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

Spore production culture medium and preparation method of bacterial spores Download PDF

Info

Publication number
CN111349592B
CN111349592B CN202010392101.3A CN202010392101A CN111349592B CN 111349592 B CN111349592 B CN 111349592B CN 202010392101 A CN202010392101 A CN 202010392101A CN 111349592 B CN111349592 B CN 111349592B
Authority
CN
China
Prior art keywords
spore
spores
culture
culture medium
bacterial spores
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202010392101.3A
Other languages
Chinese (zh)
Other versions
CN111349592A (en
Inventor
王刚
刘凤英
黄秋斌
张娟梅
张海燕
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Henan University
Original Assignee
Henan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Henan University filed Critical Henan University
Priority to CN202010392101.3A priority Critical patent/CN111349592B/en
Publication of CN111349592A publication Critical patent/CN111349592A/en
Application granted granted Critical
Publication of CN111349592B publication Critical patent/CN111349592B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

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

Spore production culture medium and preparation method of bacterial spores
Technical Field
The invention belongs to the technical field of spore preparation, and particularly relates to a preparation method of bacterial spores.
Background
The spores are dormant bodies which are round or oval, thick-walled, extremely low in water content and extremely high in stress resistance and are formed in cells due to lack of nutrition or excessive accumulation of harmful metabolites in the environment at the later growth and development stage of certain bacteria. The spore is one of the organisms with the strongest stress resistance such as heat resistance, chemical resistance, radiation resistance and the like in the whole biological world. The research on bacterial spores has important theoretical and practical significance, and is specifically shown as follows:
1. spores of different bacteria have different characteristics, and the existence, the shape, the size, the implantation position and the like of the spores are the basis or important reference for classifying and identifying the bacteria.
2. The spore has strong heat resistance, the bacterium-containing suspension is subjected to heat treatment to kill all vegetative cells, the bacterium species forming the spore can be screened out, and the screening efficiency of spore-producing bacterium species is improved.
3. The spores have strong resistance to adverse environments such as low temperature and dryness, can keep vitality for decades, is a good material for storing strains in a laboratory, and is beneficial to long-term storage of the strains.
4. Many spore-forming bacteria are strong pathogenic bacteria. Such as bacillus anthracis, clostridium botulinum and clostridium tetani, and the like, whether spores of some representative bacteria can be killed or not is a main basis for measuring and establishing various disinfection and sterilization standards.
5. Some spore-forming bacteria may produce useful products concomitantly, for example bacillus thuringiensis, which, while producing spores, may produce a biconical crystal inclusion, called parasporal crystal, which is a protein toxin with high specificity that kills larvae of certain insects, particularly lepidopterans, without being completely toxic to other animals and plants. Thus, they are ideal biopesticides for their production without the need to isolate the protein, by culturing a large number of bacteria, harvesting it as spores form and produce crystals, drying it, and making it into a powder.
6. Because of the unique mode of production of spores, the formation and development of which are strictly genetically regulated, spores are ideal materials for studying morphogenesis and genetic control.
The development of the rapid spore preparation method has important theoretical significance for researching a spore formation mechanism and a spore stress resistance mechanism, and has important application value for developing various biological agents based on a spore form, such as BT pesticides, bactericides and the like, reducing experiment and production cost and guaranteeing the health of crops and human bodies.
