CN107937330B - Application of indole or indole and calcium magnesium ions in promoting formation of bdelloplast of marine bdellovibrio bacteriovorus - Google Patents
Application of indole or indole and calcium magnesium ions in promoting formation of bdelloplast of marine bdellovibrio bacteriovorus Download PDFInfo
- Publication number
- CN107937330B CN107937330B CN201711333687.0A CN201711333687A CN107937330B CN 107937330 B CN107937330 B CN 107937330B CN 201711333687 A CN201711333687 A CN 201711333687A CN 107937330 B CN107937330 B CN 107937330B
- Authority
- CN
- China
- Prior art keywords
- indole
- bdellovibrio
- bdelloplast
- magnesium ions
- bdellovibrio bacteriovorus
- 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
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
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
-
- 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
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses an application of indole or indole and calcium magnesium ions in promoting formation of marine bdellovibrio bacteriovorus, and belongs to the technical field of bdellovibrio bacteriovorus. The invention utilizes indole or indole and calcium magnesium ions to promote the formation of Bdellovibrio bacteriovorus (Bdellovibrio sp.) BDN-1 bdelloplast; the quality guarantee period of the bdellovibrio bacteriovorus microbial preparation is obviously prolonged; and can effectively keep the activity of the thallus in the bdellovibrio bacteriovorus microbial preparation for a long time. The high-density bdelloplast provided by the invention can inhibit vibrios which can cause aquaculture biological diseases, has wide cracking spectrum property, and has better cracking effect compared with Bdellovibrio (Bdellovibrio sp.) BDN-1 mixture (containing both the bdelloplast and the swimmer). The method for culturing the leech-promoting plastid is simple and feasible, is suitable for popularization and application, and is expected to make a contribution to disease control in aquaculture industry.
Description
Technical Field
The invention belongs to the technical field of bdellovibrio, and particularly relates to application of indole or indole and calcium magnesium ions in promoting formation of bdellovibrio bacteriovorus bdelloplast of ocean.
Background
Vibrio (vibrios) is one of the most common bacterial groups in marine environments, and is widely found in sea water, seabed sediments, and marine products such as fish, shrimp, shellfish, crab, sea cucumber, kelp, and the like. 35 species of Vibrio are described in Bergey's Manual of bacteriology, 8 th edition, a significant portion of which are important pathogenic bacteria of aquaculture organisms. In the water industry, diseases of cultured organisms caused by various vibrios such as vibrio parahaemolyticus, vibrio alginolyticus, vibrio anguillarum and the like are collectively called vibriosis (vibriosis), which causes huge economic loss to the water industry every year.
At present, aiming at diseases, antibiotics and chemical drugs are still commonly used in aquaculture, so that drug-resistant strains are generated and drug residues exceed standards, and food safety problems such as the drug residue events of the turbot are caused.
Bdellovibrio bacteriovorus is widely present in natural environments such as sea and the like, and also in intestinal tracts of fish, shrimp, cattle, horses, pigs, ducks, humans and the like, and is a member of their respective microbial communities, but has no ability to infect mammalian cells such as plants, fish, shrimp and humans. In addition, it is abundant in healthy human intestinal tracts, maintaining intestinal microecological balance; it is severely reduced or even eliminated in patients suffering from enteritis and the like. It has the beneficial effects of regulating and controlling various microbial groups of human intestinal tract and maintaining ecological balance. Therefore, bdellovibrio is regarded as important in mariculture because it can cleave pathogenic vibrio, and has become one of the beneficial bacteria in culture at present. Although being used more and more widely, the products on the market today have little or no active bdellovibrio bacteriovorus.
We found by studying Bdellovibrio that the life cycle of Bdellovibrio includes the swimmer phase (free swimming phase) with flagellate movement but without proliferation and the Bdelloplast phase (Bdelloplast phase) with growth and reproduction in host cells, and the Bdelloplast only needs 4 hours to complete a life cycle. In the swimmer stage, bdellovibrio consumes huge energy and oxygen to operate life so as to meet the host. If the host is not encountered for a period of time, the energy and/or oxygen is depleted and dies. This period generally does not exceed several hours. In other words, the swimmer bdellovibrio is extremely dead and extremely difficult to survive. In the bdelloplast stage, bdellovibrio bacteriovorus enters into host cytoplasm space, under the protection of host cell wall, flagella are removed, and host cell nutrition itself is decomposed to grow rod-shaped. Then, through multi-division, the swimming body is divided into a plurality of small sections, and flagella grow out of each small section, so that the first generation of the swimming body is formed.
Studies have shown that the growth (lengthening process) can be interrupted at any time during the vermiculite phase. After the environmental conditions are proper, the leech plastid is divided and differentiated into a plurality of swimming bodies. Thus, a leech with low oxygen consumption has a strong ability to withstand harsh environments compared to a fragile and highly oxygen consuming swimmer. And the leech body and the swimmer have the same acting force. Therefore, the key point for solving the problems of low survival rate of live bacteria and short shelf life of the current bdellovibrio products is to convert the swimmer into a bdelloplast in the high-concentration fermentation process and delay the development of the bdelloplast.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the application of indole or indole and calcium magnesium ions in promoting the formation of bdelloplasts of marine bdellovibrio bacteriovorus.
