CN111471622B - 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 fermentation by using 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, host bacteria for bdellovibrio fermentation are selected from escherichia coli and aeromonas hydrophila, but the host bacteria belong to conditional pathogenic bacteria and can potentially affect the environment, animals and even human health. Rhodopseudomonas is also selected as a host bacterium for fermentation of Bdellovibrio bacteriovorus, but the viable bacteria 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 (Liao Fu, junior, research on culturing the bacteriophagic Bdellovibrio bacteriovorus by the 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: m2017826.
The Rhodobacter sphaeroides XR12 adopted in the scheme of the present invention belongs to Rhodobacter sphaeroides (Rhodobacter sphaeroides), which has been preserved in the chinese type culture collection with the preservation date: 12 months and 20 days in 2017; the preservation number is: CCTCC NO: m2017826.
For specific information about rhodobacter sphaeroides XR12, see Chinese patent application publication CN108384732A (title: rhodobacter sphaeroides for reducing the toxicity of dipterex and application thereof; application date: 2018, 02/09).
Preferably, the bdellovibrio bacteriovorus is bdellovibrio bacteriovorus F16, BDF-H16 or BDH21-02.
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 in China center for type culture Collection is CCTCC NO: M2017826) in the fermentation production of bdellovibrio bacteriovorus.
Preferably, the bdellovibrio bacteriovorus is bdellovibrio bacteriovorus F16, BDF-H16 or BDH21-02.
The invention provides a method for producing bdellovibrio bacteriovorus by fermentation by using 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.
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 bank.
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): the activation of host bacteria is as follows: inoculating host bacteria (rhodobacter sphaeroides XR 12) preserved in glycerol at-70 deg.C into Erlenmeyer flask containing 100mL of culture medium under aseptic condition, and shake culturing at 35 deg.C and 180r/min for about 24 hr until viable bacteria concentration of host bacteria reaches 1 × 10 8 ～1×10 9 cfu/mL, this is the obtained activated host bacterium;
step b): the preparation of the host bacterium seed liquid comprises the following specific steps: inoculating the activated host bacteria obtained in the step a) into a triangular flask filled with 400mL of 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 bacteria concentration of the host bacteria reaches 2.0 x 10 10 ～2.0×10 11 cfu/mL, which is the obtained host strain seed liquid.
Wherein, the concentrations of the components of the culture medium adopted in the step a) and the step b) are 4.0g/L sodium acetate, 2.0g/L yeast extract, 2.0g/L ammonium chloride, 3.0g/L peptone, 1.25g/L sodium chloride, 0.2g/L magnesium sulfate, 0.2g/L potassium dihydrogen phosphate and 1.0g/L sodium bicarbonate (the chemical reagents of the components are purchased from Shanghai's industries and developments Co., ltd.), and the pH value is 7.0-7.5; mixing, placing in a triangular flask, and autoclaving at 121 deg.C for 20 min.
Step c): the preparation of the bacterial suspension of the host bacteria comprises the following steps: inoculating the host strain seed solution obtained in step b) into 400ml of common nutrient broth medium with pH of 7.2 at 0.5% under aseptic condition, shake culturing at 30 deg.C and 200r/min for 18h, centrifuging at 6000r/min for 20min with high speed centrifuge, washing with sterile normal saline for 3 times, and making into 2.0 × 10 10 cfu/ml of a bacterial suspension of the host bacteria;
the production process of the bdellovibrio bacteriovorus F16 comprises the following steps:
step 1): the activation of Bdellovibrio bacteriovorus F16 is as follows: under the aseptic condition, inoculating bdellovibrio bacteriovorus F16 (purchased from a national aquatic animal pathogen bank) preserved in glycerol at the temperature of-70 ℃ into an Erlenmeyer flask filled with 400mL bdellovibrio bacteriovorus fermentation medium with the pH value of 7.2, simultaneously adding the bacterial suspension of the host bacteria obtained in the step c) according to the inoculation amount of 0.1 percent, and then carrying out shaking culture on a shaker at the temperature of 30 ℃ and 200r/min for 48h to prepare activated bdellovibrio bacteriovorus F16;
step 2): the preparation method of the seed liquid of the bdellovibrio bacteriovorus F16 comprises the following steps: under the aseptic condition, inoculating the activated bdellovibrio bacteriovorus F16 prepared in the step 1) into a triangular flask filled with 400mL of bdellovibrio bacteriovorus fermentation medium with the pH value of 7.2 according to the inoculation amount of 2%, simultaneously adding the bacterial suspension of the host bacteria obtained in the step c) according to the inoculation amount of 0.1%, and then carrying out shake culture at 30 ℃ and 200r/min for 48h to prepare a seed solution of the bdellovibrio bacteriovorus F16, wherein the viable bacteria concentration is about 2.0 multiplied by 10 8 pfu/mL；