The spore preparation method reported at present has the problems of long time, generally using DSM culture medium to culture thalli, transferring amount of 1:10 (v: v), needing treatment at 37 ℃ for 48 h, needing 90-100 h from strain activation to preparation of completely mature spores, often needing complicated and tedious operations, such as cold water starvation overnight treatment at 4 ℃, repeating for 2-3 times, needing differential centrifugation and the like, and increasing time and economic cost in the production process of experiments or enterprises.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide a spore production medium and a method for preparing bacterial spores, aiming at the defects of the prior art. The method well solves the problems of long spore production time, complicated operation steps and incapability of ensuring spore quality of the existing method by exploring a culture medium formula and a bacterial culture condition, improves the production benefit, further reduces the production cost of experiments and enterprises, and promotes scientific and economic development. The method for preparing the spores has the advantages of short time of about 54 hours, high yield of the spores of about 95 percent, purity of the spores of not less than 98 percent, microscopic observation and spore germination rate test, and no influence on spore forms and heat resistance.
In order to realize the technical effects, the invention adopts the technical scheme that:
a spore-forming medium for use in the preparation of bacterial spores, said spore-forming medium consisting of: 0.3-0.5 g of yeast powder, 0.3-0.5 g of peptone, 0.8-1 g of glucose and K added into per 1000 mL of distilled water2HPO4 5-7 g，KH2PO44-5 g，MgSO4·7H2O 0.01-0.1 g，NaCl 0.05-0.2 g，CaCl2 0.01-0.05 g，FeSO40.0005-0.001 g, and adjusting pH to 7.0-7.5.
Preferably, the spore-forming medium consists of: 0.3g of yeast powder, 0.4g of peptone, 0.85g of glucose and K are added into each 1000 mL of distilled water2HPO4 5.6g，KH2PO4 4.5g，MgSO4·7H2O 0.08g，NaCl 0.15g，CaCl20.04g，FeSO40.0005g, and the pH was adjusted to 7.2.
A preparation method of bacterial spores by using the spore-forming culture medium comprises the following steps:
(1) and activating strains: streak-culturing the bacteria in an activation medium;
(2) and strain culture: selecting single colony, inoculating to seed culture medium, culturing to logarithmic phase to obtain seed bacterial liquid with thallus concentration of 2 × 107-6×108CFU/mL；
(3) And spore generation: inoculating the seed bacterial liquid to the spore production culture medium for continuous culture;
(4) cracking the nutrient body: adding triton x-100 to the spore-forming culture medium, and lysing trophozites in which no spores are formed;
(5) collecting and storing spores.
Preferably, in the step (1), the activation medium consists of: adding yeast powder 4-5 g, peptone 8-10 g, NaCl 8-10 g, agar powder 18-20 g, pH7.0-7.5 into per 1000 mL of distilled water.
Preferably, in the step (1), the activation medium consists of: 5g of yeast powder, 8.5g of peptone, 9g of NaCl and 18.5g of agar powder are added into every 1000 mL of distilled water, and the pH value is 7.5.
Preferably, in the step (1), the culture temperature is 28-30 ℃ and the culture time is 12-16 h.
Preferably, the seed culture medium in step (2) consists of: every 1000 mL of distilled water is added with 4-5 g of yeast powder, 8-10 g of peptone and 8-10 g of NaCl, and the pH value is 7.0-7.5.
Preferably, the culture conditions in the step (2) are 28-30 ℃, 150-200 rpm and the culture time is 10-12 h.
Preferably, the culture conditions in step (3) are 35-37 ℃, 150-200 rpm and the culture time is 20-24 h.
Preferably, the nutrient cracking conditions in the step (4) are 37 ℃, 150-200 rpm and the cracking time is 40-60 min.
LB medium culture control in the prior art: purification of spheres on step gradients of regrografin, inMolecular Biological Methods for Bacillus，pp.391-450，1990
