CN111471622A - Fermentation production method of bdellovibrio bacteriovorus by using rhodobacter sphaeroides XR12 as host bacteria - Google Patents
Fermentation production method of bdellovibrio bacteriovorus by using rhodobacter sphaeroides XR12 as host bacteria Download PDFInfo
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Abstract
The invention relates to a method for producing bdellovibrio bacteriovorus by fermenting rhodobacter sphaeroides XR12 as host bacteria, which can obviously improve the live bacteria content and the bacteriophagic activity of the bdellovibrio bacteriovorus on the basis of good biological safety.
Description
Technical Field
The invention relates to a bdellovibrio fermentation production method using rhodobacter sphaeroides XR12 as a host bacterium, belonging to the technical field of aquatic microorganisms.
Background
Bdellovibrio is a type of arc-shaped or rod-shaped bacterium that parasitizes other bacteria (and, of course, can survive without host) and can cause lysis. It is smaller than the normal bacteria, can pass through the bacterial filter, and has the function similar to the bacteriophage. It is not a virus, but is indeed a class of bacteria that "eats" bacteria.
Due to the unique phage characteristics of bdellovibrio, the bdellovibrio bacteriovorus has great potential application value and can be used for purifying water bodies and removing harmful bacteria in the aspects of industry, agriculture, medicine and the like.
In recent years, research and development of bdellovibrio bacteriovorus preparations have been paid attention, and liquid preparations, freeze-dried powders and microcapsules thereof have been developed and commercialized successively. No matter which preparation is the bdellovibrio bacteriovorus product, the content of the live fermentation bacteria of the bdellovibrio bacteriovorus is a decisive factor for the quality of the bdellovibrio bacteriovorus preparations in various preparation forms in the production and fermentation processes of the bdellovibrio bacteriovorus.
Since Bdellovibrio is a parasitic bacterium which grows and breeds by cracking host bacteria. Therefore, the excellent host bacteria have a crucial influence on the content of live bdellovibrio fermentation bacteria. At present, colibacillus and aeromonas hydrophila are selected as host bacteria for bdellovibrio fermentation, but the host bacteria belong to conditional pathogenic bacteria and have potential influence on environment, animals and even human health. Rhodopseudomonas is also selected as a host bacterium for fermentation of bdellovibrio bacteriovorus, but the living bacterium content and the bacteriophagic activity of the bdellovibrio bacteriovorus culture solution obtained by fermentation of the Rhodopseudomonas are not different from those of escherichia coli (Leonavosa, first-class, research on the bacteriophagic bacteriovorus culture of Rhodopseudomonas). The research also shows that bacillus subtilis, bacillus licheniformis, bacillus natto, enterococcus faecalis, enterococcus faecium, bifidobacterium bifidum, lactobacillus bulgaricus, pediococcus acidilactici, streptococcus lactis, lactobacillus acidophilus, lactobacillus casei, lactobacillus lactis, lactobacillus plantarum, pediococcus pentosaceus and the like can be used as host bacteria for fermentation of bdellovibrio, but the host bacteria belong to gram-positive bacteria, and the cell wall thickness of the host bacteria.
In view of the problems of potential pathogenicity, environmental pollution, low cracking activity and the like of the host bacteria for bdellovibrio fermentation in the prior art, the technical personnel in the field hope to screen out excellent host bacteria for bdellovibrio fermentation from nontoxic and harmless gram-negative bacteria, not only has good biological safety, but also can improve the content of fermentation live bacteria and the bacteriophagic activity of bdellovibrio.
Disclosure of Invention
In view of the above problems and/or other problems of the related art, the present invention provides a method for producing bdellovibrio bacteriovorus by fermentation, wherein the host bacterium used in the method for producing bdellovibrio bacteriovorus by fermentation is rhodobacter sphaeroides XR12, which has a preservation number of CCTCC NO: and M2017826.
The rhodobacter sphaeroides XR12 used in the above-described embodiment of the present invention belongs to rhodobacter sphaeroides (rhodobacter sphaeroides), which has been deposited in the chinese typical culture collection center, and the date of deposit is: 12 months and 20 days in 2017; the preservation number is: CCTCC NO: and M2017826.
