CN108102999A - Application of the indoles in ocean bdellovibrio bdelloplast bacterial is promoted to be formed - Google Patents
Application of the indoles in ocean bdellovibrio bdelloplast bacterial is promoted to be formed Download PDFInfo
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Abstract
The present invention discloses a kind of application of indoles in ocean bdellovibrio bdelloplast bacterial is promoted to be formed, and belongs to Bdellovibrio technical field.The present invention forms the factor-indoles using bdellovibrio bdelloplast bacterial is promoted, and promotes the formation of 1 high density leech plastids of Bdellovibrio (Bdellovibrio sp.) BDE；By the study find that, indoles has apparent facilitation to the formation of bdellovibrio bdelloplast bacterial, and the cracking performance of leech plastid does not decline, and high density leech plastid group shelf-life compared with control group is obviously prolonged；High density leech plastid provided by the invention can inhibit the vibrios that people can be triggered to infect and poison by food, can inhibit again can trigger the vibrios of aquaculture organisms disease, also there is good bacteriostasis to other food-borne pathogens simultaneously, with broad fragmentation pattern, and its splitting action is constant compared with 1 mixtures of Bdellovibrio BDE；Present invention rush leech plastid cultural method is simple and practicable, can be promoted the use of.
Description
Technical field
The invention belongs to Bdellovibrio technical field, more particularly to a kind of indoles is in promoting ocean bdellovibrio bdelloplast bacterial to be formed
Application.
Background technology
Vibrios (Vibrio) is one of bacterial groups most commonly seen in marine environment, is widely present in seawater, seabed sinks
In product object and fish, shrimp, shellfish, crab, sea cucumber, kelp etc. marine product.《Primary Jie Shi bacteriology handbook》8th edition describes 35 kinds of arcs
Bacterium, quite a few in them are the important pathogenic bacterias of aquaculture organisms.In aquatic products industry, by vibrio parahaemolytious, molten algae arc
The disease for the aquaculture organism that the various vibrios such as bacterium, Vibrio anguillarum trigger is referred to as vibriosis (vibriosis), it gives aquatic products industry every year
Cause huge economic loss in boundary.At the same time, part vibrios is also the pathogenic bacteria of the mankind.They can not only cause human gastrointestinal
It is scorching, moreover it is possible to cause extra intestinal infection.In food, vibrios (Vibrio), salmonella (Salmonella), pathogenic large intestine angstrom
Uncommon bacterium (pathogenic Escherichia coli) and staphylococcus aureus (Staphylococcus aureus) etc. one
It rises, becomes the main pathogeny bacterium of food posioning.Up to the present, it is known that such as comma bacillus (Vibrio
Cholerae), vibrio parahaemolytious (V.parahaemolyticus), vibrio fluvialis (V.fluvialis) etc. can cause the intestines of people
Gastritis, and Vibrio vulnificus (V.vulnificus), vibrio alginolyticus (V.alginolyticus) etc. cause wound infection and septicemia
Wait extra intestinal infections.In recent years, as intestines problem, infectious disease and the food poisoning generation scale caused by kinds of pathogenic vibrio and people
Group's exposure scale is in apparent ascendant trend, more than salmonella food poisoning, leaps to the first place of food posioning event, into
An important factor for influence food security, cause extensive concern.
The prevention most common method of bacteriosis is to use antibiotic, but long-time service antibiotic can cause bacterium to generate
Drug resistance.Therefore, the research of effective substitute of antibiotic seems particularly urgent.Bdellovibrio monoid (Bdellovibrio-and-
Like organisms) it is a kind of newer member.It is a kind of parasitics Gram-negative bacteria.With other beneficial bacteriums by or
To inhibit harmful bacteria difference, Bdellovibrio is by works parasitic, and then the other bacteriums of cracking for site competition or secretion extracellular products
With and achieve the purpose that control harmful bacteria.Numerous researchs show that Bdellovibrio has common pathogen in cracking aquaculture of aquatic animal
Such as vibrio alginolyticus (Vibrio alginolyticus), vibrio parahaemolytious (Vibrio parahaemolyticus), comma bacillus
The ability of (Vibrio cholera) etc. by inhibiting the growth and transfer of these pernicious bacterias, can keep even increasing water
Originally there is the quantity of the probiotics such as lactic acid bacteria and Bifidobacterium of inhibitory action etc. in production animal intestinal tract to morbid vibrio, excite water
Produce Animal gut immunity system operation, enhancing immunity of organisms, premunition and anti-stress ability, so as to improve aquatic livestock enteron aisle
Bacterial community improves its survival rate.