In the prior art, spore preparation steps are complicated and long in time, and comprise 16 h of strain activation, 3 h of strain amplification culture, 20 h of cold treatment and 48 h of heat treatment, wherein the total spore preparation time is about 90 h. The specific time and operation steps are as follows: (1) activating the strain, taking out glycerol from a refrigerator at-80 ℃ for strain preservation, streaking on an LB culture medium, and culturing at 37 ℃ for 16 h. (2) Single colonies were picked and inoculated in 25 mL DSM media at 150 rpm and 37 ℃ for 2-3 h to OD values between 0.45-0.60. (3) The culture was inoculated into 250 mL DSM medium at a ratio of 1:10 and shaken at 150 rpm at 37 ℃ for 48 hours. (4) Centrifugation at 10000Xg for 10 min and discarding the supernatant suspension. (5) Suspending in sterile water at 4 deg.C, centrifuging at 10000Xg for 10 min, repeating for 1 time, and standing overnight in a 4 deg.C refrigerator. (6) Repeating the 4 th step and the 5 th step. (7) Adding 50% Renografin reagent, centrifuging at 10000Xg for 30 min, adding 4 deg.C sterile water 7000Xg, and centrifuging for 10 min. (8) Repeat step 7 3 times. (9) Suspending in sterile water at 4 deg.C for storage.
Compared with the prior art, the invention has the following advantages:
nutritional stress is an external environmental signal for the bacteria to produce spores, which are generally produced by the bacteria under conditions of insufficient nutrition, such as exhaustion of nutrients during the later stages of their growth or severe lack of nutrients in the growing environment, producing large numbers of spores to bridge adverse environmental conditions. The spore-forming culture medium contains essential nutrient elements necessary for the growth of bacteria, but the carbon and nitrogen sources are seriously insufficient and are only about one tenth of the content of the normal culture medium of the bacteria such as LB, and the bacteria can not obtain sufficient nutrition when growing in the culture medium, so that the environmental condition induces the expression of genes related to the spore formation of the bacteria, and promotes the bacteria to generate a large amount of spores. triton x-100 is a non-ionic surfactant, which can dissolve lipid to increase cell membrane permeability, facilitate bacterial and in vivo blastocyst lysis, and greatly shorten spore release process. According to the principle, the time-consuming and high-economic-cost processes such as cold treatment and heat treatment are avoided, the spore is prepared in a simple step, the strain is activated to obtain seed bacterial liquid, the strain is inoculated into a spore production culture medium for continuous culture for 20-24 hours, and then triton x-100 is added to crack nutrients which do not form spores.
The spore preparation time is shorter, the strain is activated for 12-16 h, the strain is cultured for 10-12 h, the spore is generated for 20-24 h, the nutrient is cracked for 40-60 min, the spore is collected for 8-10 min, and the total spore preparation time is 43-53 h.
Drawings
FIG. 1 is a microscopic observation result of spore morphology;
FIG. 2 shows the results of heat resistance tests of spores produced by different strains.
Detailed Description
Example 1
Bacillus cereusBacillus cereus(ATCC 14579) was streaked on an activation medium at 30 ℃ for 12 hours. The activation medium consists of: yeast powder 5g10 g of peptone, 10 g of NaCl, 20 g of agar powder and 1000 mL of distilled water, and the pH value is 7.2.
Selecting single colony, inoculating to seed culture medium, culturing at 30 deg.C and 150 rpm for 10 hr to stationary phase to obtain seed bacterial liquid with thallus concentration of 5 × 107CFU/mL. The seed culture medium comprises the following components: 5g of yeast powder, 10 g of peptone, 10 g of NaCl, 1000 mL of distilled water and pH 7.2.
Inoculating the seed bacterial liquid into a spore production culture medium according to the proportion of 1:50 (v/v) for continuous culture to obtain a large amount of spores. The culture conditions are 35 ℃, 200 rpm and 24 h, and the spore production culture medium comprises the following components: 0.5 g of yeast powder, 0.5 g of peptone, 1 g of glucose and K2HPO4 5 g，KH2PO4 5 g，MgSO4·7H2O 0.01 g，NaCl 0.05 g，CaCl2 0.01 g，FeSO40.001 g, 1000 mL of distilled water, pH 7.2.