For specific information about rhodobacter sphaeroides XR12, see Chinese patent application publication CN108384732A (title of invention: rhodobacter sphaeroides for reducing the toxicity of trichlorfon and application thereof; application date: 2018, 09.02/2018).
Preferably, the bdellovibrio bacteriovorus is Bdellovibrio bacteriovorus F16, BDF-H16 or BDH 21-02.
Preferably, the fermentation production method comprises a host bacterium culture process and a bdellovibrio production process, wherein the host bacterium culture process comprises inoculating host bacteria to a host bacterium culture medium, performing shake culture to obtain a host bacterium seed solution, and then inoculating the host bacterium seed solution to a nutrient broth, and performing shake culture to obtain a host bacterium suspension; the production process of the bdellovibrio bacteriovorus comprises the steps of inoculating the bdellovibrio bacteriovorus and the suspension of the host bacterium into nutrient broth together to carry out oscillation culture to obtain a bdellovibrio bacteriovorus seed solution, then inoculating the bdellovibrio bacteriovorus seed solution and the suspension of the host bacterium together into a bacteriovorous bdellovibrio fermentation medium, and carrying out fermentation culture in a fermentation tank to obtain a bdellovibrio bacteriovorus fermentation broth.
The invention also provides the application of rhodobacter sphaeroides XR12 (the preservation number of China center for type culture Collection is CCTCC NO: M2017826) in fermentation production of bdellovibrio bacteriovorus.
Preferably, the bdellovibrio bacteriovorus is Bdellovibrio bacteriovorus F16, BDF-H16 or BDH 21-02.
The invention provides a method for producing bdellovibrio bacteriovorus by fermenting rhodobacter sphaeroides XR12 as host bacteria, which can remarkably improve the live bacteria content and the bacteriophagic activity of bdellovibrio bacteriovorus on the basis of good biological safety.
Detailed Description
The present invention will be further described with reference to specific embodiments, but the present invention is not limited to these specific embodiments.
Materials, reagents and the like used in the following embodiments are commercially available unless otherwise specified. Where specific techniques or conditions are not indicated, according to the techniques or conditions described in the literature in the field or according to the product description.
Example 1
Fermentation production method of bdellovibrio bacteriovorus F16
Bdellovibrio bacteriovorus F16 is purchased from the national aquatic animal pathogen pool.
The method of example 1 comprises a process of culturing host bacteria and a process of producing Bdellovibrio bacteriovorus F16;
the culture process of the host bacteria comprises the following steps:
step a) activating the host bacteria, namely inoculating the host bacteria (rhodobacter sphaeroides XR12) preserved in glycerol at the temperature of 70 ℃ below zero into a triangular flask filled with a medium of L m at the temperature of 100 ℃, and then carrying out shaking culture on a shaking table at the temperature of 35 ℃ and 180r/min for about 24 hours until the viable bacteria concentration of the host bacteria reaches 1 × 108～1×109cfu/m L, this being the activated host bacterium obtained;
step b) preparing a host bacterium seed solution, namely inoculating the activated host bacterium obtained in the step a) into a triangular flask filled with a 400m L culture medium according to the inoculation amount of 0.5% under the aseptic condition, and then carrying out shaking culture on a shaking table at 35 ℃ and 180r/min for about 24 hours until the viable bacterium concentration of the host bacterium reaches 2.0 × 1010～2.0×1011cfu/m L, which is the obtained host strain seed liquid.
Wherein, the culture medium adopted in the step a) and the step b) has the concentration of 4.0 g/L sodium acetate, 2.0 g/L yeast extract, 2.0 g/L ammonium chloride, 3.0 g/L peptone, 1.25 g/L sodium chloride, 0.2g magnesium sulfate/L, 0.2 g/L potassium dihydrogen phosphate and 1.0 g/L sodium bicarbonate (chemical reagents of each component are purchased from Shanghai Aaoshan industries and developments Co., Ltd.), the pH value is 7.0-7.5, the components are fully mixed and then placed in a triangular flask, and the triangular flask is sterilized at 121 ℃ for 20min for standby.