Step 3): the preparation method of the fermentation liquor of the Bdellovibrio bacteriovorus F16 comprises the following steps: under aseptic conditions, inoculating the seed solution of Bdellovibrio bacteriovorus F16 obtained in the step 2) into a fermentation tank filled with 200L Bdellovibrio bacteriovorus fermentation medium according to the inoculation amount of 5%, simultaneously adding the bacterial suspension of the host bacteria obtained in the step c) according to the inoculation amount of 0.1%, and then controlling the temperature of the fermentation tank at 30 ℃ at 10Nm 3 Ventilating at a speed of h (air filtered by 0.22 micron filter membrane), and culturing for 48hPreparing fermentation liquor of bdellovibrio bacteriovorus F16; the detection shows that the viable bacteria concentration of the fermentation liquor of the bdellovibrio bacteriovorus F16 is 7.2 multiplied by 10 9 pfu/mL；
Wherein the bdellovibrio bacteriovorus fermentation medium is a powdery culture medium purchased from Shanghai's actual development Limited company, is prepared into a concentration of 2g/L by using sterile water, is adjusted to have a pH value of 7.2, is prepared into 200L of bdellovibrio bacteriovorus fermentation medium, is placed in a fermentation tank after being fully and uniformly mixed, 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): the activation of bdellovibrio bacteriovorus BDF-H16 is as follows: under the aseptic condition, bdellovibrio bacteriovorus BDF-H16 preserved in glycerol at-70 ℃ is inoculated into a triangular flask filled with 400mL bdellovibrio bacteriovorus fermentation culture medium with pH7.2, meanwhile, the bacterial suspension of the host bacteria obtained in the step c) is added according to the inoculation quantity of 0.2 percent, and then the mixture is subjected to shaking culture for 48 hours at 30 ℃ and 200r/min in a shaking table to prepare the activated bdellovibrio bacteriovorus BDF-H16, wherein the concentration of viable bacteria is about 5 multiplied by 10 7 pfu/mL；
Step 2): the preparation method of the bdellovibrio bacteriovorus BDF-H16 seed liquid comprises the following steps: under aseptic conditions, inoculating the activated bdellovibrio bacteriovorus BDF-H16 prepared in the step 1) into a triangular flask filled with 400mL bdellovibrio bacteriovorus fermentation culture medium with pH7.2 according to the inoculation amount of 5%, simultaneously adding the bacterial suspension of the host bacteria obtained in the step c) according to the inoculation amount of 0.2%, and then carrying out shake culture at 30 ℃ and 200r/min for 48H in a shaking table to prepare a seed solution of bdellovibrio bacteriovorus BDF-H16, wherein the viable bacteria concentration is about 5.0 multiplied by 10 8 pfu/mL；
And step 3): fermentation liquor of bdellovibrio bacteriovorus BDF-H16The preparation method comprises the following steps: under aseptic conditions, inoculating the seed solution of Bdellovibrio bacteriovorus BDF-H16 obtained in the step 2) into a fermentation tank filled with 200L Bdellovibrio bacteriovorus fermentation medium (same as example 1) according to the inoculation amount of 5%, simultaneously adding the bacterial suspension of the host bacteria obtained in the step c) according to the inoculation amount of 0.2%, controlling the temperature of the fermentation tank at 30 ℃ and adding 10Nm 3 Ventilating at a speed of/H, and culturing for 72H to prepare fermentation liquor of bdellovibrio bacteriovorus BDF-H16; the detection shows that the viable bacteria concentration of the fermentation liquor of the bdellovibrio bacteriovorus F16 is 8.6 multiplied by 10 9 pfu/mL。
Effect data
Detecting the effects of two aspects, (1) detecting the bacteriophagic activity of bdellovibrio bacteriovorus obtained by fermentation production with 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 a host bacterium;
experimental groups: example 1 production of the fermentation broth of the obtained Bdellovibrio bacteriovorus F16;
control group a: rhodobacter sphaeroides RED1 is taken as a host bacterium; similarly, a suspension of rhodobacter sphaeroides RED1 was prepared, and the viable bacteria concentration was 2.0X 10 10 cfu/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 taking escherichia coli DH5 alpha, aeromonas hydrophila S1, bacillus subtilis PNB1, bacillus licheniformis PNB3 and enterococcus faecalis X3 as host bacteria;
wherein bacterial suspensions of Escherichia coli DH5 alpha, aeromonas hydrophila S1, bacillus subtilis PNB1, bacillus licheniformis PNB3 and enterococcus faecalis X3 are respectively prepared, and the viable bacteria concentration is 2.0 × 10 10 cfu/ml; then, the bacterial suspensions of these host bacteria were used to produce fermentation broth of Bdellovibrio bacteriovorus F16 according to the conditions of steps 1) to 3) of example 1.