Up to the present, research shows that Bdellovibrio is widely present in various natural environments, such as soil, plant rhizosphere, each
Kind water body (in extra large, salty, light, sewage, enclosed cyclic culture system, etc.), exists in common eel (Eel), hybridized prussian carp
The enteron aisle of (Carassius auratus gibelio), sturgeon (Sturgeon), milk cow, horse, pig, duck and people etc. and blue crab
In the gill of (Blue crab), it is intrinsic a member of each microbiologic population, but does not have and infect plant, fish and people etc.
The ability of mammalian cell namely harmless to them.
The history of life of Bdellovibrio includes two stages (phases)：The telotroch stage that is dynamic with flagellum operation but not being proliferated
(free swimming phase) and the leech plastid stage (Bdelloplast phase) in host cell expert's growth and breeding.
Completing a life cycle only needs 4 hours.In the telotroch stage, Bdellovibrio need to consume huge energy and the dynamic life of oxygen operation,
Host is obtained to meet.As do not met host whithin a period of time, then energy and/or oxygen depletion and die.This stage is usually no more than
A few hours.In other words, telotroch Bdellovibrio is easily dead, it is extremely difficult to survive.In the leech plastid stage, Bdellovibrio has enter into host
Periplasmic space (periplasmic space) under the protection of host cell wall, has sloughed flagellum, is sought by decomposing host cell
Itself is supported, into elongated rod shape.Afterwards, by dividing (multiple fission) more, it is divided into multiple segments, whip is grown per segment
Hair, first filial generation telotroch.
Some researches show that the leech plastid stage of Bdellovibrio, its growth (elongated process) can be interrupted at any time.Treat environment
After condition is suitable, leech plastid again line splitting, be divided into multiple telotroches.Therefore, compared to the telotroch of fragile and high oxygen consumption, low consumption
The leech plastid tolerance adverse circumstances ability of oxygen is strong.
Although Bdellovibrio is used more and more widely, " detection of Bdellovibrio class biological agent used for aquiculture "
Research discloses, and product on the market there's almost no active Bdellovibrio at present or quantity is few.We study similary table
Bright, Bdellovibrio affects the market development of product there is survival rate is extremely low, shelf-life extremely short problem at present.
It can be seen that solving the problems, such as that Viable detection present in current Bdellovibrio product is low, the shelf-life is short, there is weight
The economic value wanted.And it solves the problems, such as this key and is that during the high gravity fermentation of Bdellovibrio it is leech to change telotroch
Plastid, while delay leech development of plastid, then the high concentration Bdellovibrio in the leech plastid stage is freeze-dried or is directly protected
It hides, thus extends the shelf-life of Bdellovibrio, maintain the survival rate of Bdellovibrio product Bdellovibrio before being taken into use, and carry out leech
The market development of vibrios products application makes prominent tribute in Bdellovibrio to hope in terms of culture fishery prevents bacteriosis
It offers.
The content of the invention
The shortcomings that in order to overcome the prior art and deficiency, it is a primary object of the present invention to provide a kind of indoles to promote sea
Foreign bdellovibrio bdelloplast bacterial formed in application.
Another object of the present invention is to provide a kind of ocean bdellovibrio bdelloplast bacterial microorganism formulation.
The purpose of the present invention is achieved through the following technical solutions：
The present invention provides a kind of application of indoles in ocean bdellovibrio bdelloplast bacterial is promoted to be formed, and especially indoles is extending
Application in the shelf-life of ocean bdellovibrio bdelloplast bacterial.
Preferably, the final concentration of 1~3mmol/L of the use of the indoles；More preferably 2~3mmol/L, most preferably
3mmol/L。
A kind of ocean bdellovibrio bdelloplast bacterial microorganism formulation, is prepared by the following method to obtain：In the DNB containing host strain
Bdellovibrio is accessed in fluid nutrient medium, 36~48h is cultivated in 20~35 DEG C, 150~300r/min, adds host strain again, and
Indoles is added in, continues 20~28h of culture, culture solution is after 4 DEG C, 6000~8000r/min centrifuge 10~15min, and removal is containing leech
The supernatant of vibrios telotroch, the aseptic distillation of precipitation use quality volume ratio (g/mL) 10~30 ‰ (being preferably 20 ‰) salinity
Suspension is played in water suction, adjusts its concentration to 1011PFU/mL is ocean bdellovibrio bdelloplast bacterial microorganism formulation.
Preferably, the Bdellovibrio is Bdellovibrio (Bdellovibrio sp.) BDE-1.
Described Bdellovibrio (Bdellovibrio sp.) BDE-1, is derived from the seawater of Hainan aquaculture, warp
Artificial enrichment culture isolates and purifies to obtain, be one plant with bacillus subtilis (Bacillus subtilis) for parasitic host
Bdellovibrio.
