3) ×100%

where M1 is the number of spores, M2 is the number of bacteria, and M3 is the number of spores that have been formed but not completely released.

After adding 0.02% triton x-100 for 50 min to lyse the vegetative body, samples were taken for spore staining, and 10 visual fields were counted for the oil microscope observation to calculate the average value. Spore purity=N1/(N1+N2+N3)×100%

Among them, N1 is the number of spores, N2 is the number of bacteria, and N3 is the number of spores that have been formed but not completely released.

Spore heat resistance test: After adding 0.02% triton x-100 to lyse the vegetative body for 40 minutes, the spores were collected by centrifugation, resuspended in sterile water and diluted to ^10-6 times, and then divided into two equal parts, one directly The count of the coated plate is A; the count of another plate after 80 °C water bath for 10 min is B; the germination rate of spores=(B/A)×100%. The spore germination rate obtained from LB culture was used as the control, and three parallel samples were used in each group. Figure 2 shows that the germination rate of spores obtained by this method is not significantly different from the control, indicating that this method does not affect the heat resistance of B. subtilis BGSC168 spores.

This example effectively solves the problems of long time, cumbersome process and low purity of spores prepared by conventional methods through the exploration of medium formula and culture conditions, and reduces the cost of experiments and enterprise production. The total time required for spore formation of Bacillus subtilis was 49 h, the spore yield was 93%, and the spore purity was 100%. Microscopic examination and heat resistance experiments showed that the prepared spore morphology and heat resistance remained unchanged.

Example 3

Bacillus cereus B. cereus 0-9 (the preservation number is CCTCC No.M209041) was streaked on the activated medium, and the culture temperature was 29 °C for 14 h. The activation medium is composed of: yeast powder 4.5 g, peptone 9 g, NaCl 9 g, agar powder 19 g, distilled water 1000 mL, pH 7.5.

A single colony was picked and inoculated into the seed medium, and cultured at 29 °C and 200 rpm for 11 h to obtain a seed bacterial liquid with a bacterial concentration of 5×10 ⁷ CFU/mL. The seed medium was composed of: 4.5 g of yeast powder, 9 g of peptone, 9 g of NaCl, 1000 mL of distilled water, pH 7.5.

The seed bacterial liquid was inoculated into the spore-forming medium at a ratio of 1:50 (v/v) and continued to cultivate to obtain a large number of spores. The culturing conditions were 36°C, 180 rpm, and 22 h, and the sporulation medium consisted of: yeast powder 0.3 g, peptone 0.4 g, glucose 0.9 g, K ₂ HPO ₄ 7 g, KH ₂ PO ₄ 4.5 g, MgSO ₄ ·7H ₂ O 0.1 g, NaCl 0.15 g, CaCl ₂ 0.05 g, FeSO ₄ 0.0005 g, distilled water 1000 mL, pH 7.5.

Triton x-100 was added to a final concentration of 0.02% (v/v) to lyse vegetative bodies in which no spores were formed. The vegetative lysis conditions were 37 °C, 200 rpm, and 45 min for lysis time.

Spore collection and preservation. The collection conditions described were centrifuging the spore-forming medium after the lysate of the vegetative body at 7000 rpm for 10 min, and discarding the supernatant. Described preservation condition is 4 ℃ of preservations.

Observation of spore morphology and calculation of yield and purity: After adding 0.02% triton x-100 lysate for 20 minutes, samples were taken for spore staining, smears were made and dried and fixed, first stained with 5% malachite green aqueous solution for 5 minutes, and then used 0.5% safranin solution was counterstained for 2 min, and the tablets were dried and observed with an oil microscope. The spores were green and the bacteria were red. The spores that were not released were red rod-shaped bacteria containing green spores. Spore staining and microscopic observation were carried out with the cells cultured in LB in the prior art as a control. The granular shape is the spore, and the rod shape is the bacterial body. Figures 1E-F show that there is no difference in the morphology of the spores formed by this method and the conventional culture in LB medium. It shows that this method does not affect the basic morphology of Bacillus cereus 0-9 spores.

After 22 hours of culture in the spore-forming medium, samples were taken for staining of spores, and 10 visual fields were counted to calculate the average value under oil microscope observation. Spore yield=(M1+M3)/(M1+M2+M3) ×100%

where M1 is the number of spores, M2 is the number of bacteria, and M3 is the number of spores that have been formed but not completely released.

After adding 0.02% triton x-100 for 50 min to lyse the vegetative body, samples were taken for spore staining, and 10 visual fields were counted for the oil microscope observation to calculate the average value. Spore purity=N1/(N1+N2+N3)×100%

Among them, N1 is the number of spores, N2 is the number of bacteria, and N3 is the number of spores that have been formed but not completely released.