For the detection of the aspect (1), the culture solution of Bdellovibrio bacteriovorus F16 produced in the above experimental group, control group A, and control groups 1-5 was filtered through a 0.45 μm microfiltration membrane to obtain a filtrate. The filtrate was tested by tap water double-layer agar plate method to obtain a concentration of 2.0X 10 10 cfu/ml ofBacteriophagic activity of Vibrio cholerae QH, 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 generation, bdellovibrio bacteriovorus F16 produced by fermentation with rhodobacter sphaeroides XR12 as host bacteria has the earliest plaque generation, 12 hours earlier than rhodobacter sphaeroides RED1 (conventional rhodobacter sphaeroides) and 12 hours earlier than Escherichia coli DH5 alpha and Aeromonas hydrophila S1 (conventional host bacteria of Bdellovibrio), respectively. In view of the maximum diameter of plaque production, the maximum diameter of plaque produced by Bdellovibrio bacteriovorus F16 produced by fermentation using rhodobacter sphaeroides XR12 as a host bacterium is 2.5mm, compared with the values of rhodobacter sphaeroides RED1, escherichia coli DH5 alpha and Aeromonas hydrophila S1 of 1.0, 1.5 and 1.0, respectively, which are much smaller than that of rhodobacter sphaeroides XR 12. From the number of plaques produced after 120h of culture, the number of plaques produced by Bdellovibrio bacteriovorus F16 fermented and produced by using rhodobacter sphaeroides XR12 as a host strain is 186pfu, while the numbers of rhodobacter sphaeroides RED1, escherichia coli DH5 alpha and Aeromonas hydrophila S1 are 32, 38 and 36pfu, respectively, which are much smaller than that of rhodobacter sphaeroides XR 12.
As can be seen from the data in Table 1, the Bdellovibrio bacteriovorus F16 produced by fermentation with rhodobacter sphaeroides XR12 as host bacteria has the strongest bacteriophagic activity (time for generating plaques, maximum diameter and number); the bacteriophagic activity of the bdellovibrio produced by fermentation is not only obviously superior to that of the conventional bdellovibrio host bacteria (escherichia coli DH5 alpha and aeromonas hydrophila S1) but also superior to that of the conventional rhodobacter sphaeroides (rhodobacter sphaeroides RED 1).
For the detection of the aspect (2), the live bacteria content of the bdellovibrio bacteriovorus F16 is determined by ten times dilution method combined with tap water double-layer agar plate method by using the bdellovibrio bacteriovorus F16 culture solution produced by the experimental group, the control group A and the control groups 1-5.
See table 2 below for results.
TABLE 2
From the results shown in Table 2 above, it can be seen that the viable bacteria content of Bdellovibrio bacteriovorus F16 produced by fermentation using rhodobacter sphaeroides XR12 as host bacteria is the highest, higher than that of conventional rhodobacter sphaeroides (rhodobacter sphaeroides RED 1) and higher than that of other conventional vibrio bacteriovorus host bacteria.
Comprehensively, by adopting the method, the bdellovibrio bacteriovorus is produced by fermenting with rhodobacter sphaeroides XR12 as host bacteria, and the live bacteria content and the phagocytic 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 BDH21-02.
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. The application of rhodobacter sphaeroides XR12 as a host bacterium in the fermentation production of bdellovibrio bacteriovorus is characterized in that: the preservation number of the rhodobacter sphaeroides XR12 in the China center for type culture Collection is CCTCC NO: m2017826.
5. The use of claim 4, wherein:
the bdellovibrio bacteriovorus is Bdellovibrio bacteriovorus F16, BDF-H16 or BDH21-02.
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