The preservation information of described Bdellovibrio (Bdellovibrio sp.) BDE-1：Depositary institution：Guangdong Province's microbial bacteria
Kind collection (GDMCC), preservation date：On November 27th, 2017, preservation address：The compound 59 of Xianlie Middle Road, Guangzhou City 100
Number Guangdong Microbes Inst of 5 building, building, deposit number：GDMCC NO：60292.
The cultivation temperature of the Bdellovibrio BDE-1 be preferably 20~35 DEG C (being most preferably 30 DEG C), PH is preferably 6.0~
7.0 (being most preferably 7.0), salinity are preferably 10~30 ‰ (being most preferably 20 ‰)；
The culture medium of the culture Bdellovibrio BDE-1 is preferably DNB culture mediums；
The Bdellovibrio BDE-1 has following morphological feature and physio-biochemical characteristics：
A, the Bdellovibrio BDE-1 Gram's staining screened is feminine gender, and rod-shaped, no gemma holds raw single flagellum；
B, the morphological feature of plaque is：The Bdellovibrio BDE-1 is in 30 DEG C of DNB bilayer solid plating mediums tablet
After culture 4 days, plaque is rounded, transparent, is recessed, moistening, neat in edge shape, 1~3mm of diameter.
The present invention is had the following advantages compared with the prior art and effect：
(1) present invention forms the factor --- indoles using bdellovibrio bdelloplast bacterial is promoted, and promotes Bdellovibrio (Bdellovibrio
Sp.) the formation of BDE-1 high density leech plastid；By the study find that, indoles has significantly the formation of bdellovibrio bdelloplast bacterial
Facilitation, the cracking performance of leech plastid do not decline；Present invention rush leech plastid cultural method is simple and practicable, and can carry out promoting makes
With.
(2) high density leech plastid provided by the invention can inhibit can to trigger people infect and the vibrios of food poisoning and
Inhibition can trigger the vibrios of aquaculture organisms disease, while also have good bacteriostasis to other food-borne pathogens,
With wide cracking spectral property, and the same Bdellovibrio of its splitting action (Bdellovibrio sp.) BDE-1 mixtures were (both containing leech matter
Body contains telotroch again) compared to constant；
(3) Bdellovibrio (Bdellovibrio sp.) BDE-1 high density leech plastid groups provided by the invention are the same as control group phase
It is obviously prolonged than shelf-life.
Description of the drawings
Fig. 1 is host strain bacillus subtilis (Bacillus subtilis) 100 × oil mirror microscopy figure.
Fig. 2 is the DNB bilayer solid culture medium flat plate method separating resulting figures of Bdellovibrio BDE-1
Fig. 3 is the transmission electron microscope observing result figure of Bdellovibrio BDE-1.
Fig. 4 is action diagram of the various concentration indoles in embodiment 2 to Bdellovibrio BDE-1 leech plastid forming amounts.
Fig. 5 is that Bdellovibrio under the different conditions in embodiment 4 number of days of shelf-life at 4 DEG C and 10 DEG C compares figure.
Specific embodiment
With reference to embodiment and attached drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited
In this.
The culture medium arrived involved in embodiment：
The formula (g/L) of DNB fluid nutrient mediums：The nutrition human body 0.8, acid hydrolyzed casein 0.5, yeast extract powder 0.1, chlorine
Change sodium 20；Final pH 7.0；
DNB upper stratas culture medium prescription：The agar powder that mass ratio is 0.8%, final pH are added in the sterile water of 20 ‰ salinity
7.0；
DNB lower floors culture medium prescription：The agar powder that mass ratio is 1.5%, final pH are added in DNB fluid nutrient mediums
7.0；
Nutrient broth (NB) Liquid Culture based formulas (g/L)：Peptone 10；Beef extract powder 3；Sodium chloride 5, final pH
7.0；
Nutrient broth (NB) solid culture based formulas：The fine jade that mass ratio is 1.5% is added in nutrient broth fluid nutrient medium
Cosmetics, final pH 7.0；
The formula of indoles mother liquor：0.1g indoles powder is weighed to be dissolved in the sterile distilled water of 100mL.
Bacillus subtilis (Bacillus subtilis GIM 1.136) purchase is in Guangdong Province microorganism in the present embodiment
Research institute.