Triton x-100 was added to a final concentration of 0.02% (v: v) to lyse the nutrients in which no spores were formed. The cracking condition of the nutrient body is 37 ℃, 200 rpm, and the cracking time is 40 min.
Collecting spores, drying and storing. The collection condition is to centrifuge the spore-forming culture solution after nutrient body cracking at 7000 rpm for 10 min and discard the supernatant. The preservation condition is preservation at 4 ℃.
Spore morphology observation and yield and purity calculation: sampling for spore staining when 0.02% triton x-100 lysis nutrient is added for 25 min, making smear, drying and fixing, staining with 5% malachite green water solution for 5 min, re-staining with 0.5% safranin solution for 2 min, flaking, drying, observing with oil lens, wherein the spore is green, the bacterium is red, and the spore is not released and contains green spore in red bacilliform bacteria. In the figure, the granular form is green spores, the rod form is a bacterial body, and spore staining and microscopic observation were performed using LB-cultured cells of the prior art as a control. As can be seen, FIGS. 1A-B show that the spore morphology formed by this method treatment was indistinguishable from that of conventional LB medium. Shows that the method does not influence the bacillus cereusBacillus cereus(ATCC 14579) basic morphology of spores.
After the spore production culture medium is cultured for 24 hours, a sample is taken for spore staining, 10 visual fields are taken for oil-mirror observation, and the counting and the average value are respectively calculated. 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 formed but not completely released.
After 0.02% triton x-100 lysis nutrient is added for 40 min, sampling is carried out for spore staining, 10 visual fields are taken for oil-mirror observation, and the counting and the average value are respectively calculated. Spore purity = N1/(N1+ N2+ N3). times.100%
Where N1 is the number of spores, N2 is the number of bacteria, and N3 is the number of spores that have formed but not completely released.
Spore heat resistance test: adding 0.02% triton x-100 to lyse the nutrient for 40 min, centrifuging, collecting spores, resuspending with sterile water and diluting to 10-6Dividing into two equal parts after doubling, and counting one part directly coated on a flat plate as A; another 80 ℃ water bath is carried out for 10 min, and then the plate is coated to count as B; spore germination rate = (B/a) × 100%. The germination rate of spores obtained by LB culture in the prior art is used as a control, and each group adopts three parallel samples. FIG. 2 shows that the germination rate of the spores obtained by the method is not significantly different from that of the control, which indicates that the method does not influence the Bacillus cereusBacillus cereus(ATCC 14579) heat resistance of spores.
In the embodiment, by exploring the formula of the culture medium and the culture conditions, the problems of long time and complicated process of spores prepared by a conventional method are effectively solved, the production cost of experiments and enterprises is reduced, the total time of spores of bacillus cereus prepared by the method is 47 h, the yield of the spores is 98%, the purity of the spores is 92%, and microscopic examination and heat resistance experiments show that the shapes and the heat resistance of the prepared spores are unchanged.
Example 2
Bacillus subtilisB.subtilisBGSC168 was streaked onto activated medium at 28 ℃ for 16 h. The activation medium consists of: 4g of yeast powder, 8 g of peptone, 8 g of NaCl, 18 g of agar powder and 1000 mL of distilled water, and the pH value is 7.0.
Selecting single colony, inoculating to seed culture medium, culturing at 28 deg.C and 180 rpm for 12 hr toObtaining seed bacterial liquid in stationary phase, the bacterial concentration is 5X 108CFU/mL. The seed culture medium comprises the following components: 4g of yeast powder, 8 g of peptone, 8 g of NaCl, 1000 mL of distilled water and pH 7.0.