The method is characterized in that the formation of bdelloplast of the marine bdellovibrio bacteriovorus is promoted by adding indole or indole and calcium magnesium ions, and the concentration of the bdellovibrio bacteriovorus is improved. The method overcomes the defect of short shelf life of the microbial preparation of Arctobacterium in the experimental technology.
The invention also aims to provide a bdelloplast microbial preparation of the marine bdellovibrio bacteriovorus.
The purpose of the invention is realized by the following technical scheme:
the invention provides an application of indole or indole and calcium magnesium ions in promoting formation of bdelloplasts of marine bdellovibrio bacteriovorus, in particular an application of indole or indole and calcium magnesium ions in prolonging the shelf life of bdelloplasts of marine bdellovibrio bacteriovorus.
Preferably, the indole is used at the final concentration of 1-3 mM; more preferably 2 to 3mM, most preferably 3 mM.
Preferably, the molar ratio of the calcium ions to the magnesium ions is 2: 1.
preferably, the indole and the calcium magnesium ions are used to the final concentration of 1-3 mM, (4-40)/(2-20) mM respectively; more preferably 2-3 mM, (4-20)/(2-10) mM; most preferably 3mM, 20/10 mM.
A bdelloplast microbial preparation of marine bdellovibrio bacteriovorus is prepared by inoculating bdellovibrio bacteriovorus into a DNB liquid culture medium containing host bacteria, culturing at 25-35 ℃ and 150-300 rpm for 36-48 h, adding the host bacteria again, adding indole or indole and calcium magnesium ions, continuing culturing for 20-28 h, centrifuging the culture solution at 4 ℃ and 6000-8000 rpm for 10-15 min, removing supernatant containing bdellovibrio bacteriovorus swimmers, sucking and suspending precipitates with sterilized distilled water with a mass-to-volume ratio (g/m L) of 10-25 per mill (preferably 15 per mill) of salinity, and adjusting the concentration to 1012PFU/m L, namely the microbial preparation of bdelloplast of sea bdellovibrio bacteriovorus.
Preferably, the Bdellovibrio bacteriovorus is Bdellovibrio bacteriovorus (Bdellovibrio sp.) BDN-1.
The Bdellovibrio (Bdellovibrio sp.) BDN-1 is obtained from seawater of certain aquiculture in Fujian by artificial enrichment culture, separation and purification, and is a Bdellovibrio taking Bacillus subtilis as a parasitic host.
The preservation information of Bdellovibrio bacteriovorus (Bdellovibrio sp.) BDN-1: the preservation unit: guangdong province culture Collection (GDMCC), preservation date: 11/27/2017, deposit address: the microbial research institute of Guangzhou province, No. 59 building, No. 5 building, Guangdong province, of the Zhonglu-Jieli, Guangzhou city, the preservation number: GDMCC NO: 60291.
the culture temperature of bdellovibrio bacteriovorus BDN-1 is preferably 25-35 ℃ (more preferably 30 ℃), the pH is preferably 6.5-8.0 (more preferably 7.2), and the salinity is preferably 10-25 per thousand (more preferably 15 per thousand).
The culture medium for culturing the bdellovibrio bacteriovorus BDN-1 is preferably DNB culture medium;
the bdellovibrio bacteriovorus BDN-1 has the following morphological characteristics and physiological and biochemical characteristics:
a. the screened bdellovibrio bacteriovorus BDN-1 is negative in gram stain, oval, free of spores and terminated by a single flagellum;
b. the morphological characteristics of plaques are: after the bdellovibrio bacteriovorus BDN-1 is cultured for 3 days at 30 ℃ in a DNB double-layer solid medium plate, plaques are round, transparent, moist, neat in edge and 2-3 mm in diameter;
the mechanism of the invention is as follows:
according to the existing research, the invention discloses a method for prolonging the quality guarantee period of a bdellovibrio bacteriovorus bdelloplast reagent by adding various calcium-magnesium ions and indole combinations in the high-concentration fermentation process to promote the transformation of a bdelloplast and delay the development of the bdelloplast, and then concentrating the bdellovibrio bacteriovorus bdelloplast reagent and freeze-drying or directly preserving the bdellovibrio bacteriovorus bdelloplast reagent, so that the survival rate of the bdellovibrio bacteriovorus bdelloplast reagent is ensured.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention utilizes the combination of calcium and magnesium ions and indole to promote the formation of bdellovibrio bacteriovorus (Bdellovibrio sp.) BDN-1 bdelloplast; the quality guarantee period of the bdellovibrio bacteriovorus microbial preparation is obviously prolonged; and can effectively keep the activity of the thallus in the bdellovibrio bacteriovorus microbial preparation for a long time. The method for culturing the leech-promoting plastid is simple and feasible, and is suitable for popularization and use.
(2) The high-density bdelloplast provided by the invention can inhibit vibrios which can cause aquaculture biological diseases, has wide cracking spectrum property, and has better cracking effect compared with Bdellovibrio (Bdellovibrio sp.) BDN-1 mixture (containing both the bdelloplast and the swimmer);
(3) compared with a control group, the Bdellovibrio bacteriovorus BDN-1 high-density bdelloplast group provided by the invention has prolonged shelf life.
Drawings
FIG. 1 is a diagram showing the result of the plate separation of Bdellovibrio BDN-1 by a DNB double-layer solid medium.
FIG. 2 is a transmission electron microscope observation result picture of bdellovibrio bacteriovorus BDN-1.
FIG. 3 is a graph showing the results of bdellovibrio bacteriovorus BDN-1 bdelloplast density of groups of indoles of different concentrations in example 2.