Step c) preparation of bacterial suspension of host bacteria, which comprises, taking the seed liquid of the host bacteria obtained in the step b) under aseptic condition, inoculating 0.5% of the seed liquid into 400ml of ordinary nutrient broth culture medium with pH7.2, shaking and culturing for 18h at 30 ℃ and 200r/min, then centrifuging for 20min at 6000r/min by a high-speed centrifuge, washing 3 times by aseptic normal saline, and finally preparing 2.0 × 1010cfu/ml of a bacterial suspension of the host bacteria;
the production process of bdellovibrio bacteriovorus F16 comprises the following steps:
step 1) activation of bdellovibrio bacteriovorus F16, which is characterized in that bdellovibrio bacteriovorus F16 (purchased from a national aquatic animal pathogen library) preserved in glycerol at-70 ℃ is inoculated into a triangular flask filled with 400m L of bdellovibrio bacteriovorus fermentation medium with pH7.2, meanwhile, bacterial suspension of the host bacteria obtained in the step c) is added according to 0.1 percent of inoculation amount, and then shaking culture is carried out at 30 ℃ and 200r/min for 48h to prepare activated bdellovibrio bacteriovorus F16;
step 2) preparation of seed solution of Bdellovibrio bacteriovorus F16, which comprises inoculating the activated Bdellovibrio bacteriovorus F16 prepared in step 1) into a triangular flask containing 400m L pH7.2 Bdellovibrio bacteriovorus fermentation medium under aseptic condition, adding 0.1% inoculum size into the bacterial suspension of the host bacteria obtained in step c), and shake culturing at 30 deg.C and 200r/min for 48h to obtain seed solution of Bdellovibrio bacteriovorus F16, wherein the viable bacteria concentration is about 2.0 × 108pfu/mL；
Step 3): preparation of fermentation broth of Bdellovibrio bacteriovorus F16The method comprises inoculating the seed solution of Bdellovibrio bacteriovorus F16 obtained in step 2) into a fermenter containing 200L Bdellovibrio bacteriovorus fermentation medium under aseptic condition, adding the bacterial suspension of the host bacteria obtained in step c) at 0.1%, controlling the temperature of the fermenter at 30 deg.C, and controlling the fermentation temperature at 10Nm3Ventilating at a speed of/h (air filtered by a 0.22 micron filter membrane), culturing for 48h to obtain fermentation broth of Bdellovibrio bacteriovorus F16, wherein the viable bacteria concentration of the fermentation broth of Bdellovibrio bacteriovorus F16 is 7.2 × 109pfu/mL；
Wherein the bdellovibrio bacteriovorus fermentation culture medium is a powdery culture medium purchased from Shanghai's actual development Limited company, is prepared into a concentration of 2 g/L by using sterile water, is adjusted to have a pH value of 7.2, is prepared into a bdellovibrio bacteriovorus fermentation culture medium of 200L, is fully and uniformly mixed and then is placed in a fermentation tank, and is sterilized under high pressure at 121 ℃ for 20min for later use.
Example 2
Fermentation production method of bdellovibrio bacteriovorus BDF-H16
Bdellovibrio bacteriovorus BDF-H16 is purchased from the national aquatic animal pathogen bank.
The method of example 2 comprises a process of culturing host bacteria and a process of producing Bdellovibrio bacteriovorus BDF-H16;
the process of culturing the host bacteria is described in example 1 above, and will not be described in detail.