Inoculating the seed bacterial liquid into a spore production culture medium according to the proportion of 1:50 (v/v) for continuous culture, so that a large amount of spores are produced by the thalli. The culture conditions are 37 ℃, 150 rpm and 20 h, and the spore production culture medium comprises the following components: 0.4g of yeast powder, 0.3g of peptone, 0.8 g of glucose and K2HPO4 6 g，KH2PO4 4 g，MgSO4·7H2O 0.05 g，NaCl 0.1 g，CaCl20.03 g，FeSO40.0008 g, 1000 mL of distilled water, pH 7.0.
Triton x-100 was added to a final concentration of 0.02% (v/v) to lyse nutrients in which no spores were formed. The cracking condition of the nutrient body is 37 ℃, 200 rpm, and the cracking time is 50 min.
Collecting and storing spores. The collection condition is that the spore-forming culture solution after nutrient body cracking is centrifuged for 8 min at 8000 rpm, and the supernatant is discarded. The preservation condition is preservation at 4 ℃.
Spore morphology observation and yield and purity calculation: sampling and carrying out spore staining when 0.02% triton x-100 is added to lyse the nutrient for 30 min, preparing a smear, drying and fixing, firstly staining for 5 min by using a 5% peacock green aqueous solution, then re-staining for 2 min by using a 0.5% safranin solution, flaking and drying, and observing by using an oil mirror, wherein spores are green, bacteria are red, and the undelivered spores are formed and contain green spores in red bacilli. Spore staining and microscopic observation were performed using LB-cultured cells as a control in the prior art. The particles are spores and the rods are bacterial bodies, and FIGS. 1C-D show that the spore morphology formed by the method treatment is not different from that of the prior art control LB culture medium in the conventional culture. Shows that the method does not influence the bacillus subtilisB.subtilisBasic morphology of BGSC168 spores.
After the spore-forming culture medium is cultured for 20 h, a sample is taken for spore staining, 10 visual fields are respectively counted and the average value is calculated by oil-scope 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 formed but not completely released.
After 0.02% triton x-100 lysis nutrient is added for 50 min, sampling is carried out for spore staining, 10 visual fields are taken for oil-mirror observation, and the counting and the average value are respectively calculated. Spore purity = N1/(N1+ N2+ N3). times.100%
Where N1 is the number of spores, N2 is the number of bacteria, and N3 is the number of spores that have formed but not completely released.
Testing spore heat resistance: adding 0.02% triton x-100 to lyse the nutrient for 40 min, centrifuging, collecting spores, resuspending with sterile water and diluting to 10-6Dividing into two equal parts after doubling, and counting one part directly coated on a flat plate as A; another 80 ℃ water bath is carried out for 10 min, and then the plate is coated to count as B; spore germination rate = (B/a) × 100%. With LB cultures or resulting spore germination rates as controls, three replicates were used for each group. FIG. 2 shows that the spore germination rate obtained by the method has no significant difference from the control, which indicates that the method does not influence the Bacillus subtilisB.subtilisThermotolerance of BGSC168 spores.
In the embodiment, by exploring a culture medium formula and culture conditions, the problems of long time, complicated process and low spore purity of spores prepared by a conventional method are effectively solved, the production cost of experiments and enterprises is reduced, the total time of bacillus subtilis spores prepared by the method is 49 hours, the spore yield is 93 percent, the spore purity is 100 percent, and microscopic examination and heat resistance experiments show that the prepared spores are unchanged in shape and heat resistance.
Example 3
Bacillus cereusB. cereus0-9 (CCTCC No. M209041) is streaked on an activation medium, the culture temperature is 29 ℃, and the culture time is 14 h. The activation medium consists of: 4.5 g of yeast powder, 9g of peptone, 9g of NaCl, 19 g of agar powder and 1000 mL of distilled water, and the pH value is 7.5.