FIG. 4 is a graph showing the results of bdellovibrio bacteriovorus BDN-1 bdelloplast density of the calcium ion groups of different concentrations in example 3.
FIG. 5 is a graph showing the results of bdellovibrio bacteriovorus BDN-1 bdelloplast density of the magnesium ion groups of different concentrations in example 4.
FIG. 6 is a graph showing the results of bdellovibrio bacteriovorus BDN-1 bdelloplast density of the combination groups of calcium and magnesium ions of different concentrations in example 5.
FIG. 7 is a comparison of shelf life effects of the bdelloplast preparation of the preferred indole group Bdellovibrio in example 7 reduced to 1% of the original concentration.
FIG. 8 is a comparison of the shelf life effect of the Bdellovibrio bacteriovorus preparation of the preferred indole and calcium magnesium ion group of example 7 reduced to 1% of the original concentration.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The media referred to in the examples:
formulation (g/L) of DNB broth, nutritious meat 0.8, acid hydrolyzed casein 0.5, yeast extract powder 0.1, sodium chloride 15, final pH 7.2;
DNB upper medium formula: adding 0.8% agar powder into sterile water with 15% salinity, and final pH7.2;
DNB lower layer medium formula: adding agar powder with the mass ratio of 1.5% into a DNB liquid culture medium, and finally, adjusting the pH to 7.2;
nutrient Broth (NB) liquid medium formula (g/L), peptone 10, beef extract powder 3, sodium chloride 5, final pH 7.2;
nutrient Broth (NB) solid medium formulation: adding agar powder with the mass ratio of 1.5% into a nutrient broth liquid culture medium, and finally adjusting the pH to 7.2;
formulation of indole mother liquor 0.1g of indole powder was weighed out and dissolved in 100m L of sterile distilled water.
The formula of the calcium chloride mother liquor is that 0.55g of calcium chloride powder is weighed and dissolved in 50m L of distilled water, and the mixture is sterilized;
magnesium sulfate mother liquor is prepared by weighing 12.32g magnesium sulfate heptahydrate powder, dissolving in 50m L distilled water, and sterilizing.
Bacillus subtilis GIM1.136 in this example was purchased from the institute of microorganisms, Guangdong province.
Among the indicator strains of the present example 26 strain, Vibrio alginolyticus (Vibrio alginolyticus)1, Vibrio alginolyticus (V.alginolyticus)2, Vibrio alginolyticus (V.algolyticus) 3, Vibrio alginolyticus (V.algolyticus) 4, Vibrio parahaemolyticus (V.parahaemolyticus)8, Vibrio parahaemolyticus (V.parahaemolyticus)9, Vibrio alginolyticus (V.algolyticus) 10, Vibrio alginolyticus (V.algolyticus) 11, Vibrio putrescentilis (Shewanella alginolyticus) 12, Serratia odorifera fimbriae (Serratia ficia) 15, Vibrio algicidal (V.algicidalus) 16, Pseudomonas aeruginosa (Pseudomonas aeruginosa)17, Vibrio alginolyticus (V.algicidal) 19, Vibrio aerolyticus 19, Pseudomonas aeruginosa (P.20, Pseudomonas aeruginosa (P.31, Pseudomonas aeruginosa) 20, Pseudomonas aeruginosa (P.31. faecalis, P.20, Pseudomonas aeruginosa, P35, and P35, or P35, and P35, or P35, and P35, or P35, or P35, or P, P;
providencia rettgeri (Providencia rettgeri)32 is provided by The university of southern China university college of light industry and food (The protective effect of bdellovibrio-and-like organisms (BA L O) on a delayed filing of acquired Salmonella enterica ssp. enteca servo Typhimurium [ J ]. L u F, Cai J. L ets in Applied Microbiology,2010,51: 625. 631.);
meanwhile, in the embodiment, 26 indicator bacteria are disclosed in the patent Z L201410752144.2, a lactic acid bacterium with broad-spectrum bacteriostatic activity and application thereof.
EXAMPLE 1 isolation and purification of Bdellovibrio BDN-1
Taking a sample from a seawater body area of certain aquaculture of Fujian, centrifuging a sample liquid 10m L at 20000rpm for 20min for enrichment, suspending with a sterilized DNB liquid culture medium, adding into a sterile-treated DNB liquid nutrient medium in a bottle of 50m L, culturing at 30 ℃ at 200r/min for 48h, centrifuging a bacterial liquid at 6000rpm for 10min, filtering a supernatant with a 0.8-micron cellulose acetate filter membrane, centrifuging a filtrate at 20000rpm for 20min, suspending a precipitate with sterile water, and diluting the suspension into 10-1、10-2、10-3、10-4Detecting the condition of spots by using each dilution gradient inverted DNB double-layer solid medium plate, culturing for 3-4 days at 30 ℃, selecting single plaques which are consistent in size, round, transparent and neat in edge from inoculating loops, continuously culturing in a 50m L DNB liquid medium, repeating inverted DNB double-layer solid medium plates until the plaques on the double-layer plates are basically consistent in size, round, transparent and neat in edge, preliminarily determining bdellovibrio bacteriovorus, and performing transmission electron microscope observation and strain identification;
identifying the purified bdellovibrio bacteriovorus, wherein the identification result is as follows:
a. the size of the screened bdellovibrio bacteriovorus is 0.82 × 0.43.43 μm, the bdellovibrio bacteriovorus is rod-shaped, the flagellum is generated at the end, and the flagellum length is about 2.1 μm;
b. the morphological characteristics of plaques are: after the DNB double-layer solid medium is cultured for 4 days at 30 ℃, the plaques are round, transparent, concave, moist in surface, neat in edge and 2-3 mm in diameter;
DNB double layer solid medium plate separation results are shown in FIG. 1.