In the present embodiment in 39 plants of indicator bacterias, staphylococcus aureus (Staphylococcus aureus) AS
1.1865th, escherichia coli (Escherichia coli) AS 1.2420 and salmonella typhimurium (Salmonella
Typhimurium) CMCC 50115 is bought in China General Microbiological preservation administrative center；
Vibrio anguillarum (Vibrio anguillarum) ATCC 43305, vibrio fluvialis (Vibrio fluvialis) ATCC
33809th, vibrio parahaemolytious (V.parahaemolyticus) (tdh+) ATCC17802 is purchased from American Type Culture collection
(American Type Culture Collection)；
Vibrio alginolyticus (Vibrio alginolyticus) 1, vibrio alginolyticus (V.alginolyticus) 2, vibrio alginolyticus
(V.alginolyticus) 3, vibrio alginolyticus (V.alginolyticus) 4, comma bacillus (V.cholera) 6, vibrio parahaemolytious
(V.parahaemolyticus) 8, vibrio parahaemolytious (V.parahaemolyticus) 9, vibrio alginolyticus
(V.alginolyticus) 10, vibrio alginolyticus (V.alginolyticus) 11, Shewanella putrefaciens (Shewanella
Putrefaciens) 12, vibrio alginolyticus (V.alginolyticus) 13, smell sand thunder bacterium (Serratia ficaria) 15, molten
Algae vibrios (V.alginolyticus) 16, pseudomonas aeruginosa (Pseudomonas aeruginosa) 17, pseudomonas aeruginosa
(P.aeruginosa) 18, vibrio alginolyticus (V.alginolyticus) 19, smell sand thunder bacterium (Serratia ficaria) 20,
Vibrio parahaemolytious (V.parahaemolyticus) 21, pseudomonas aeruginosa (P.aeruginosa) 22, vibrio alginolyticus
(V.alginolyticus) 23, Shewanella putrefaciens (Shewanella putrefaciens) 24, vibrio parahaemolytious
(V.parahaemolyticus) 25, vibrio parahaemolytious (V.parahaemolyticus) 26, Shewanella putrefaciens
(Sh.putrefaciens) 27, Shewanella putrefaciens (Sh.putrefaciens) 28, pseudomonas aeruginosa
(P.aeruginosa) 29, pantoea agglomerans (Pantoea agglomerans) 30, acid-producing Klebsiella bacterium (Klebsiella
Oxytoca) 31, aeromonas salmonicida (Aeromonas salmonicida) 33, Shewanella putrefaciens
(Sh.putrefaciens) 34, pseudomonas aeruginosa (P.aeruginosa) 35 is by South China Science ＆ Engineering University's light industry and Foodstuffs Academy
(biological characterization of two marine bdellovibrio-and-like are provided
organisms isolated from Daya bay of Shenzhen,China and their application in
the elimination of Vibrio parahaemolyticus in oyster[J].Li H,Liu C,Chen L et
al.International Journal of FoodMicrobiology,2011,151:36–43.)；
Providencia rettgeri (Providencia rettgeri) 32 is by South China Science ＆ Engineering University's light industry and Foodstuffs Academy
(The protective effect ofbdellovibrio-and-like organisms (BALO) on tilapia are provided
fish fillets against Salmonella enterica ssp.enterica serovar Typhimurium[J]
.Lu F,Cai J.Letters inAppliedMicrobiology,2010,51:625–631.)；
Vibrio anguillarum (Vibrio anguillarum) MVM425 is by East China University of Science's bioreactor state key experiment
Room give (minimums of marine fishes pathogenic bacteria Vibrio anguillarum MVM425 virulence plasmid pEIB1 replication regions analysis [J] Wus sea is precious,
Zhang Huizhan, Li Gang, Ye Jiang, Ma Yue, Zhang Yuanxing food and drug, 2007,04:1-4.)
Meanwhile in the present embodiment 39 plants of indicator bacterias patent " ZL201410752144.2, one plant have broad-spectrum antibacterial live
Disclosed in the lactic acid bacteria of property and its application ".