Selecting single colony, inoculating to seed culture medium, culturing at 29 deg.C and 200 rpm for 11 hr to stationary phase to obtain seed bacterial liquid with thallus concentration of 5 × 107CFU/mL. The seed culture medium comprises the following components: 4.5 g of yeast powder, 9g of peptone, 9g of NaCl, 1000 mL of distilled water and pH 7.5.
Inoculating the seed bacterial liquid into a spore production culture medium according to the proportion of 1:50 (v/v) for continuous culture to obtain a large amount of spores. The culture conditions are 36 ℃, 180 rpm and 22 h, and the spore production culture medium comprises the following components: 0.3g of yeast powder, 0.4g of peptone, 0.9 g of glucose and K2HPO4 7 g，KH2PO4 4.5 g，MgSO4·7H2O 0.1 g，NaCl 0.15 g，CaCl2 0.05 g，FeSO40.0005g, 1000 mL of distilled water, pH 7.5.
Triton x-100 was added to a final concentration of 0.02% (v/v) to lyse the nutrients in which no spores were formed. The cracking condition of the nutrient body is 37 ℃, 200 rpm, and the cracking time is 45 min.
Collecting and storing spores. The collection condition is to centrifuge the spore-forming culture solution after nutrient body cracking at 7000 rpm for 10 min and discard the supernatant. The preservation condition is preservation at 4 ℃.
Spore morphology observation and yield and purity calculation: sampling for spore staining when 0.02% triton x-100 lysis nutrient is added for 20 min, making smear, drying and fixing, staining with 5% malachite green water solution for 5 min, re-staining with 0.5% safranin solution for 2 min, flaking, drying, observing with oil lens, wherein the spore is green, the bacterium is red, and the spore is not released and contains green spore in red bacilliform bacteria. Spore staining and microscopic observation were performed using the prior art LB-cultured cells as a control. The particles are spores and the rods are bacterial bodies, and FIGS. 1E-F show that the treatment of this method does not differ from the spore morphology formed by conventional cultivation in LB medium. The method does not influence the basic morphology of the spores of the bacillus cereus from 0 to 9.
After the spore-forming culture medium is cultured for 22 h, a sample is taken for spore staining, 10 visual fields are respectively counted and the average value is calculated by oil-scope 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 formed but not completely released.
After 0.02% triton x-100 lysis nutrient is added for 50 min, sampling is carried out for spore staining, 10 visual fields are taken for oil-mirror observation, and the counting and the average value are respectively calculated. Spore purity = N1/(N1+ N2+ N3). times.100%
Where N1 is the number of spores, N2 is the number of bacteria, and N3 is the number of spores that have formed but not completely released.
Testing spore heat resistance: adding 0.02% triton x-100 to lyse the nutrient for 40 min, centrifuging, collecting spores, resuspending with sterile water and diluting to 10-6Dividing into two equal parts after doubling, and counting one part directly coated on a flat plate as A; another 80 ℃ water bath is carried out for 10 min, and then the plate is coated to count as B; spore germination rate = (B/a) × 100%. With LB cultures or spore germination rates obtained as controls, three parallel samples were taken for each group. FIG. 2 shows that the germination rate of the spores obtained by the method is not significantly different from that of the control, which indicates that the method does not influence the Bacillus cereusB. cereus0-9 spore heat resistance.
In the embodiment, by exploring the formula of the culture medium and the culture conditions, the problems of long time and complicated process of spores prepared by a conventional method are effectively solved, the production cost of experiments and enterprises is reduced, the total time of the spores of the bacillus cereus prepared by the method is 48 hours, the yield of the spores is 95%, the purity of the spores is 99%, and microscopic examination and heat resistance experiments show that the shapes and the heat resistance of the prepared spores are unchanged.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, changes and equivalents of the above embodiments according to the technical spirit of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims (10)