The transmission electron microscope observation result chart is shown in FIG. 2.
Molecular biology identification results:
inoculating a bottle of bdellovibrio bacteriovorus bacterial liquid to a plaque-carrying shaking table for culturing, firstly centrifuging at 6000rpm for 10min, then centrifuging the supernatant at 20000rpm for 20min, enriching and precipitating by using TE buffer solution of 500 mu L, and sending the precipitate to a sequencing company (Shanghai Bioengineering Co., Ltd.) for sequencing, wherein primers used for PCR amplification are as follows:
63F：5'-CAGGCCTAACACATGCAAGTC-3'；
842R: 5'-CGWCACTGAAGGGGTCAA-3', wherein degenerate base W represents A/T;
the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 deg.C for 1 min; annealing at 56 deg.C for 45 s; extending for 1min at 72 ℃; 35 cycles; final extension at 72 deg.C for 10 min; the invention adopts a monoclonal identification method, two fragments are obtained by amplification of a forward primer 63F and a reverse primer 842R, and a target fragment of about 800bp is obtained by splicing two sequences of DNAStar.
The sequence of the 16S rDNA gene sequencing results were identified by NCBI B L AST homology alignment analysis in GenBank database, the RDP classificator search was classified as Bdellovibrio sp, and further NCBI B L AST analysis revealed that the Bdellovibrio sp and their closest relatives Bdellovibrio sp.BDH12 and Bdellovibrio bacterium BDHSH06 have 99% similarity, and thus strain BDN-1 was identified as Bdellovibrio sp.
In review, the Bdellovibrio bacteriovorus separated and purified by the invention is named Bdellovibrio bacteriovorus (Bdellovibrio sp.) BDN-1, and the preservation information thereof is as follows: the preservation unit: guangdong province culture Collection (GDMCC), preservation date: 11/27/2017, deposit address: the microbial research institute of Guangzhou province, No. 59 building, No. 5 building, Guangdong province, of the Zhonglu-Jieli, Guangzhou city, the preservation number: GDMCCNO: 60291.
the sequence of 16S rDNA of Bdellovibrio bacteriovorus (Bdellovibrio sp.) BDN-1 is shown in SEQ ID NO: 3 (758 bp).
Example 2 cultivation of indole on Bdellovibrio BDN-1 Bdelloplastid
(1) Culturing host, namely taking newly cultured host bacterium Bacillus subtilis (Bacillus subtilis GIM1.136), burning a bottle mouth under the outer flame of an alcohol lamp in a sterile operating platform, sucking 3m L host bacterium seed liquid, transferring the host bacterium seed liquid into a 250m L bottled NB liquid culture medium, culturing the inoculated host bacterium in a constant-temperature shaking table at about 31 ℃ for about 15 hours, putting the cultured host bacterium into a refrigerated centrifuge, centrifuging the cultured host bacterium for 10 minutes at 6000rpm and 4 ℃, removing supernatant, adding sterile distilled water with 1m L15 per mill of salinity into each tube, scattering and uniformly mixing the sterile distilled water, transferring the mixture into a centrifuge tube to obtain host bacterium concentrated solution, bagging the host bacterium concentrated solution, and putting the host bacterium concentrated solution into a refrigerator at 4 ℃ for storage;
(2) BDN-1 seed liquid culture, selecting several rounder plaques on the separated plate, inoculating into 50m L DNB liquid culture medium, adding 250 mu L of MSG (sodium glutamate) with the concentration of 1 mol/L, adding 1m L of host bacteria concentrated solution, and performing shake culture at 31 ℃ for the third day to obtain BDN-1 seed liquid.
(3) And (3) inoculating and culturing the indole group, namely inoculating 1m L BDN-1 seed liquid in the step (2) into four bottles of 50m L DNB liquid culture medium, adding 250 mu L MSG with the concentration of 1 mol/L, adding 1m L host bacterium concentrated solution, carrying out shake culture at 31 ℃ for 48h, supplementing 500 mu L host bacterium concentrated solution, supplementing 1m L host bacterium concentrated solution again after culturing for 72h, adding indole which is subjected to filtration sterilization by a 0.22 mu m filter membrane to ensure that the final concentrations are 1mM, 2mM and 3mM respectively, taking the rest group as a control group, adding no indole, and continuing culturing for 24h after inoculation, so as to facilitate the formation of leech plastid.
(4) Separating and inverting the two layers, centrifuging all culture solution at 6000rpm to precipitate Hirudo plasmid, discarding supernatant, suspending the precipitate with sterile distilled water with salinity of 1m L15 ‰ and then diluting to 10 μ L-1、10-2And 10-3Pouring the gradient vermiculite suspension into a DNB double-layer solid medium flat plate, and placing the flat plate in an incubator at 30 ℃ for static culture;
(5) and (4) observing results: observing the plates every 12h, observing the spot forming conditions of all the plates of bdellovibrio bacteriovorus, counting the number of plaques after the plaques are formed, and calculating the corresponding concentrations (see figure 3).
As a result, the indole concentration was increased by 73%, 83% and 87% in the 1mM, 2mM and 3mM groups, respectively, as compared with the control group. The bdellovibrio bacteriovorus has higher bdelloplast density with the increase of indole concentration. The indole has good culture promoting effect and obvious amplification, and the culture promoting factor for improving the bdelloplast density of the bdellovibrio bacteriovorus is preferably 3mM indole.
Example 3 cultivation promotion of calcium ions on Bdellovibrio BDN-1 Bdellovibrio plastid
(1) Inoculating and culturing a host: same as example 2, step (1);
(2) BDN-1 seed liquid culture: same as example 2, step (2);
(3) calcium ion group inoculation and culture: in the same manner as in step (3) of example 2, the indole variables were changed to calcium chloride at final concentrations of 4mM, 20mM and 40 mM;
(4) separation and reverse bilayer of hirulog: same as example 2, step (4);
(5) and (4) observing results: same as example 2, step (5) (see fig. 4).
As a result, calcium ion concentrations of 4mM, 20mM and 40mM were reduced by 91%, 93% and 99%, respectively, as compared with the control group. The bdellovibrio bacteriovorus has lower bdelloplast density with the increase of calcium ion concentration.
Example 4 cultivation promotion of magnesium ions on Bdellovibrio BDN-1 Bdellovibrio plastid
(1) Inoculating and culturing a host: same as example 2, step (1);
(2) BDN-1 seed liquid culture: same as example 2, step (2);
(3) magnesium ion group inoculation and culture: in the same manner as in step (3) of example 2, the indole concentration was changed to magnesium sulfate to final concentrations of 2mM, 10mM and 20 mM;
(4) separation and reverse bilayer of hirulog: same as example 2, step (4);
(5) and (4) observing results: same as example 2, step (5) (see fig. 5).
As a result, the magnesium ion concentration was reduced by 13%, 51% and 95% in the 2mM, 10mM and 20mM groups, respectively, as compared with the control group. The bdellovibrio bacteriovorus has lower bdelloplast density with the increase of magnesium ion concentration.
Example 5 cultivation promotion of calcium and magnesium ion combinations on Bdellovibrio BDN-1 Bdelloplastid
(1) Inoculating and culturing a host: same as example 2, step (1);
(2) BDN-1 seed liquid culture: same as example 2, step (2);
(3) and (3) inoculating and culturing the calcium-magnesium ion combined group: in the same manner as in step (3) of example 2, the indole variable was changed to calcium/magnesium combinations at final concentrations of 4/2mM, 20/10mM and 40/20 mM;
(4) separation and reverse bilayer of hirulog: same as example 2, step (4);
(5) and (4) observing results: same as example 2, step (5) (see fig. 6).
As a result, the calcium/magnesium ion concentration was increased by 89%, 92% and 54% in the 4/2mM, 20/10mM and 40/20mM, respectively, compared with the control group. Along with the increase of the calcium and magnesium ion combination concentration, the bdellovibrio bacteriovorus bdelloplast density is increased and then decreased.
The calcium and magnesium ions are combined to discuss the cracking capability and the quality guarantee period of high-density leech bodies.
Example 6 application of broad cleavage Spectrum Properties of Bdellovibrio BDN-1 Bdelloplastids
(1) Preparation of 26 indicator strains A single colony of 26 indicator strains (Table 1) was inoculated into a nutrient broth liquid medium, cultured at 37 ℃ at 200r/min for 8h, and the concentration was adjusted to 1 × 106CFU/m L, storing at 4 ℃ for later use;
TABLE 126 different Experimental indicator strains
(2) The preparation method of bdelloplast of bdellovibrio bacteriovorus comprises mixing bdellovibrio bacteriovorus BDN-1, host bacteria concentrated solution and DNB liquid culture medium in a volume ratio of 1:1:50, adding MSG with a concentration of 250 mu L of 1 mol/L (making the final concentration of MSG 5 mmol/L), culturing at constant temperature, adding host concentrated solution every 48h, culturing for 72h, adding culture promoting factors of indole, indole and calcium magnesium ions with optimal concentration, adding host bacteria concentrated solution again, culturing for 24h to obtain culture solution, centrifuging the culture solution at 6000rpm and 4 ℃ for 10min, discarding supernatant, collecting precipitate, sucking and suspending with sterile distilled water with a mass volume ratio of (g/m L) of 15% salinity, adjusting the concentration, and the final concentration of bdelloplast of bdellovibrio bacteriovorus is 5.7 × 1012PFU/mL、6.2×1012PFU/m L, 4 ℃ preservation this concentrated hirulogThe leech plastid is used for the subsequent accelerated test.
(3) (Bdellod) lysis experiment, namely, 500 mu L of each indicator bacterium and the Bdellod prepared in the step (1) are taken together to be mixed with the upper layer culture medium of 3m L DNB, the mixture is evenly shaken and is paved on the lower layer culture medium of the DNB, and after the upper layer culture medium of the DNB is coagulated, the mixture is kept still and cultured for 3 days at 30 ℃.
The bacterial liquid of the bdellovibrio mixture (containing both bdelloplast and swimmer) is used as a reference in the experiment, so as to test whether the cracking capacity of the bdelloplast relative to the mixture is weakened. The results of the bdelloplast lysis experiment are shown in Table 2, and 26 pathogenic bacteria of different sources are used as indicator bacteria in the experiment.
Control group: the cracking rate of Bdellovibrio bacteriovorus (Bdellovibrio sp.) BDN-1 mixture to 26 indicator strains reaches 73 percent. Among the 26 test strains, BDN-1 strain has a lysis rate of 75% for Vibrio alginolyticus (V.algolylyticus) and 67% for Vibrio parahaemolyticus (V.parahaemolyticus).
Indole group: compared with a mixture, the Bdellovibrio bacteriovorus (Bdellovibrio sp.) BDN-1 bdelloplast has improved cracking capability, the cracking rate of 26 indicator strains reaches 88 percent, and the cracking capability is not influenced. Among the 26 test strains, BDN-1 strain has a cracking rate of 100% for Vibrio alginolyticus (V.algolylyticus) and a cracking rate of 100% for Vibrio parahaemolyticus (V.parahaemolyticus).
Indole and calcium magnesium ion group: the Bdellovibrio bacteriovorus (Bdellovibrio sp.) BDN-1 bdelloplast and indole group have consistent cracking capability, and the cracking rate of 26 indicator strains reaches 88 percent. Among the 26 test strains, BDN-1 strain showed a cleavage rate of 100% for Vibrio alginolyticus (V.algolylyticus) and 100% for Vibrio parahaemolyticus (V.parahaemolyticus).
TABLE 2 lysis of 26 pathogenic indicator bacteria from different sources by Bdelloplast BDN-1
Note: + indicates that the bdellovibrio bacteriovorus bdelloplast can crack the corresponding strain
Example 7 shelf life Studies on preservation of Bdellovibrio BDN-1 Bdellovibrio plastids
(1) Preparing a bdellovibrio bdelloplast: see example 6, step (2); the control group was not supplemented with any proculture factors.
(2) Placing the prepared bdellovibrio bdelloplast at a gradient temperature for an acceleration test;
(3) sampling periodically, and detecting the concentrations of bdellovibrio bdelloplast of the control group and the group added with indole, indole and calcium magnesium ions;
(4) calculating the shelf life of the microbial preparation by using an Arrhenius equation and the data recorded in the step (3), and determining whether the shelf life is prolonged or not by comparing the shelf lives of the optimal culture promoting factors of indole, indole and calcium magnesium ions.
The gradient temperature in the step (2) is preferably 25 ℃, 37 ℃, 45 ℃, 55 ℃, 65 ℃, 75 ℃, 85 ℃ and 95 ℃;
the accelerated test in the step (2) is that the length of the accelerated time is decreased with the increase of the temperature;
the detection interval time of the periodic sampling described in the step (2) is preferably as shown in Table 3.
TABLE 3 sampling test intervals and sampling times at different temperatures
| Temperature of | Duration/sample interval time | Total number of samples |
| 25 |
0～72h/12h | 6 |
| 37 |
0～48h/8h | 6 |
| 45 |
0～24h/4h | 6 |
| 55 |
0～8h/40min | 12 |
| 65℃ | 0～2h/20min | 6 |
| 75 |
0～1h/10min | 6 |
| 85 |
0～30min/5min | 6 |
| 95 |
0～10min/ |
10 |
The concentration of bdellovibrio in the control group, the indole group and the indole and calcium magnesium ion group in the step (3) is preferably detected by a DNB double-layer solid medium plate detection method;
the arrhenius equation in step (4) is: law of exponents of Arrhenius equation (Arrhenius formula) k ═ Ae-E/RTIn logarithmic form: lgk ═ E/2.303RT + lgA;
wherein k is an inactivation rate constant, E is an apparent activation energy, R is a molar gas constant, T is a thermodynamic temperature, and A is a frequency factor; the initial concentration of bdellovibrio in the mixed solution is C0The measured bdellovibrio concentration is C, and the relative activity after different storage times at different temperatures is determined (Cr ═ C/C)0) The inactivation rate constant (k) of bdellovibrio at each temperature is obtained by regression analysis of lgCr to time (T), lgk is subjected to regression analysis to 1/T × 103 to obtain an Allenioux equation, and then the quality guarantee period of the bdellovibrio mixed solution and the bdellovibrio plastid is obtained when the concentration is reduced to 1% of the original concentration at different temperatures (4 ℃ and 10 ℃) (see Table 4 and figures 7 and 8).
TABLE 4 shelf life of the normal and high density hirudin groups at different temperatures
| Bdellovibrio bacteriovorus with different treatment modes | Shelf life at 4 ℃/d | Shelf life at 10 ℃/d |
| Control group | 49.537 | 21.682 |
| Indole group | 70.225 | 32.497 |
| Indole and calcium magnesium ion group | 74.425 | 34.505 |
The Bdellovibrio bacteriovorus is preferably Bdellovibrio bacteriovorus (Bdellovibrio sp.) BDN-1, and a high-density bdelloplast group and a control group of the Bdellovibrio bacteriovorus BDN-1 are subjected to quality guarantee period research by using a classical constant temperature acceleration test for researching the quality guarantee period, so that the quality guarantee period of the strain bdelloplast is prolonged compared with that of the control group, and the aim of improving the quality guarantee period of the Bdellovibrio bacteriovorus BDN-1 by using the method is fulfilled.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> university of southern China's science
Application of indole or indole and calcium magnesium ions in promoting formation of bdelloplast of marine bdellovibrio bacteriovorus
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>63F
<400>1
caggcctaac acatgcaagt c 21
<210>2
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>842R
<400>2
cgwcactgaa ggggtcaa 18
<210>3
<211>758
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> sequence of 16S rDNA of Bdellovibrio sp BDN-1
<400>3
gtggcgcacg ggtgagtaac gcgtaggtga cgtgcctttt agtgggggac aacatcgtga 60
aaacggtgct aataccgcat aagttaagcg acattgaaaa agcttaagaa agtgggcttc 120
ggctcacgct gaaagatcgg cctgcgtatc attagcttgt tggtggggta acggcctacc 180
atggctacga tgattaactg gtctgagagg atgatcagtc acactggaac tgagacacgg 240
tccagactcc tacgggaggc agcagtaggg aatattgcgc aatgggggaa accctgacgc 300
agcaatgcca cgtgagtgag gaaggccctt gggttgtaaa gctctgtcct atgggaagaa 360
ctgcattacg gttaataccc gtagtgtttg acggtaccat agaagaaagc accggctaac 420
tccgtgccag cagccgcggt aatacggagg gtgcaagcgt tgttcggatt tactgggcgt 480
aaagcgcgcg caggcggatt ggcaagtcag atgtgaaatc tcggggctca accccgaaac 540
tgcgtctgaa actatcagtc tagagtctca tagggggcag gggaatttca cgtgtagggg 600
taaaatccgt agagatgtga aggaacaccc gtggcgaagg cgcctgcctg gatgagcact 660
gacgctgagg cgcgaaagcg tggggagcaa acaggattat ataccctggt actccacgcc 720
gtaaacgatg agtactagcc cttggaggta ttgacccc 758
Claims (9)
1. The application of indole or indole and calcium magnesium ions in promoting formation of bdelloplast of marine bdellovibrio bacteriovorus is characterized in that: the indole is used at the final concentration of 1-3 mM;
the molar ratio of the calcium ions to the magnesium ions is 2: 1, the final use concentrations of indole and calcium magnesium ions are respectively 1-3 mM, (4-40)/(2-20) mM;
the marine bdellovibrio is bdellovibrio bacteriovorus (Bdellovibrio)Bdellovibriosp.) BDN-1, deposited in 27 months 11 and 2017 at the Guangdong province microbial strain collection center of Guangzhou province microbial research institute of No. 59 building of No. 5 building of Midduo No. 100, Middui, Guangzhou city, with the collection number: GDMCC NO: 60291.
2. use according to claim 1, characterized in that:
the application of indole or indole and calcium magnesium ions in prolonging the shelf life of a bdelloplast of marine bdellovibrio bacteriovorus.
3. Use according to claim 1 or 2, characterized in that:
the indole is used at the final concentration of 2-3 mM.
4. Use according to claim 1 or 2, characterized in that:
the final use concentrations of the indole and the calcium magnesium ions are respectively 2-3 mM and (4-20)/(2-10) mM.
5. Use according to claim 4, characterized in that:
the indole and the calcium magnesium ions are respectively used at the final concentrations of 3mM and 20/10 mM.
6. A bdelloplast microbial preparation of marine bdellovibrio is characterized by being prepared by the following steps: inoculating bdellovibrio bacteriovorus into a DNB liquid culture medium containing host bacteria, culturing at 25-35 ℃ and 150-300 rpm for 36-48 h, adding the host bacteria again, adding indole or indole and calcium magnesium ions, continuously culturing for 20-28 h, centrifuging the culture solution at 4 ℃ and 6000-8000 rpm for 10-15 min, removing supernatant containing bdellovibrio bacteriovorus swimmers, sucking and suspending precipitates by using sterilized distilled water with the salinity of 10-25 per mill by mass-volume ratio, and adjusting the concentration to 1012PFU/m L, namely a bdelloplast microbial preparation of marine bdellovibrio bacteriovorus;
the indole is used at the final concentration of 1-3 mM;
the molar ratio of the calcium ions to the magnesium ions is 2: 1, the final use concentrations of indole and calcium magnesium ions are respectively 1-3 mM, (4-40)/(2-20) mM;
the bdellovibrio is bdellovibrio (Bdellovibrio)Bdellovibriosp.) BDN-1, deposited in 27 months 11 and 2017 at the Guangdong province microbial strain collection center of Guangzhou province microbial research institute of No. 59 building of No. 5 building of Midduo No. 100, Middui, Guangzhou city, with the collection number: GDMCC NO: 60291.
7. the bdelloplast microbial preparation of marine bdellovibrio bacteriovorus according to claim 6, which is characterized in that:
the indole is used at the final concentration of 2-3 mM.
8. The bdelloplast microbial preparation of marine bdellovibrio bacteriovorus according to claim 6, which is characterized in that:
the final use concentrations of the indole and the calcium magnesium ions are respectively 2-3 mM and (4-20)/(2-10) mM.
9. The bdelloplast microbial preparation of marine bdellovibrio bacteriovorus according to claim 8, which is characterized in that:
the indole and the calcium magnesium ions are respectively used at the final concentrations of 3mM and 20/10 mM.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711333687.0A CN107937330B (en) | 2017-12-14 | 2017-12-14 | Application of indole or indole and calcium magnesium ions in promoting formation of bdelloplast of marine bdellovibrio bacteriovorus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711333687.0A CN107937330B (en) | 2017-12-14 | 2017-12-14 | Application of indole or indole and calcium magnesium ions in promoting formation of bdelloplast of marine bdellovibrio bacteriovorus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107937330A CN107937330A (en) | 2018-04-20 |
| CN107937330B true CN107937330B (en) | 2020-07-28 |
Family
ID=61943063
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711333687.0A Active CN107937330B (en) | 2017-12-14 | 2017-12-14 | Application of indole or indole and calcium magnesium ions in promoting formation of bdelloplast of marine bdellovibrio bacteriovorus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107937330B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101638629A (en) * | 2009-08-28 | 2010-02-03 | 华南理工大学 | Bdellovibrio bacteriovorus for preventing and curing rice bacterial diseases and application thereof |
| CN102038714A (en) * | 2010-08-31 | 2011-05-04 | 华南理工大学 | Application of bdellovibrio bdelloplast in preparing bacterial agent for preventing and controlling red mouth disease of scophthalmus maximus |
-
2017
- 2017-12-14 CN CN201711333687.0A patent/CN107937330B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101638629A (en) * | 2009-08-28 | 2010-02-03 | 华南理工大学 | Bdellovibrio bacteriovorus for preventing and curing rice bacterial diseases and application thereof |
| CN102038714A (en) * | 2010-08-31 | 2011-05-04 | 华南理工大学 | Application of bdellovibrio bdelloplast in preparing bacterial agent for preventing and controlling red mouth disease of scophthalmus maximus |
Non-Patent Citations (5)
| Title |
|---|
| Dependence of Marine Bdellovibrios on Potassium, Calcium，and Magnesium Ions;A. MARBACH等;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;19780731;第36卷(第1期);第169-177页 * |
| Indole negatively impacts predation by Bdellovibrio bacteriovorus and its release from the bdelloplast;Mohammed Dwidar等;《Environmental Microbiology》;20140428;第17卷(第4期);第1009-1022页 * |
| Influence of NaCl, Ca2+ and Mg2+ on the growth of a Marine Bdellovibrio sp.;R.G.Bell等;《Estuarine and Coastal Marine Science》;19751231;第3卷;第381-384页 * |
| 淡水蛭弧菌的分离鉴定及其裂解大肠杆菌作用机制的初步研究;彭宗辉等;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20110315(第03期);第E059-85页 * |
| 蛭弧菌的生长特性及其培养条件的优化;韩晓宁等;《现代食品科技》;20091231;第25卷(第1期);第19-23页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107937330A (en) | 2018-04-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109355223B (en) | Bacillus subtilis N2 with ammonia nitrogen degradation function and application thereof | |
| CN112195158B (en) | Vibrio parahaemolyticus bacteriophage RDP-VP-19003 and application thereof | |
| CN107686832B (en) | Novel vibrio parahaemolyticus bacteriophage, and composition, preparation method and application thereof | |
| CN112940982B (en) | Snakehead source Bacillus belezii and application thereof | |
| Li et al. | Bdellovibrio and like organisms enhanced growth and survival of Penaeus monodon and altered bacterial community structures in its rearing water | |
| CN114621885B (en) | Bacillus subtilis for efficiently removing ammoniacal nitrogen and nitrite nitrogen and application of bacillus subtilis in aquaculture | |
| CN112481165B (en) | Rhodopseudomonas palustris P-3 and screening method and application thereof | |
| CN112760296A (en) | Vibrio parahaemolyticus bacteriophage RDP-VP-19010 and application thereof | |
| CN106754559A (en) | A kind of probiotics preparation method for effectively purifying water | |
| CN116904376B (en) | High-salt-tolerance bacillus cereus strain, microbial agent and application thereof | |
| CN107974418B (en) | Marine bdellovibrio bacteriovorus and application thereof in promoting formation of bdelloplast under ampicillin | |
| CN111334454B (en) | Microbacterium PT3 with protein degradation function and application thereof | |
| CN111088181B (en) | Bifidobacterium breve strain BK55 and application thereof in inhibiting clostridium difficile | |
| CN107937330B (en) | Application of indole or indole and calcium magnesium ions in promoting formation of bdelloplast of marine bdellovibrio bacteriovorus | |
| CN107988135B (en) | Application of magnesium ions in promoting formation of bdelloplast of marine bdellovibrio bacteriovorus | |
| CN107988136B (en) | Marine bdellovibrio bacteriovorus and application thereof in promoting formation of bdelloplast under ampicillin | |
| CN106148218B (en) | Photosynthetic bacteria microecology for aquatic products and application thereof | |
| Sandlund et al. | Screening and characterisation of potentially pathogenic bacteria associated with Atlantic cod Gadus morhua larvae: bath challenge trials using a multidish system | |
| CN107974417B (en) | Application of calcium ions in promoting formation of bdelloplast of marine bdellovibrio bacteriovorus | |
| CN108102999B (en) | Application of indole in promoting formation of bdelloplast of marine bdellovibrio | |
| CN114621884B (en) | Bacillus subtilis and application thereof in water quality purification | |
| CN111363690B (en) | Bdellovibrio bacteriovorus mutant strain with strong cracking performance and application thereof | |
| JP3675824B2 (en) | Methods for culturing bacteria that produce proteins expressed in a temperature-controlled manner | |
| CN110885872A (en) | Screening and identifying method for strain with high sinapiside degradation capability | |
| CN112646785B (en) | High-temperature-resistant virulent proteobacterium bacteriophage RDP-SA-20018 and application thereof |
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 |