The production process of bdellovibrio bacteriovorus BDF-H16 comprises the following steps:
step 1) activation of Bdellovibrio bacteriovorus BDF-H16, which comprises inoculating Bdellovibrio bacteriovorus BDF-H16 preserved in glycerol at-70 ℃ in a triangular flask filled with a Bdellovibrio bacteriovorus fermentation medium with the pH of 7.2 and the inoculation quantity of 400m L, simultaneously adding the bacterial suspension of the host bacteria obtained in the step c) according to the inoculation quantity of 0.2 percent, and then carrying out shaking culture for 48 hours at 30 ℃ and 200r/min to prepare the activated Bdellovibrio bacteriovorus BDF-H16, wherein the concentration of viable bacteria is about 5 × 107pfu/mL；
Step 2): the preparation method of the bdellovibrio bacteriovorus BDF-H16 seed liquid comprises the following steps: inoculating the activated Bdellovibrio bacteriovorus BDF-H16 obtained in the step 1) into a container in an inoculation amount of 5 percent under aseptic conditionsAdding 0.2 percent of inoculation amount of the bacterial suspension of the host bacteria obtained in the step c) into a triangular flask containing 400m L of a Bdellovibrio bacteriovorus fermentation medium with the pH of 7.2, and performing shaking culture at 30 ℃ and 200r/min for 48 hours to prepare a seed solution of the Bdellovibrio bacteriovorus BDF-H16, wherein the concentration of viable bacteria is about 5.0 × 108pfu/mL；
Step 3) preparation of fermentation broth of Bdellovibrio bacteriovorus BDF-H16, which comprises inoculating the seed solution of Bdellovibrio bacteriovorus BDF-H16 obtained in step 2) into a fermenter filled with 200L Bdellovibrio bacteriovorus fermentation medium (same as example 1) at an inoculum size of 5%, simultaneously adding the bacterial suspension of the host bacteria obtained in step c) at an inoculum size of 0.2%, controlling the temperature of the fermenter at 30 deg.C, and performing fermentation at 10Nm3Ventilating at a speed of/H, culturing for 72H to obtain fermentation broth of Bdellovibrio bacteriovorus BDF-H16, and detecting that the viable bacteria concentration of the fermentation broth of Bdellovibrio bacteriovorus F16 is 8.6 × 109pfu/mL。
Effect data
Detecting the effects of two aspects, (1) detecting the bacteriophagic activity of bdellovibrio bacteriovorus obtained by fermentation production by taking rhodobacter sphaeroides XR12 as host bacteria; (2) detecting the content of viable bacteria in bdellovibrio bacteriovorus fermentation liquor obtained by fermentation production by using rhodobacter sphaeroides XR12 as host bacteria;
experimental groups: example 1 the fermentation broth of the obtained Bdellovibrio bacteriovorus F16 was produced;
control group A, using rhodobacter sphaeroides RED1 as host bacteria, and preparing bacterial suspension of rhodobacter sphaeroides RED1 with viable bacteria concentration of 2.0 × 1010cfu/ml; then producing a fermentation liquid of the bdellovibrio bacteriovorus F16 according to the conditions of the steps 1) to 3) in the example 1;
control groups 1-5, respectively using Escherichia coli DH5 α, Aeromonas hydrophila S1, Bacillus subtilis PNB1, Bacillus licheniformis PNB3 and enterococcus faecalis X3 as host bacteria;
wherein bacterial suspensions of escherichia coli DH5 α, aeromonas hydrophila S1, bacillus subtilis PNB1, bacillus licheniformis PNB3 and enterococcus faecalis X3 are respectively prepared, and the concentration of viable bacteria is 2.0 × 1010cfu/ml; further following the conditions of steps 1) to 3) in example 1The bacterial suspensions of these host bacteria are used to produce fermentation liquor of Bdellovibrio bacteriovorus F16.
For the test of the aspect (1), the culture solution of Bdellovibrio bacteriovorus F16 obtained from the experimental group, the control group A, and the control groups 1-5 was filtered through a 0.45 μm microfiltration membrane to obtain a filtrate, and the filtrate was subjected to a test using a tap water double-layer agar plate method to determine the concentration of 2.0 × 1010Bacteriophagic activity of vibrio cholerae QH cfu/ml, in particular: the plaque formation is observed after the tap water double-layer agar plates are respectively put into a biochemical incubator and incubated at the constant temperature of 33 ℃ for 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h, 108h and 120h, and the earliest time, the maximum diameter and the number of the plaque generation are used as evaluation criteria of the plaque activity.
See table 1 below for results.
TABLE 1
In Table 1, "ND" means "none".
Analyzing the data in Table 1, from the earliest time of plaque production, the number of plaques produced by fermentation of Bdellovibrio bacteriovorus F16 produced by fermentation of the host strain Bdellovibrio bacteriovorus XR12 was 12 hours earlier than that of the host strain Bdellovibrio bacteriovorus RED1 (conventional Bdellovibrio bacteriovorus strain), the number of plaques produced by fermentation of the host strain Bdellovibrio bacteriovorus XR12 was 12 hours earlier and 24 hours earlier than that of the host strain Bdellovibrio bacteriovorus S1 (conventional Bdellovibrio bacteriovorus host strain), the number of plaques produced by fermentation of the host strain Bdellovibrio bacteriovorus F16 was 2.5mm larger than that of the host strain Bdellovibrio bacteriovorus DH5 α and Bdellovibrio hydrophila 1 (conventional Bdellovibrio bacteriovorus host strain), the number of plaques produced by fermentation of the host strain Bdellovibrio bacteriovorus XR12 was 1.5 mm larger than that of the host strain Bdellovibrio bacteriovorus RED1, the number of the host strain Bdellovorus 5 α and the host strain Bdellovorus S1 were 1.0, 1.5 larger than that of the number of the host strain Bdellovorus bacteriovorus lysosome of the host strain XR 3638 produced by the host strain XR 3638, the host strain H3638, the host strain Escherichia coli strain produced by.
As can be seen from the data in Table 1, the bacteriophagic activity of Bdellovibrio bacteriovorus F16 produced by fermentation with rhodobacter sphaeroides XR12 as host bacteria is the strongest (the time, the maximum diameter and the number of generated plaques), and the bacteriophagic activity of the Bdellovibrio bacteriovorus produced by fermentation is not only obviously better than that of the conventional Bdellovibrio bacteriovorus host bacteria (Escherichia coli DH5 α and Aeromonas hydrophila S1) but also better than that of the conventional rhodobacter sphaeroides (rhodobacter sphaeroides RED 1).
For the detection of the aspect (2), the live bacteria content of bdellovibrio bacteriovorus F16 is determined by ten times dilution method in combination with tap water double-layer agar plate method with bdellovibrio bacteriovorus F16 culture solutions produced by the experimental group, the control group A and the control groups 1-5.
See table 2 below for results.
TABLE 2
As can be seen from the results in Table 2 above, the viable cell content of Bdellovibrio bacteriovorus F16 produced by fermentation using rhodobacter sphaeroides XR12 as host bacteria is highest, higher than that of conventional rhodobacter sphaeroides (rhodobacter sphaeroides RED1) and higher than that of other conventional vibrio bacteriovorus host bacteria.
Comprehensively, by adopting the method, the bdellovibrio bacteriovorus is produced by fermenting the rhodobacter sphaeroides XR12 serving as a host bacterium, and the live bacteria content and the bacteriophagic activity of the bdellovibrio bacteriovorus can be obviously improved.
It should be understood that although the present description refers to embodiments, not every embodiment contains only a single technical solution, and such description is for clarity only, and those skilled in the art should make the description as a whole, and the technical solutions in the embodiments can also be combined appropriately to form other embodiments understood by those skilled in the art.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.
Claims (5)
1. A fermentation production method of bdellovibrio bacteriovorus is characterized by comprising the following steps:
the host bacterium adopted by the fermentation production method of bdellovibrio bacteriovorus is rhodobacter sphaeroides XR12, and the preservation number of the host bacterium in China center for type culture collection is CCTCC NO: m2017826.
2. The fermentative production method according to claim 1, wherein:
the bdellovibrio bacteriovorus is Bdellovibrio bacteriovorus F16, BDF-H16 or BDH 21-02.
3. The fermentative production method according to claim 1, wherein:
the fermentation production method comprises the culture process of host bacteria and the production process of bdellovibrio bacteria,
inoculating the host bacteria into a host bacteria culture medium, performing shake culture to obtain a host bacteria seed solution, and then inoculating the host bacteria seed solution into nutrient broth, and performing shake culture to obtain a host bacteria suspension;
the production process of the bdellovibrio bacteriovorus comprises the steps of inoculating the bdellovibrio bacteriovorus and the suspension of the host bacterium into nutrient broth together to carry out oscillation culture to obtain a bdellovibrio bacteriovorus seed solution, then inoculating the bdellovibrio bacteriovorus seed solution and the suspension of the host bacterium together into a bacteriovorous bdellovibrio fermentation medium, and carrying out fermentation culture in a fermentation tank to obtain a bdellovibrio bacteriovorus fermentation broth.
4. Rhodobacter sphaeroides XR12, which has a preservation number of CCTCC NO: m2017826, in the fermentation production of Bdellovibrio.
5. The use of claim 4, wherein:
the bdellovibrio bacteriovorus is Bdellovibrio bacteriovorus F16, BDF-H16 or BDH 21-02.
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