1. A spore-forming culture medium for preparing bacterial spores is characterized by comprising the following components: 0.3-0.5 g of yeast powder, 0.3-0.5 g of peptone, 0.8-1 g of glucose and K added into per 1000 mL of distilled water2HPO4 5-7 g，KH2PO4 4-5 g，MgSO4·7H2O 0.01-0.1 g，NaCl 0.05-0.2 g，CaCl2 0.01-0.05 g，FeSO40.0005-0.001 g, and adjusting pH to 7.0-7.5.
2. As in claimThe spore-forming culture medium used in the preparation of bacterial spores of claim 1, wherein the spore-forming culture medium comprises: 0.3g of yeast powder, 0.4g of peptone, 0.85g of glucose and K are added into each 1000 mL of distilled water2HPO4 5.6g，KH2PO4 4.5g，MgSO4·7H2O 0.08g，NaCl 0.15g，CaCl2 0.04g，FeSO40.0005g, and the pH was adjusted to 7.2.
3. A method for preparing bacterial spores using the spore-forming medium of claim 1, comprising the steps of:
(1) and activating strains: streak culturing the bacteria in an activation medium; the bacteria are Bacillus cereus B.cereus 0-9 with the preservation number of CCTCC No. M209041 or Bacillus cereus with the preservation number of ATCC 14579;
(2) and strain culture: selecting single colony, inoculating to seed culture medium, culturing to logarithmic phase to obtain seed bacterial liquid with thallus concentration of 2 × 107-6×108CFU/mL；
(3) And spore generation: inoculating the seed bacterial liquid to the spore production culture medium for continuous culture;
(4) cracking the nutrient body: adding triton x-100 to the spore-forming culture medium, and lysing trophozites in which no spores are formed;
(5) collecting and storing spores.
4. The method of preparing bacterial spores as defined in claim 3, wherein in step (1), the activation medium consists of: adding yeast powder 4-5 g, peptone 8-10 g, NaCl 8-10 g, agar powder 18-20 g, pH7.0-7.5 into per 1000 mL of distilled water.
5. The method of preparing bacterial spores as defined in claim 3, wherein in step (1), the activation medium consists of: every 1000 mL of distilled water is added with 5g of yeast powder, 8.5g of peptone, 9g of NaCl, 18.5g of agar powder and pH 7.5.
6. The method for producing bacterial spores as claimed in claim 3, wherein in the step (1), the culture temperature is 28 to 30 ℃ and the culture time is 12 to 16 hours.
7. The method of preparing bacterial spores of claim 3, wherein the seed medium in step (2) consists of: every 1000 mL of distilled water is added with 4-5 g of yeast powder, 8-10 g of peptone and 8-10 g of NaCl, and the pH value is 7.0-7.5.
8. The method for preparing bacterial spores as claimed in claim 3, wherein the culture conditions in step (2) are 28-30 ℃, 150-200 rpm, and the culture time is 10-12 h.
9. The method of preparing bacterial spores as defined in claim 3, wherein in step (3), the bacterial spores are prepared by the method of preparing bacterial spores
The culture conditions are 35-37 ℃, 150-.
10. The method for preparing bacterial spores as claimed in claim 3, wherein the nutrient lysis conditions in step (4) are 37 ℃, 150 ℃ and 200 rpm, and the lysis time is 40-60 min.
CN202010392101.3A 2020-05-11 2020-05-11 Spore production culture medium and preparation method of bacterial spores Active CN111349592B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010392101.3A CN111349592B (en) 2020-05-11 2020-05-11 Spore production culture medium and preparation method of bacterial spores