The separation and purifying of 1 Bdellovibrio BDE-1 of embodiment
Sample is fetched from the Marine water region of Hainan aquaculture, with bacillus subtilis (Bacillus
Subtilis) as parasitic host bacterium, sample liquids 10mL is taken to add in the 50mL DNB fluid nutrient mediums of aseptic process,
Bacterium solution is first centrifuged 10min by 200r/min, 30 DEG C of enrichment culture 30h under 6000r/min, then by supernatant in 20000r/
After centrifuging 20min under min, a small amount of bacterium of centrifugation bottom of the tube with sterile aqueous suspension, and suspension is diluted to 10-1、10-2、10-3、
10-4, each dilution gradient bilayer solid of falling DNB culture medium flat plate successively, 30 DEG C are cultivated 3~4 days, select plaque number for 30~
300 tablet, transfer needle picking size is basically identical, circular, transparent and neat in edge single plaque to 50mL DNB
Continue to cultivate in fluid nutrient medium, the centrifugation dilution bilayer solid of falling DNB culture medium flat plate is then continued to, until on double-layer plate
Plaque size is basically identical, rounded transparent and neat in edge, you can tentatively confirms as Bdellovibrio, and carries out transmission electron microscope sight
It examines and strain idenfication；
The Bdellovibrio that purifying obtains is identified that qualification result is as follows：
A, the Bdellovibrio size screened is 1.03 × 0.45 μm, rod-shaped, holds raw flagellum, flagellum length is about 4.8 μm；
B, the morphological feature of plaque is：After the culture 4 days of 30 DEG C of DNB bilayer solids culture medium flat plate, plaque is in circle
Shape, transparent, recess, surface wettability, neat in edge, 1~3mm of diameter；
Host strain bacillus subtilis (Bacillus subtilis) 100 × oil mirror microscopy figure, as shown in Figure 1.
DNB bilayer solid culture medium flat plate method separating resulting figures, as shown in Figure 2.
Transmission electron microscope observing result figure, as shown in Figure 3.
Molecular biology identification result：
Tablet plaque one bottle of Bdellovibrio bacterium solution of shaking table culture is inoculated with, 10min is first centrifuged under 6000r/min, then will be upper
Clear liquid centrifuges 20min under 20000r/min, is precipitated with the TE buffering liquid enrichments of 500 μ L, send in sequencing company (Shanghai life work life
Object Engineering Co., Ltd) it is sequenced；Wherein, PCR amplification the primer is：
63F：5'-CAGGCCTAACACATGCAAGTC-3'；
842R：5'-CGWCACTGAAGGGGTCAA-3', wherein, degeneracy base W represents A/T；
PCR reaction conditions are：95 DEG C of pre-degeneration 3min；94 DEG C, it is denatured 1min；56 DEG C, anneal 45s；72 DEG C, extension
1min；35 Xun Huans；72 DEG C, extend 10min eventually；The present invention through forward primer 63F and is reversely drawn using monoclonal identification method
Object 842R expands to obtain two bar segments, and DNAStar splicing two sequences obtain about 800bp target fragments.
16S rDNA gene sequencing result sequences are subjected to homologous comparison by NCBI BLAST in GenBank databases
It analyzes and identifies, RDP classifier classifications for search are Bdellovibrio, and further NCBI BLAST analyses are found, the Bdellovibrio
(Bdellovibrio sp.) and they with nearest affiliation-Bdellovibrio sp.BDH12,
Bdellovibrionales bacterium BDHSH06, uncultured bacterium clone Bms_CK248 have
98% similarity, therefore, bacterial strain BDE-1 are accredited as Bdellovibrio (Bdellovibrio sp.).
Described in summary, the Bdellovibrio that the present invention isolates and purifies is named as Bdellovibrio (Bdellovibrio sp.) BDE-
1, preservation information：Depositary institution：Guangdong Province's Culture Collection (GDMCC), preservation date：November 27 in 2017
Day, preservation address：5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100 Guangdong Microbes Inst, deposit number：GDMCC
NO：60292.
The sequence such as SEQ ID NO of the 16S rDNA of described Bdellovibrio (Bdellovibrio sp.) BDE-1：Shown in 3
(774bp)。
2 indoles of embodiment acts on the rush culture of Bdellovibrio BDE-1 leech plastids
(1) it is inoculated with host and cultivates：Take host strain bacillus subtilis (the Bacillus subtilis GIM newly cultivated
1.136) one bottle of host's seed liquor, the burned bottleneck under alcolhol burner flame envelope in aseptic operating platform are drawn 3mL host strains seed liquor and are turned
Enter in the bottled NB fluid nutrient mediums of 250mL, the host strain being inoculated with is placed on progress shaking table training in 31 DEG C or so of constant-temperature table
After supporting about 15h, it is dispensed into 4 100mL centrifuge tubes, is put into refrigerated centrifuge and is centrifuged at 6000r/min rotating speeds, 4 DEG C
Then 10min abandons supernatant, often pipe adds in the sterile distilled water suspension of 5 ‰ salinity of 1mL, breaks up and moves into one after mixing
In centrifuge tube, sterile lid is covered, bagging, sack need to be sterile, is then placed in 4 DEG C of refrigerators and saves backup, and obtains host strain concentration
Liquid；
(2) BDE-1 seed liquors culture：Take BDE-1 tablets after purification, picking circular non-opaque plaque access 50mL DNB
In fluid nutrient medium, while MSG (sodium glutamate) solution that 250 μ L concentration are 1mol/L is accessed, add the host of 1mL concentrations
Bacterium concentrate is uniformly mixed, is positioned in shaking table, is cultivated under the conditions of 30 DEG C, 200r/min, every 500 μ L of 48h plus host,
It cultivates to after the 5th day, it is spare to be put into refrigerator after addition 1mL host's continuation shaking table 30min；
(3) indoles group is inoculated with：Take the BDE-1 seed liquors in step (2), 100 μ L BDE-1 of inoculation will be to will contain 0,1,2 and
In four bottles of 50mL DNB fluid nutrient mediums of 3mmol/L indoles, while 1mL host's bacterium concentrate is added in, 250 μ L MSG liquid are put
Shaking table culture is carried out in 30 DEG C of shaking tables；
(4) access indoles and promote medium exchange：After the BDE-1 shaking table cultures of three groups of shaking table cultures are 4 days full, it will configure in advance
It gets well and is respectively connected to corresponding blake bottle by the amount calculated with the indoles mother liquor of 0.22 μm of membrane filtration degerming, make indoles group
Final concentration is respectively 1mmol/L, 2mmol/L and 3mmol/L, connects after promoting medium exchange, blake bottle is placed in shaking table and is continued
It cultivates for 24 hours, convenient for the formation of leech plastid；
(5) separation of leech plastid and the bilayer that falls：The former culture bacterium solutions of all 50mL are centrifuged with 6000r/min, convenient for leech
Plastid settles, and then pours out supernatant in sterile centrifugation tube, takes 1mL sterile waters that leech plastid is precipitated to the suction that suspends and beats uniformly,
And it is diluted to 10-1、10-2With 10-3Then three concentration gradients take 500 μ L dilutions bacterium solution to carry out the bilayer solid of falling DNB culture medium
Tablet, whole are marked and classified with anti-water-color paintbrush after getting well, and after the top-layer agar condensation of double-layer plate, use fresh-keeping plastic bag
It wraps, is placed in quiescent culture in 30 DEG C of incubators；
(6) result is observed：A tablet is observed every 12h, sees that the Bdellovibrio of all tablets goes out spot situation, spot to be gone out
Afterwards, count plaque number and calculate each respective concentration.
As a result as shown in table 1 and Fig. 4, to be compared with control group, indoles concentration adds 1.3log for 1mmol/L groups,
2mmol/L groups add 1.45log, and 3mmol/L groups amplification is maximum, adds 1.72log.It can be seen that with indoles addition concentration
Increase, the leech plastid density in the former culture bacterium solution of Bdellovibrio is higher.
Influence that 1 various concentration indoles of table forms bdellovibrio bdelloplast bacterial (concentration to represent numerical value)
Note：a:Average ± standard deviation, n=3
The application of the wide fragmentation pattern of 3 Bdellovibrio BDE-1 leech plastids of embodiment
The preparation of (1) 39 plant of indicator bacteria
In 39 plants of indicator bacteria (table 2) single bacterium colony inoculation nutrient broth fluid nutrient mediums, 37 DEG C, 200r/min culture 8h are adjusted
Its concentration is saved to 1 × 106CFU/mL, 4 DEG C of preservations are spare；
The different experiment indicator strain of 2 39 plants of table
(2) preparation of bdellovibrio bdelloplast bacterial
The mono- plaques of the Bdellovibrio identified in embodiment 1 (Bdellovibrio sp.) BDE-1 are inoculated in 50mL
DNB fluid nutrient mediums, are cultivated 3 days, cultivate its concentration to 1 × 10 by 30 DEG C, 2000r/min8PFU/mL；6000r/min, 4 DEG C,
10min centrifuges Bdellovibrio culture solution, and enrichment precipitation (leech plastid) tests spare, 4 DEG C of preservations, spare (concentration system for bacteria lysis
The Bdellovibrio concentration of standby mixed liquor is 7.6 × 1014PFU/mL, the Bdellovibrio concentration of leech plastid is 8.1 × 1011PFU/mL)；
(3) (leech plastid) cracking experiment
Cracking experiment：Take the indicator bacteria that step (1) is prepared and each 500 μ L of leech plastid that step (2) obtains together with
3mL DNB upper stratas culture medium mixing shaken well is laid in and has shifted to an earlier date on Dao Hao DNB lower floors culture medium, treats DNB upper stratas culture medium
After condensation, cultivated 3~4 days at 30 DEG C, check whether each tablet plaque can occurs, and record；
Interpretation of result：
Experiment (had not only contained leech plastid but also had contained telotroch) bacterium solution as control using this plant of bdellovibrio mixture, to examine
Test the possibility whether leech plastid is present with decrease compared with the cracking ability of mixture.Leech plastid cracks experimental result such as table 3
It is shown, the indicator bacteria of the pathogenic bacteria of 39 plants of separate sources as this experiment, Bdellovibrio (Bdellovibrio sp.) BDE-1 leech matter
Body and mixture cracking ability are still consistent, and to the cleavage rates of 39 plants of indicator bacterias up to 100%, this just illustrates Bdellovibrio
The leech plastid of (Bdellovibrio sp.) BDE-1 still remains the cracking ability of the almost identical intensity of same mixture, does not go out
The situation that existing cracking ability weakens.
Therefore, in the present invention, Bdellovibrio (Bdellovibrio sp.) BDE-1 leech plastid fragmentation patterns are relatively broad, can
Inhibition can trigger vibrio parahaemolytious (Vibrio parahaemolyticus) of the vibrios of people's enteritis such as containing tdh toxin genes,
Can inhibit again can trigger the vibrios of aquaculture organisms disease, such as vibrio alginolyticus (Vibrio alginolyticus).
3 leech plastid of table cause a disease to 39 plants of separate sources indicator bacteria cracking situation
Note：+ represent that the bdellovibrio bdelloplast bacterial can crack corresponding bacterium
The shelf-life research of 4 Bdellovibrio BDE-1 leech plastids of embodiment
(1) preparation of bdellovibrio bdelloplast bacterial：See step (2) in embodiment 3；Control group does not add any rush medium exchange.
(2) control group and high density leech plastid group cultivated this plant of Bdellovibrio are placed under gradient temperature and carry out acceleration examination
It tests；
(3) periodically sampling detects the concentration of control group and high density leech plastid group Bdellovibrio；
(4) data recorded with Arrhenius equation and step (3) calculate control group and high density leech plastid group
Shelf-life, by than determining whether the high density leech plastid group shelf-life more permanent to the shelf-life；
Two bottles of mixed liquors are cultivated in step (2) under the same conditions, specific cultural method is shown in embodiment 2, wherein one bottle adds
The leech plastid for entering optium concentration promotees medium exchange --- indoles, and after culture is good, precipitation of this bottle after 6000r/min is centrifuged is i.e.
For leech plastid；
The Bdellovibrio concentration of mixed liquor prepared by concentration is 7.6 × 10 in step (2)14PFU/mL, the Bdellovibrio of leech plastid
Concentration is 8.1 × 1011PFU/mL；
Gradient temperature described in step (3) is preferably 25 DEG C, 37 DEG C, 45 DEG C, 55 DEG C, 65 DEG C, 75 DEG C, 85 DEG C, 95 DEG C；
The Bdellovibrio mixed liquor 3mL prepared in step (2) and leech plastid 3mL are respectively charged into the sterile centrifugation tube of 4mL
In, sealing carries out constant temperature and accelerates in fact under conditions of being subsequently placed in 25 DEG C, 37 DEG C, 45 DEG C, 55 DEG C, 65 DEG C, 75 DEG C, 85 DEG C, 95 DEG C
It tests.
Accelerated test described in step (2) is that the length of acceleration time is successively decreased with the rise of temperature；
The assay intervals time periodically sampled described in step (3) is preferably as shown in table 4.
Assay intervals time and sampling number are sampled at a temperature of 4 difference of table
The concentration of Bdellovibrio is preferably double-deck solid by DNB in control group and high density leech plastid group described in step (3)
Body culture medium flat plate detection method is detected；
Arrhenius equation described in step (4) is：The finger of Arrhenius equation (Arrhenius formula)
Number law k=Ae-E/RT, logarithmic form is：Lgk=-E/2.303RT+lgA；
Wherein k is deactivation rate constant, and E is apparent activation energy, and R is mol gas constant, and T is thermodynamic temperature, and A is frequency
The rate factor；Using the initial concentration of Bdellovibrio in mixed liquor as C0, the Bdellovibrio concentration surveyed is C, is obtained and preserves at each temperature not
With the relative activity (C after the timer=C/C0)；With lgCrRegression analysis is carried out to the time (t), draws Bdellovibrio at each temperature
Deactivation rate constant (k), by lgk to 1/T × 103Regression analysis is carried out, obtains Arrhenius equation；Hereafter, this kind of leech is obtained
The shelf-life (being shown in Table 5 and Fig. 5) of vibrios mixed liquor and leech plastid under different temperatures (4 DEG C and 10 DEG C).
The shelf-life of control group and high density leech plastid group under 5 different temperatures of table
| The Bdellovibrio of Different treatments | Shelf-life/d at 4 DEG C | Shelf-life/d at 10 DEG C |
| Control group | 39.433 | 19.356 |
| High density leech plastid group | 70.685 | 32.674 |
The Bdellovibrio is preferably Bdellovibrio (Bdellovibrio sp.) BDE-1, by using the research shelf-life
Classical thermostatic accelerated experiment carries out shelf-life research to the high density leech plastid group and control group of Bdellovibrio BDE-1, to determine to be somebody's turn to do
Bacterial strain leech plastid realizes to reach by this method and improves Bdellovibrio compared to the effectual time of extended shelf-life for control group
The purpose of BDE-1 shelf-lifves.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention and from above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Sequence table
<110>South China Science ＆ Engineering University
<120>Application of the indoles in ocean bdellovibrio bdelloplast bacterial is promoted to be formed
<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> 774
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Bdellovibrio（Bdellovibrio sp.）The sequence of the 16S rDNA of BDE-1
<220>
<221> misc_feature
<222> (760)..(760)
<223> n is a, c, g, t or u
<400> 3
gtggcgcacg ggtgagtaac gcgtaggtga cgtgcctttt agtgggggac aacatcggga 60
aaccggtgct aataccgcat aagttaagcg acattgaaaa agcttaagaa agtgggcttc 120
ggctcacgct gaaagatcgg cctgcgttac attagcttgt tggtggggta acggcctacc 180
aaggctacga tgtataactg gtctgagagg atgatcagtc acactggaac tgagacacgg 240
tccagactcc tacgggaggc agcagtaggg aatattgcgc aatgggggaa accctgacgc 300
agcaatgcca cgtgagtgag gaaggccctt gggttgtaaa gctctgtcct atgggaagaa 360
ctgcattacg gttaataccc gtagtgtttg acggtaccat agaagaaagc accggcatac 420
tccgtgccag cagccgcggt aatacggagg gtgcaagcgt tgttcggatt tactgggcgt 480
aaagcgcgcg caggcggatt ggcaagtcag atgtgaaatc tcggggctca accccgaaac 540
tgcgtctgaa actatcagtc tagagtctca tagggggcag gggaatttca cgtgtagggg 600
taaaatccct agagatgtga aggaacaccc gtggcgaagg cgcctgcctg gatgagcact 660
gacgctgagg cgcgaaagcg tggggagcaa acaggattag ataccctggt agtccacgcc 720
ctaaacgatg agtactagcc cttggaggta ttgccccccn tccagtgacc gaaa 774
Claims (7)
1. application of the indoles in ocean bdellovibrio bdelloplast bacterial is promoted to be formed.
2. application according to claim 1, it is characterised in that：
Application of the indoles in the shelf-life for extending ocean bdellovibrio bdelloplast bacterial.
3. application according to claim 1 or 2, it is characterised in that：
Final concentration of 1~the 3mmol/L of use of the indoles.
4. application according to claim 3, it is characterised in that：
Final concentration of 2~the 3mmol/L of use of the indoles.
5. application according to claim 3, it is characterised in that：
The final concentration of 3mmol/L of use of the indoles.
6. a kind of ocean bdellovibrio bdelloplast bacterial microorganism formulation, it is characterised in that be prepared by the following method to obtain：Containing place
Bdellovibrio is accessed in the DNB fluid nutrient mediums of main bacterium, 36~48h is cultivated in 20~35 DEG C, 150~300r/min, adds again
Host strain, and add in indoles continues 20~28h of culture, culture solution after 4 DEG C, 6000~8000r/min centrifuge 10~15min,
The supernatant containing Bdellovibrio nectophore is removed, the sterile purified water suction of 10~30 ‰ salinity of precipitation use quality volume ratio, which is beaten, hangs
It is floating, its concentration is adjusted to 1011PFU/mL is ocean bdellovibrio bdelloplast bacterial microorganism formulation.
7. bdellovibrio bdelloplast bacterial microorganism formulation in ocean according to claim 6, it is characterised in that：
The Bdellovibrio is Bdellovibrio (Bdellovibrio sp.) BDE-1, and Guangzhou is preserved on November 27th, 2017
Guangdong Province's Culture Collection of 5 building, the building of compound the 59th of martyr Road 100 Guangdong Microbes Inst, preservation are compiled
Number：GDMCC NO：60292.
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Citations (1)
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103320345A (en) * | 2010-08-31 | 2013-09-25 | 华南理工大学 | Bdellovibrio bacteriovorus preparation, and fermentation method and applications thereof |
Non-Patent Citations (1)
| Title |
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| DWIDAR M等: "Indole negatively impacts predation by Bdellovibrio bacteriovorus and its release from the bdelloplast", 《ENVIRON MICROBIOL》 * |
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