Spore heat resistance test: After adding 0.02% triton x-100 to lyse the vegetative body for 40 minutes, the spores were collected by centrifugation, resuspended in sterile water and diluted to ^10-6 times, and then divided into two equal parts, one directly The count of the coated plate is A; the count of another plate after 80 °C water bath for 10 min is B; the germination rate of spores=(B/A)×100%. The spore germination rate obtained from LB culture was used as the control, and three parallel samples were used in each group. Figure 2 shows that the germination rate of spores obtained by this method has no significant difference with the control, indicating that this method does not affect the heat resistance of B. cereus 0-9 spores.

This example effectively solves the problems of long time and cumbersome process for spores prepared by conventional methods, and reduces the cost of experiments and enterprise production by exploring the medium formula and culture conditions. The Bacillus cereus spores prepared by this method The total time required was 48 hours, the spore yield was 95%, and the spore purity was 99%. Microscopic examination and heat resistance experiments showed that the prepared spores remained unchanged in morphology and heat resistance.

The above are only preferred embodiments of the present invention, and do not limit the present invention. Any simple modifications, changes and equivalent replacements made to the above embodiments according to the technical essence of the invention still belong to the technical solutions of the present invention. within the scope of protection.

Claims (10)

1. a sporulation medium used in the preparation of bacterial spores, is characterized in that, described sporulation medium consists of: yeast powder 0.3-0.5 g added in every 1000 mL of distilled water, peptone 0.3-0.5 g, glucose 0.8-1 g, K ₂ HPO ₄ 5-7 g, KH ₂ PO ₄ 4-5 g, MgSO ₄ 7H ₂ O 0.01-0.1 g, NaCl 0.05-0.2 g, CaCl ₂ 0.01-0.05 g, FeSO ₄ 0.0005- 0.001 g, pH adjusted to 7.0-7.5. 2. the sporulation medium used in the preparation of a kind of bacterial spore as claimed in claim 1, is characterized in that, described sporulation medium consists of: the yeast powder 0.3g added in every 1000 mL of distilled water, peptone 0.4g , Glucose 0.85g, K ₂ HPO ₄ 5.6g, KH ₂ PO ₄ 4.5g, MgSO ₄ ·7H ₂ O 0.08g, NaCl 0.15g, CaCl ₂ 0.04g, FeSO ₄ 0.0005g, pH was adjusted to 7.2. 3. the preparation method of the bacterial spore of applying the sporulation medium described in claim 1, is characterized in that, comprises the steps as follows: (1) Activation of strains: streak the bacteria on the activation medium; the bacteria are Bacillus cereus B. cereus0-9, the preservation number is CCTCC No.M209041 or Bacillus cereus, the preservation number is ATCC14579; (2) Bacterial culture: pick a single colony and inoculate it into the seed medium, cultivate to logarithmic phase to obtain seed bacterial liquid, and the bacterial concentration is 2×10 ⁷ -6×10 ⁸ CFU/mL; (3), spore production: seed bacterial liquid is inoculated in the described sporulation medium to continue to cultivate; (4), lysing vegetative bodies: adding triton x-100 to the spore-forming medium to lyse vegetative bodies without spores; (5), spore collection and preservation. 4. The method for preparing bacterial spores according to claim 3, wherein in the step (1), the activation medium is composed of: adding 4-5 g of yeast powder per 1000 mL of distilled water, peptone 8-10 g, NaCl 8-10 g, agar powder 18-20 g, pH 7.0-7.5. 5 . The method for preparing bacterial spores according to claim 3 , wherein in the step (1), the activation medium consists of: adding 5 g of yeast powder and 8.5 g of peptone per 1000 mL of distilled water. 6 . , NaCl 9g, agar powder 18.5g, pH7.5. 6. The method for preparing bacterial spores according to claim 3, wherein in the step (1), the cultivation temperature is 28-30 °C, and the cultivation time is 12-16 h. 7. The method for preparing bacterial spores according to claim 3, characterized in that, in the step (2), the seed culture medium consists of: adding 4-5 g of yeast powder, 8-10 g of peptone to every 1000 mL of distilled water, NaCl 8-10 g, pH 7.0-7.5. 8. The method for preparing bacterial spores according to claim 3, wherein in the step (2), the culture conditions are 28-30 °C, 150-200 rpm, and the culture time is 10-12 h. 9. The method for preparing bacterial spores according to claim 3, wherein in the step (3) The culture conditions are 35-37 °C, 150-200 rpm, and the incubation time is 20-24 h. 10 . The method for preparing bacterial spores according to claim 3 , wherein the vegetative lysis conditions in the step (4) are 37° C., 150-200 rpm, and the lysis time is 40-60 min. 11 .

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