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010392101.3A CN111349592B (en) 2020-05-11 2020-05-11 Spore production culture medium and preparation method of bacterial spores

Publications (2)

Publication Number Publication Date
CN111349592A CN111349592A (en) 2020-06-30
CN111349592B true CN111349592B (en) 2022-06-03

Family

ID=71193559

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010392101.3A Active CN111349592B (en) 2020-05-11 2020-05-11 Spore production culture medium and preparation method of bacterial spores

Country Status (1)

Country Link
CN (1) CN111349592B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113462573A (en) * 2021-07-12 2021-10-01 河北科技大学 Preservation method of agricultural bacillus liquid microbial inoculum
CN114410536B (en) * 2022-01-28 2023-12-26 北京四良科技有限公司 Method for releasing intracellular enzyme by bacterial culture

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1471789B1 (en) * 2002-02-05 2009-04-15 Council of Scientific and Industrial Research Novel composition for early and profuse sporulation in fungi and a method thereof
CN101812412B (en) * 2009-05-12 2012-11-14 河南大学 Bacillus cereus and preparation method and application thereof
WO2012022032A1 (en) * 2010-08-17 2012-02-23 中国水产科学研究院黄海水产研究所 Endogenous antiviral complex-probiotic-preparation for prawn
CN102154181B (en) * 2011-02-18 2012-07-04 厦门大学 Culture medium for vibrio BS02 and preparation method thereof
CN103232948B (en) * 2013-05-10 2015-07-15 天津科技大学 High-temperature resistant saccharomyces cerevisiae strain and breeding method thereof
CN104560766B (en) * 2013-10-16 2017-07-28 浙江海正药业股份有限公司 A kind of Actinoplanes bacteria strain and its application
CN105462870B (en) * 2014-08-22 2020-09-11 北京大北农科技集团股份有限公司 Culture medium for fast spore production of spore bacteria
CN104928345A (en) * 2015-06-29 2015-09-23 苏州东辰林达检测技术有限公司 Detecting method of bacillus cereus in food
CN109722392B (en) * 2017-10-30 2020-01-03 中粮生物科技股份有限公司 Culture medium capable of promoting bacillus subtilis to produce spores and application thereof
CN109468259B (en) * 2018-10-24 2022-04-05 亚太星原农牧科技海安有限公司 Culture medium for promoting spore generation
CN109880786B (en) * 2019-03-15 2020-12-18 河南科技大学 Method for promoting sporulation of bacillus coagulans
CN110438060A (en) * 2019-07-22 2019-11-12 南京巨鲨显示科技有限公司 A kind of preparation of Geobacillus stearothermophilus gemma, enrichment method

Also Published As

Publication number Publication date
CN111349592A (en) 2020-06-30

Similar Documents

Publication Publication Date Title
Lacroix et al. Fermentation technologies for the production of probiotics with high viability and functionality
CN111349592B (en) Spore production culture medium and preparation method of bacterial spores
Chu et al. Enhanced removal of nitrate in the maize rhizosphere by plant growth-promoting Bacillus megaterium NCT-2, and its colonization pattern in response to nitrate
CN108949618A (en) A kind of algae-lysing bacterium and its application
CN110747147B (en) Escherichia coli with improved itaconic acid stress resistance
CN113528395B (en) Myxococcus xanthus prey on tomato bacterial wilt and application thereof in biological prevention and control of tomato bacterial wilt
CN107674850B (en) Pseudomonadaceae fragrans Sneb811 for killing root-knot nematodes, metabolite and application
Hewlett et al. In vitro culture of Pasteuria penetrans
KR102201035B1 (en) Method for isolating and culturing microorganism difficult to culture
KR101489865B1 (en) Novel strain Burkholderia cepacia JBK9 having antifungial activity against phytopathogenic fungi, its culture, and composition containg them for controlling phytopathogenic fungi
CN115386510A (en) Bacillus cereus and application thereof in degradation of tea saponin in oil-tea camellia cake
CN111876350B (en) Sphingomyelina gracilis for high yield of carbohydrate and application thereof
Boyko et al. Estimation of productivity Bacillus thuringiensis on different media
Kamal et al. Potential allelopathic effects of sunflower (Helianthus annuus L.) on microorganisms
CN109593701A (en) A kind of acidproof recombinant lactic acid bacteria and its construction method
CN110616177A (en) Bacillus with high fermentation density and fermentation production method thereof
Abbasli MORPHO-CULTURAL AND PHYSIOLOGICAL CHARACTERISTICS OF BACTERIA ISOLATED FROM SOME THERMAL SPRINGS OF THE REPUBLIC OF AZERBAIJAN
EP4438716A1 (en) Improving microbial activity
CN112920962B (en) ARTP-NTG composite mutagenesis bacillus thuringiensis mutant strain and application thereof
CN114634889B (en) Bacillus licheniformis nmxc2-1, fermentation product, microbial inoculum and application thereof
RU2828723C1 (en) Method of isolating uncultured forms of streptococcus pneumoniae
CN114381380B (en) Metarhizium anisopliae rejuvenation method
CN110283756B (en) Preparation method and application of colloidal lysobacter fermentation broth
RU2108384C1 (en) Strain bacillus thuringiensis - a producer of endotoxin to coleopterus insects and a method of its culturing
Balasim et al. Studying some of the optimal conditionals for the best bacterial isolates from soil and its effects on their growth rates

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant