CN106544296B - Promote the growth of Gambia algae, photosynthesis and microorganism, method and the kit of secreting ciguatoxin - Google Patents
Promote the growth of Gambia algae, photosynthesis and microorganism, method and the kit of secreting ciguatoxin Download PDFInfo
- Publication number
- CN106544296B CN106544296B CN201610920840.9A CN201610920840A CN106544296B CN 106544296 B CN106544296 B CN 106544296B CN 201610920840 A CN201610920840 A CN 201610920840A CN 106544296 B CN106544296 B CN 106544296B
- Authority
- CN
- China
- Prior art keywords
- algae
- gambia
- preparation
- ciguatoxin
- microorganism
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/075—Bacillus thuringiensis
-
- 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
- C12N1/205—Bacterial isolates
-
- 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
- C12N1/12—Unicellular algae; Culture media therefor
-
- 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
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/06—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using actinomycetales
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
Abstract
The invention proposes a kind of promotion Gambia algae growth, photosynthesis and microorganism, method and the kits of secreting ciguatoxin.Microorganism is bacillus thuringiensis (Bacillus thuringiensis sp.), and Guangdong Province's Culture Collection was preserved on September 5th, 2016, and deposit number is GDMCC No.60074.Microorganism of the invention can not only increase the biomass and photosynthetic efficiency of Gambia algae, moreover it is possible to improve the secretory volume of ciguatoxin, obtain new method for the source of ciguatoxin standard items, facilitate the scrutiny of the toxin.
Description
Technical field
The present invention relates to biological fields.In particular it relates to promote the growth of Gambia algae, photosynthesis and secretion snow
Microorganism, method and the kit of card toxin.
Background technique
It avenges card ichthyotoxin (Ciguatera fish poisoning, CFP), also known as ciguatoxin, is a kind of serious harm people
The algae toxin of class health is mainly generated by Di Qi Gambia algae (Gambierdiscus sp.).The toxin is tired with food chain
Product effect, toxic Gambia algae are enriched in fish body after fish, and the flesh of fish flows to dining table through food chain and finally threatens
Human health.With the expansion of global fishing trade scale, poisoning increases year by year, and edible ciguatoxin is easy to cause nerve
The dysfunction of system, cardiovascular system and gastrointestinal system, it is extremely disadvantageous to the health of human body.In addition ciguatoxin has rouge
Dissolubility feature, is difficult metabolite clearance in vivo, so treatment is extremely difficult.Currently, detection and prevention and control to ciguatoxin at
The hot spot of global concern, there is an urgent need to establish ciguatoxin detection method and efficient treatment means to ensure that food and life are pacified
Entirely.
Currently, restricting, ciguatoxin is quickly detected and the maximum bottleneck of Depth Study is the shortage of ciguatoxin standard items.
Because it is reference that the formulation of food sanitation standard, medicinal specification and detection method, which is dependent on standard items,.Instantly, ciguatoxin standard items
Source is mostly derived from the high toxicity fish in nature production contaminated area domain and extracts toxin, but the toxin amount that this mode obtains has very much
Limit, and expensive (the conservative market price is about 20,000-35,000 RMB/μ g), are unable to satisfy the need of world market
It asks.The whole world only has a few experiments room and possesses the standard items in terms of microgram.
As main producers-Gambia algae (Gambierdiscus sp.) of ciguatoxin, how from Gambia
The ciguatoxin that high yield is obtained in algae still requires study.
Summary of the invention
The present invention is directed to solve one of the technical problems existing in the prior art at least to a certain extent.
It should be noted that the present invention is the following discovery based on inventor and completes:
Inventor carries out different snow card toxicity intensity areas the study found that there are Gambia algaes in the region of high-intensitive toxicity
Symbiotic microorganism, the symbiotic microorganism can promote the growth of Gambia algae, photosynthesis and produce ciguatoxin.Further,
By carrying out screening discovery to symbiotic microorganism, bacillus thuringiensis is for the growth of Gambia algae, photosynthesis and production
Ciguatoxin plays facilitation.
For this purpose, in one aspect of the invention, the invention proposes a kind of microorganisms.According to an embodiment of the invention, institute
Stating microorganism is bacillus thuringiensis (Bacillus thuringiensis sp.), is preserved in Guangdong on September 5th, 2016
Culture Collection is saved, deposit number is GDMCC No.60074.Microorganism of the invention can not only increase ridge ratio
The biomass and photosynthetic efficiency of sub- algae, moreover it is possible to improve the secretory volume of ciguatoxin, be obtained for the source of ciguatoxin standard items
New method facilitates the scrutiny of the toxin.
In another aspect of this invention, the invention proposes a kind of preparations.According to an embodiment of the invention, in the preparation
Metabolite containing microorganism described above.Preparation of the invention can promote Gambia algae growth, photosynthesis and
Secrete at least one of ciguatoxin.
According to an embodiment of the invention, the preparation can also have following additional technical feature:
According to an embodiment of the invention, the metabolite obtains in the following manner: the microorganism is carried out
Activation culture obtains activation products；The activation products are subjected to the first fermented and cultured, to obtain the metabolite；Or
Obtained first tunning of first fermented and cultured is handled, to obtain the metabolite.Basis as a result,
The preparation of the embodiment of the present invention can promote at least one of the growth of Gambia algae, photosynthesis and secretion ciguatoxin.
According to an embodiment of the invention, the temperature of first fermented and cultured is 28~35 DEG C.It is according to the present invention another
A embodiment, the time of first fermented and cultured are 24~36h.According to still another embodiment of the invention, first hair
Ferment culture is that rotation shake culture is carried out with the radius of turn of the revolving speed of 200~220rpm, 13.5cm.It is according to the present invention again
One embodiment, the culture medium of first fermented and cultured are LB liquid medium.Preparation according to an embodiment of the present invention as a result,
It can promote at least one of the growth of Gambia algae, photosynthesis and secretion ciguatoxin.
According to an embodiment of the invention, the processing includes: to be centrifuged first tunning, supernatant is collected
Liquid, to obtain the metabolite；Or first tunning is subjected to ultrasonication, it is produced to obtain the metabolism
Object.Preparation according to an embodiment of the present invention can promote the growth of Gambia algae, photosynthesis and secretion ciguatoxin extremely as a result,
It is one of few.
According to an embodiment of the invention, the processing includes: that first tunning is carried out ultrasonication, so as to
To the metabolite.Preparation according to an embodiment of the present invention can promote the growth of Gambia algae, photosynthesis and secretion as a result,
At least one of ciguatoxin.
In still another aspect of the invention, the invention proposes a kind of growth of promotion Gambia algae, photosynthesis and secretion snow
The method of at least one card toxin.According to an embodiment of the invention, the described method includes: the Gambia algae is carried out the second hair
Ferment culture is added described second after a certain period of time, by the metabolite of microorganism described above or preparation described above
In the fermentation liquid of fermented and cultured, continue second fermented and cultured.It can promote according to the method for the embodiment of the present invention as a result,
Into at least one of the growth of Gambia algae, photosynthesis and secretion ciguatoxin.
According to an embodiment of the invention, the temperature of second fermented and cultured is 26 DEG C, intensity of illumination 3800Lux,
12h:12h Dark-light cycle.It can promote the growth of Gambia algae, photosynthesis according to the method for the embodiment of the present invention as a result, and divide
Secrete at least one of ciguatoxin.
According to an embodiment of the invention, the metabolite of the microorganism or the preparation promote the Gambia algae raw
It is long, which comprises when the Gambia algae is carried out second fermented and cultured to logarithmic phase, by the generation of the microorganism
It thanks to product or the preparation is added in the fermentation liquid of second fermented and cultured, continue second fermented and cultured.As a result,
It can promote at least one of the growth of Gambia algae, photosynthesis and secretion ciguatoxin according to the method for the embodiment of the present invention.
According to an embodiment of the invention, after the fermentation liquid is added, the metabolite of the microorganism or the preparation
Final concentration of 5 × 104~5 × 106A cell/mL, more preferable 1 × 106~5 × 106A cell/mL.As a result, according to the present invention
The method of embodiment can promote at least one of the growth of Gambia algae, photosynthesis and secretion ciguatoxin.
According to an embodiment of the invention, the metabolite of the microorganism or the preparation promote the Gambia algae photosynthetic
Effect, which comprises when the Gambia algae is carried out second fermented and cultured to logarithmic phase, by the microorganism
Metabolite or the preparation are added in the fermentation liquid of second fermented and cultured, continue second fermented and cultured.By
This, can promote according to the method for the embodiment of the present invention Gambia algae growth, photosynthesis and secretion ciguatoxin at least it
One.
According to an embodiment of the invention, after the fermentation liquid is added, the metabolite of the microorganism or the preparation
Final concentration of 5 × 104~5 × 106A cell/mL, more preferable 1 × 106~5 × 106A cell/mL.As a result, according to the present invention
The method of embodiment can promote at least one of the growth of Gambia algae, photosynthesis and secretion ciguatoxin.
According to an embodiment of the invention, the metabolite of the microorganism or the preparation promote the Gambia algae secretion
Ciguatoxin, which comprises when the Gambia algae is carried out second fermented and cultured to plateau, by micro- life
The metabolite of object or the preparation are added in the fermentation liquid of second fermented and cultured, continue second fermented and cultured
To 40~55 days.It can promote the growth of Gambia algae, photosynthesis and secretion snow card according to the method for the embodiment of the present invention as a result,
At least one of toxin.
According to an embodiment of the invention, after the fermentation liquid is added, the metabolite of the microorganism or the preparation
Final concentration is not higher than 2 × 106A cell/mL, more preferable 5 × 105~1 × 106A cell/mL.Implement according to the present invention as a result,
The method of example can promote at least one of the growth of Gambia algae, photosynthesis and secretion ciguatoxin.
In still another aspect of the invention, the invention proposes microorganisms described above or preparation to promote Gambia algae
Purposes at least one of growth, photosynthesis and secretion ciguatoxin.
In still another aspect of the invention, the invention proposes a kind of kits.According to an embodiment of the invention, the reagent
Box includes at least one of microorganism described above or preparation described above.As a result, using implementing according to the present invention
The kit of example can promote at least one of the growth of Gambia algae, photosynthesis and secretion ciguatoxin.
In still another aspect of the invention, the invention proposes a kind of kits described above to promote Gambia algae raw
Purposes at least one of long, photosynthesis and secretion ciguatoxin.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Detailed description of the invention
Above-mentioned and/or additional aspect of the invention and advantage will become from the description of the embodiment in conjunction with the following figures
Obviously and it is readily appreciated that, in which:
Fig. 1 shows bacillus thuringiensis according to an embodiment of the invention (Bacillus thuringiensis
Sp.GQS09) the screening of GDMCC No.60074；
Fig. 2 shows the microscope photograph of Gambia algae form according to an embodiment of the invention, wherein left hand view
Amplification factor is 40 times, and the amplification factor of right part of flg is 10 times；
Fig. 3 shows logarithmic phase addition bacillus thuringiensis (Bacillus according to an embodiment of the invention
Thuringiensis sp.GQS09) GDMCC No.60074 influence that Gambia algae is grown；
Fig. 4 shows logarithmic phase addition bacillus thuringiensis (Bacillus according to an embodiment of the invention
Thuringiensis sp.GQS09) GDMCC No.60074 is on the photosynthetic influence of Gambia algae；
Fig. 5 shows plateau addition bacillus thuringiensis (Bacillus according to an embodiment of the invention
Thuringiensis sp.GQS09) GDMCC No.60074 influence that Gambia algae is grown；And
Fig. 6 shows bacillus thuringiensis according to an embodiment of the invention (Bacillus thuringiensis
Sp.GQS09) influence of the addition of GDMCC No.60074 to ciguatoxin yield.
Specific embodiment
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair
It is bright, and be not considered as limiting the invention.
It should be noted that term " first ", " second " are used for description purposes only, it is not understood to indicate or imply phase
To importance or implicitly indicate the quantity of indicated technical characteristic.Define " first " as a result, the feature of " second " can be with
Explicitly or implicitly include one or more of the features.Further, in the description of the present invention, unless otherwise saying
Bright, the meaning of " plurality " is two or more.
The invention proposes a kind of microorganism, preparation, microorganism or preparation promote Gambia algae growth, photosynthesis and
Secrete the purposes at least one of ciguatoxin, promote the growth of Gambia algae, photosynthesis and secretion ciguatoxin at least it
One method, kit and its use at least one of the growth of promotion Gambia algae, photosynthesis and secretion ciguatoxin
On the way, it will be described in greater detail respectively below.
Microorganism
In one aspect of the invention, the invention proposes a kind of microorganisms.The microorganism is bacillus thuringiensis
(Bacillus thuringiensis sp.) was preserved in Guangdong Province's Culture Collection, ground on September 5th, 2016
Location are as follows: 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, postcode: 510075, deposit number is GDMCC No.60074.
Inventor carries out different snow card toxicity intensity areas the study found that there are Gambia algaes in the region of high-intensitive toxicity
Symbiotic microorganism, the symbiotic microorganism can promote the growth of Gambia algae, photosynthesis and produce ciguatoxin.
Research in recent years has shown that, there are information interchange between bacterium, many bacteriums can synthesize and discharge a kind of claimed
For the signaling molecule of self-induction substance (autoinducer, AI), extracellular AI concentration can increase with the increase of bacterial density,
When reaching a critical concentration, AI can start the expression of related gene in thallus, regulate and control the biobehavioral of bacterium.Such as generate poison
Element, formation biomembrane, generation antibiotic, generation spore, generation fluorescence etc., to adapt to the variation of environment, this phenomenon is known as group
Body-sensing should adjust (quorum sensing.QS).This induction only can just be sent out after bacterial density reaches certain threshold value
It is raw, so this phenomenon to be also known as to gene expression (the cell density dependent control of cell density dependence
of gene expression)。
Research confirms that the embodiment of microbial ecological effect depends on certain quantity and ecological niche (such as biofilm
Biofilm), and Microflora and Biofilm by colony induction signaling (quorum sensing, QS) adjusting.It is total in phycomycete
In raw body, the embodiment of microbial ecological function relies on certain group structure and quantity, and population density is adjusted by QS signal.
In view of this, inventor by screening the study found that can generate the thallus of QS signal, then by itself and ridge
Than sub- algae symbiosis culture, further screening can promote the growth of Gambia algae, photosynthesis and the bacterial strain for secreting ciguatoxin.Into
And inventor is promoted the growth of Gambia algae, photosynthesis and the fungal component-Soviet Union for secreting ciguatoxin by many experiments
Cloud gold bacillus, the bacterium is by generating QS signal, so that population density is adjusted, a large amount of metabolites effect for generating thallus
In Gambia algae, to promote the growth of Gambia algae, photosynthesis and secretion ciguatoxin.
Preparation
In another aspect of this invention, the invention proposes a kind of preparations.According to an embodiment of the invention, containing in preparation
The metabolite of previously described microorganism.Inventors have found that microorganism described above is promoted by its metabolite
Growth, photosynthesis and/or the production ciguatoxin of Gambia algae.Preparation according to an embodiment of the present invention can promote ridge as a result,
Than the growth of sub- algae, photosynthesis and/or produce ciguatoxin.
According to an embodiment of the invention, metabolite obtains in the following manner: microorganism is subjected to activation culture,
Obtain activation products；Activation products are subjected to the first fermented and cultured, to obtain metabolite；Or it will be obtained by the first fermented and cultured
To the first tunning handled, to obtain metabolite.Inventors have found that promoting the growth, photosynthetic of Gambia algae
Effect and/or the substance for producing ciguatoxin are obtained by fermentation.As a result, by carrying out fermentation process to microorganism, thus
Metabolite needed for obtaining.
According to an embodiment of the invention, the temperature of the first fermented and cultured is 28~35 DEG C, preferably 30 DEG C.It is according to the present invention
Another embodiment, the time of the first fermented and cultured are 24~36h.According to still another embodiment of the invention, the first fermentation training
Support is that rotation shake culture, preferably 220rpm are carried out with the radius of turn of the revolving speed of 200~220rpm, 13.5cm.According to this
Another embodiment of invention, the culture medium of the first fermented and cultured are LB liquid medium.Inventors have found that fermentation culture conditions
Significantly affect growth, photosynthesis and the secretion ciguatoxin ability of subsequent Gambia algae.Further, inventor is through excessive
Amount experiment obtains above-mentioned optimal fermentation condition, can obtain the growth, the photosynthesis that largely promote Gambia algae with this condition
And the metabolite of secretion ciguatoxin.Under other fermentation conditions, bacillus thuringiensis can not generate or generate on a small quantity
Metabolite, or activity is lower, leads to that the biomass of Gambia algae is lower, photosynthetic efficiency is lower and/or secretion
Ciguatoxin amount is less.
According to an embodiment of the invention, processing includes: to be centrifuged the first tunning, supernatant is collected, so as to
To metabolite；Or the first tunning is subjected to ultrasonication, to obtain the metabolite.
Preferred embodiment in accordance with the present invention, processing include: that first tunning is carried out ultrasonication, so as to
To the metabolite.Microorganism according to an embodiment of the present invention can promote the growth of Gambia algae, photosynthesis and divide as a result,
Secrete at least one of ciguatoxin.
Inventors have found that metabolite can be obtained by the way that tunning to be centrifuged to and collected supernatant, but for ridge
Facilitation than the growth of sub- algae, photosynthesis and secretion ciguatoxin is relatively low.It is found by further investigation, may advantageously facilitate ridge
Metabolite than the growth of sub- algae, photosynthesis and secretion ciguatoxin is most probably not only present in extracellularly, is also present in thin
It is intracellular.In turn, by the way that tunning is carried out cell ultrasonication, while obtaining Extracellular metabolism, additionally it is possible to release
Metabolite intracellular keeps it preferable to the facilitation of the growth of Gambia algae, photosynthesis and secretion ciguatoxin.
A method of promoting at least one the growth of Gambia algae, photosynthesis and secretion ciguatoxin
In still another aspect of the invention, the invention proposes a kind of growth of promotion Gambia algae, photosynthesis and secretion snow
The method of at least one card toxin.According to an embodiment of the invention, this method comprises: Gambia algae is carried out the second fermented and cultured
After a certain period of time, the second fermented and cultured is added in the metabolite of microorganism described above or preparation described above
In fermentation liquid, continue the second fermented and cultured.
According to an embodiment of the invention, the temperature of the second fermented and cultured is 26 DEG C, intensity of illumination 3800Lux, 12h:12h
Dark-light cycle.Inventor obtains above-mentioned optimal fermentation condition by many experiments, with this condition the growth of Gambia algae, photosynthetic work
With and secretion ciguatoxin effect it is preferable.
According to an embodiment of the invention, microorganism metabolite or preparation promote Gambia algae growth, method include: by
When Gambia algae carries out the second fermented and cultured to logarithmic phase, the second fermented and cultured is added in the metabolite of microorganism or preparation
In fermentation liquid.
Inventor it was unexpectedly observed that the time that metabolite or preparation is added significantly affect the growth of Gambia algae.Work as ridge
When fermenting than sub- algae to logarithmic phase (such as cell quantity is no more than 1000 cell/mL), metabolite or preparation is added, continues
After fermentation, the growth of Gambia algae can effectively promote.In contrast to this, when Gambia algae was fermented to plateau, metabolism is added
Product or preparation, after continuing fermentation, the yield of Gambia algae is relatively relatively low.
According to an embodiment of the invention, after fermentation liquid is added, the metabolite of microorganism or preparation final concentration of 5 ×
104~5 × 106A cell/mL, more preferable 1 × 106~5 × 106A cell/mL.Inventors have found that additional amount significantly affects ridge
Than the yield of sub- algae.Specifically, when additional amount is 5 × 104~5 × 105It is limited to the promotion of algae when a cell/mL, fail to compare
According to being significantly improved；And works as and be increased to 1 × 106~5 × 106The growth of Gambia algae is promoted significantly, still when a cell/mL
When additional amount is higher than 5 × 106A cell/mL, then will appear inhibiting effect, and the yield of Gambia algae is caused to reduce.
It should be noted that term " final concentration " used in the present invention refer to based on be added microorganism metabolite or
The total volume of preparation post-fermentation liquid, the metabolite of microorganism or the cell concentration of preparation.
It should also be noted that, for through the obtained metabolite of ultrasonication, term " cell used in the present invention
Concentration " is mainly for the cell number before broken.
According to an embodiment of the invention, the metabolite or preparation of microorganism promote Gambia algae photosynthesis, method packet
It includes: when Gambia algae is carried out the second fermented and cultured to logarithmic phase, the second fermentation is added in the metabolite of microorganism or preparation
In the fermentation liquid of culture, continue second fermented and cultured.
Inventor it was unexpectedly observed that the time that metabolite or preparation is added significantly affect the photosynthesis of Gambia algae.
When Gambia algae is fermented to logarithmic phase (such as cell quantity is no more than 1000 cell/mL), metabolite or preparation is added,
After continuing fermentation, Gambia's algae photosynthesis can effectively promote.In contrast to this, when Gambia algae was fermented to plateau,
Metabolite or preparation is added, after continuing fermentation, photosynthetic efficiency is relatively relatively low.
According to an embodiment of the invention, after fermentation liquid is added, the metabolite of microorganism or preparation final concentration of 5 ×
104~5 × 106A cell/mL, more preferable 1 × 106~5 × 106A cell/mL.Inventors have found that additional amount significantly affects ridge
Than the photosynthesis of sub- algae.Specifically, when additional amount is 5 × 104~5 × 106A cell/mL, to the photosynthesis of Gambia algae
There is the aobvious effect of different degrees of promotion, when additional amount is 1 × 106~5 × 106A cell/mL, to the photosynthesis of Gambia algae
Promote significantly, but when additional amount is higher than 5 × 106A cell/mL, then will appear inhibiting effect, lead to the photosynthetic of Gambia algae
Effect reduces.
According to an embodiment of the invention, the metabolite or preparation of microorganism promote Gambia algae to secrete ciguatoxin, side
Method includes: that the metabolite of microorganism or preparation are added second when Gambia algae is carried out the second fermented and cultured to plateau
In the fermentation liquid of fermented and cultured, continue second fermented and cultured to 40~55 days.
Inventor blocks poison it was unexpectedly observed that the time of addition metabolite or preparation significantly affects Gambia algae secretion snow
Element.When Gambia algae was fermented to plateau (such as 6000-8000 cell/mL of cell quantity), metabolite or system is added
Agent after continuing fermentation, can effectively promote Gambia algae secretion ciguatoxin, and obtained ciguatoxin yield is higher.With
This is compared, and when Gambia algae is fermented to logarithmic phase, metabolite or preparation, after continuing fermentation, the yield of ciguatoxin is added
It is relatively relatively low.
According to an embodiment of the invention, the metabolite of microorganism or the final concentration of preparation are not higher than 2 after fermentation liquid is added
×106A cell/mL, more preferable 5 × 105~1 × 106A cell/mL.Inventors have found that additional amount significantly affects ciguatoxin
Yield.Specifically, when additional amount is not higher than 2 × 106A cell/mL promotes significantly Gambia algae secretion ciguatoxin, but
It is when additional amount is higher than 2 × 106A cell/mL, then will appear inhibiting effect, lead to the reduction of ciguatoxin yield.
It will be appreciated to those of skill in the art that above for feature and advantage described in microorganism and preparation, together
The method that sample is suitable for promoting at least one the growth of Gambia algae, photosynthesis and secretion ciguatoxin, details are not described herein.
Microorganism or preparation are promoting the use at least one of the growth of Gambia algae, photosynthesis and secretion ciguatoxin
On the way
In still another aspect of the invention, the invention proposes a kind of microorganisms described above or preparation to promote ridge ratio
Purposes at least one of sub- algae growth, photosynthesis and secretion ciguatoxin.
It will be appreciated to those of skill in the art that above for feature and advantage described in microorganism and preparation, together
Sample is suitable for the microorganism or preparation during promoting at least one of the growth of Gambia algae, photosynthesis and secretion ciguatoxin
Purposes, details are not described herein.
Kit
In still another aspect of the invention, the invention proposes a kind of kits.According to an embodiment of the invention, the kit
At least one including microorganism described above or preparation.
It will be appreciated to those of skill in the art that above for feature and advantage described in microorganism and preparation, together
Sample is suitable for the kit, and details are not described herein.
Kit is promoting the purposes at least one of the growth of Gambia algae, photosynthesis and secretion ciguatoxin
In still another aspect of the invention, the invention proposes a kind of kits described above to promote Gambia algae raw
Purposes at least one of long, photosynthesis and secretion ciguatoxin.
It will be appreciated to those of skill in the art that above for feature and advantage described in kit, it is equally applicable
In the purposes, details are not described herein.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment
Part, it described technology or conditions or is carried out according to the literature in the art according to product description.Agents useful for same or instrument
Production firm person is not specified in device, and being can be with conventional products that are commercially available.
Rhizobium reporting bacterial strain (A136) is given by Chinese Academy of Science Nanhai Ocean Research Institute Wang Xiaoxue researcher.
Gambia's algae algae (Gambierdiscus 1022M2C12, M2) is by marine pollution country, City University of Hong Kong weight
Point laboratory separates and give.
LB solid medium: peptone 1g, yeast extract 0.5g, sodium chloride 1g, deionized water 100ml, agar 1.5g,
pH7.0。
LB liquid medium: peptone 1g, yeast extract 0.5g, sodium chloride 1g, deionized water 100ml, pH7.0.
K medium algae culturing liquid: (75 grams of sodium nitrate solids are dissolved in 1 liter of distilled water sodium nitrate solution 1.0ml, are expressed as
75g/L ddH20)；Ammonium chloride, 1.0ml (2.68g/L ddH2O)；Sodium β-glycerophosphate, 1.0ml (2.16g/L ddH2O)；Nine
Water sodium metasilicate, 1.0ml (30.0g/L ddH2O)；Selenous acid, 1.0ml (1.29g/L ddH2O)；Tris-Base (pH 7.2),
1.0ml(121.1g/L ddH2O)；K Trace Metal solution, 1.0ml；F/2 vitamin solution, 0.5ml；Filter Chen Haishui, constant volume
To 1L；121 DEG C high pressure steam sterilization 20 minutes after mixing.
Zobell 2216E culture medium: yeast extract Yeast Extract, 5.0g；Tryptone Tryptone,
1.0g；Ferric trichloride FeCl3,0.01g；Chen Haishui seawater, 1.0L；Technology agar powder, 15g (solid medium)；It mixes
115 DEG C high pressure steam sterilization 20 minutes afterwards.
The separation and identification of 1 bacterial strain of embodiment
One, the separation of bacterial strain
The selection triumphant reef atoll of Republic of Kiribati's horse traction (Marakei) four different sampled points of toxicity are sampled, and are wrapped
Two strong snow card virulence region M1 (2 ° of 01.394'N, 173 ° of 15.383'E) and M2 (1 ° of 59.879'N, 173 ° of 15.032'E) are included,
And two weak snow card virulence region M3 (1 ° of 58.246'N, 173 ° of 16.268'E) and M4 (2 ° of 00.150'N, 173 ° of 18.010'
E).Sample is collected from the region that the Gambia such as large-scale algae, coral, seaweed algae easily adheres to, is separately cultured for algae and is sieved with bacterium
Choosing.(Shenzhen Graduate School of Tsinghua University marine ecology laboratory) sterile working after this Preservation in sterile condition takes back laboratory is sampled to apply
It is distributed in 12cm medium size solid medium tablets containing 2216E, 30 DEG C of overnight stand cultures, until growing high-visible Dan Ke
It is grand.
Then from about 200 plants of monoclonals of picking on culture plate in 2ml EP pipe, the Liquid Culture of LB containing 1ml in EP pipe
This 200 plants of monoclonals are put into constant-temperature table 30 DEG C, 220r/min overnight incubation by base.The screening of QS signal, tool are carried out respectively
Body process is as follows:
Primary dcreening operation: by A136 reporting bacterial strain (A.tumefaciens 136) and 200 plants of monoclonal bacterial strains to be measured in 96 orifice plates
It is mixed according to 1 ︰, 1 ratio, 30 DEG C of shake culture 6h, X-GAL (the 20 μ g mL of 5 μ L are added dropwise in every hole–1), 30 DEG C are protected from light culture 3h, if
Bacterium solution becomes blue, then is possible QS bacterial strain.
Secondary screening: configuration 100mL semisolid LB culture medium, after high pressure steam sterilization, to be cooled to 45 DEG C or so (attention is kept away
Exempt from agar to be condensed into blocks), 1mL A136 bacterial strain is accessed semisolid culturemedium by sterile working, and the nothing of 90mm diameter is poured into after shaking up
Bacterium plate.After cooling, using 1.5cm as interval, the aseptic filter paper piece that radius is 0.3cm is spread in plate to culture medium.With
Pipettor is added drop-wise to the 5 μ L of bacterium solution for the possible QS bacterial strain that primary dcreening operation obtains on filter paper, 30 DEG C of culture 6h.In each filter paper
Upper X-GAL (the 20 μ g mL that 5 μ L are added dropwise–1), 30 DEG C be protected from light culture 3h after observe as a result, strain to be tested lawn periphery generate blue
Its generation AHL signal (one of QS signal) of the explanation of phenomenon.
Experimental result is as shown in Figure 1, in the 200 plants of bacterial strains screened, about 10% QS positive strain, Wo Mencong
In to pick one plant of coloration ability stronger, i.e. GQS09 carries out subsequent experimental.
Two, the identification of bacterial strain GQS09 bacterium
The bacterial strain GQS09 that step 1 obtains is identified in terms of form and 16s rDNA sequence homology two.
1, Morphological Identification
It will be in logarithmic growth phase, and bacterium colony size is stablized, the isolated bacterial strain GQS09 of above-mentioned steps one carries out single bacterium
Fall state description, the main size including bacterium colony, color, transparency, wettability, bacterium colony surface state (whether flat, protrusion,
Fold, recess etc.), colony edge state (whether neat, irregular, radial etc.).
The result shows that: the isolated bacterial strain of above-mentioned steps one is Gram-negative bacteria, and for bacterium colony at faint yellow, surface is wet
Profit, edge are flat.
2,16s rDNA sequence homology analysis
The isolated bacterial strain GQS09 of conventional method culture above-mentioned steps one, extracts the total DNA of bacterial strain as gene magnification
Template, using universal primer 27f:5 '-AGAGTTTGATCCTGGCTCAG-3 ', 1492r:5 '-
TACGGTTACCTTGTTACGACTT-3 ' amplification bacterial 16 S rRNA gene conserved region.25 μ L amplification systems include: 1 × PCR anti-
Answer buffer, 200 μm of ol/L dNTPs, upstream and each 0.2 μm of ol/L of downstream primer, 1U Taq archaeal dna polymerase, 1 μ L template
DNA.Reaction condition: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 40s, 72 DEG C of extension 30s, totally 25 recycle；72
DEG C extend 10min.1% detected through gel electrophoresis of PCR product, positive findings 27f:5 '-AGAGTTTGATCCTGGCTCAG-
3 ', 1492r:5 '-TACGGTTACCTTGTTACGACTT-3 ' carry out bidirectional sequencing.Sequence assembly and similarity analysis use
DNAStar software complete, sequence alignment by National Center for Biotechnology Information ncbi database (http: //
Www.ncbi.nlm.nih.gov) online to complete.
See Table 1 for details for the sequence of the 16s rDNA of bacterial strain GQS09.
1 nucleic acid sequence table of table
In view of above-mentioned morphological feature and 16s rDNA sequence homology analysis as a result, the bacterium that step 1 separation screening is obtained
Strain GQS09 is accredited as bacillus thuringiensis (Bacillus thuringiensis serovar konkukian).The Su Yun
Golden bacillus (Bacillus thuringiensis serovar konkukian) has been preserved on September 5th, 2016 extensively
East saves Culture Collection, address are as follows: 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, postcode: and 510075, it protects
Hiding number is GDMCC No.60074.
Embodiment 2, bacillus thuringiensis (Bacillus thuringiensis serovar konkukian) GDMCC
No.60074 promotes the growth of Gambia algae and photosynthetic detection
1, the preparation of algae is tested
According to the source of separating obtained Gambia's algae algae and toxicity, the stronger Gambia algae of production ciguatoxin has been selected
M2 is studied bacillus thuringiensis (Bacillus thuringiensis serovar konkukian) as experimental subjects
The facilitation that GDMCC No.60074 grows Gambia algae.
The culture of algae: the growing state of 40 times of each Kong Zhong Gambia algaes of 96 orifice plate of optical microphotograph sem observation is used.It will be at
Function breeding and the good Gambia algae of upgrowth situation, which are transferred in the fresh K medium of 10ml, expands culture.It, will after culture 10 days
5ml Gambia algae is transferred to the healthy and free from worry 25cm equipped with the fresh K medium of 20ml2It is expanded culture in ventilative Tissue Culture Flask,
Frustule concentration is counted every three days primary.
Method of counting is as follows: jog Tissue Culture Flask is evenly distributed on Gambia algae in algae solution, and pipettor draws 100 μ L
Algae solution is laid on 20 × 20mm phytoplankton tally, and counter is utilized at 16 FA of conventional optical microscope Leica MA
Gambia algae is counted.Algae form and growth conditions are as shown in Figure 2.It can be seen that frustule form is complete, color is equal
It is even, Individual Size is consistent, embody preferable physiological status.
When frustule concentration is more than 2000cell/ml, 20ml Gambia algae algae solution is transferred to fresh equipped with 80ml
The healthy and free from worry 75cm of Kmedium2Ventilative Tissue Culture Flask expands culture culture 72 hours, finally all shifts the bacterium solution after culture
To the healthy and free from worry 225cm that the fresh K medium of 400ml is housed2Gas-permeable flasks in cultivated, Gambia's algae cultivating system
For follow-up test.
2, bacterium adds experiment-growth
Tunning acquisition pattern:
1) fermented and cultured stimulation bacterium (bacillus thuringiensis), cultivating system are 500mL conical flask, and liquid amount is
200mL, cultivation temperature are 30 degree, and revolving speed is 220 revs/min.
2) bacterium solution is ultrasonically treated 5 minutes in cell crushing instrument under condition of ice bath, obtains tunning.
According to experimental design, stimulation test is divided into two parts, respectively early stage stimulation test and mid-term stimulation test.It is early
In phase stimulation test, tunning be added time be Gambia algae growth logarithmic phase (cell concentration is no more than
1000cells/mL)；In mid-term stimulation test, the time of addition be the plateau of Gambia algae (cell concentration be 6000~
8000cells/mL), at this point, frustule concentration kept stable.Irritaiting concentration is arranged in microorganism early stage stimulation test
To be three groups basic, normal, high, wherein the final concentration for adding bacterium in each concentration stimulation group is respectively 5 × 104Cells/mL, 5 ×
105Cells/mL and 5 × 106cells/mL。
3, bacterium adds experiment-photosynthesis
Experiment condition is the same as above-mentioned steps 2.
The measurement of chlorophyll content using chlorophyll fluorescence analyzer Phytoplankton Analyzer (PHYTO-PAM,
Walz it) carries out.Specific step is as follows:
It is careful to shake algae solution, 3ml algae solution is shifted rapidly into glass cuvette, is placed in PHYTO-PAM.It opens
PHYTO-PAM software kit records chlorophyll parameter in Report-window, obtains photosynthetic data.
It is as shown in Figure 3 that logarithmic phase adds influence result of the bacterium to algal grown.It can be seen from the figure that bacterium is low dense
Degree group 5 × 104Cells/mL and middle concentration group 5 × 105Cells/mL, it is limited to the growth-promoting effect of algae, fail to show
It has a clear superiority than control group.And when concentration increases to 5 × 106When cells/mL, then apparent facilitation is shown, this table
It is bright when stimulating algal grown, the most suitable additive amount of bacterium need to guarantee 106The cells/mL order of magnitude, when concentration be 1~5 ×
106It is best when cells/mL.
It is as shown in Figure 4 that logarithmic phase adds influence result of the bacterium to algae photosynthesis.It can be seen from the figure that bacterium exists
Low concentration group 5 × 104Cells/mL and middle concentration group 5 × 105Cells/mL improved 1.7 Hes than control group respectively at 27 days
2.1 times (P < 0.05).When bacterial concentration is increased to 5 × 106Cells/mL becomes apparent photosynthetic facilitation, from
Start within 17 days to be more than comprehensively control group, continue up to the 52nd day, and peak is 4.2 times of control group.
4, bacterium addition experiment and the preparation (mid-term stimulation) of stimulant
In mid-term stimulation test, high concentration group, double high concentration group and four times of high concentration groups are set by irritaiting concentration.
Final concentration after stimulating bacterium that Gambia's algae culturing liquid is added is respectively 5 × 105cells/mL、1×106Cells/mL and 2 ×
106cells/mL.Three kinds of different microorganism stimulants are devised in mid-term stimulation test:
The first is inoculum living, preparation method:
1) 10 μ L bacillus thuringiensis are inoculated in 1ml LB culture medium, temperature be 30 degrees Celsius, revolving speed 220
It is activated overnight under conditions of rev/min.
2) using 50ml liquid 2216E expand culture activation after bacillus thuringiensis, condition of culture be 30 degrees Celsius,
Revolving speed is 220 revs/min, obtains inoculum living.
Second be bacterial fermentation object, preparation method:
Inoculum living obtained above is ultrasonically treated 5 minutes in cell crushing instrument under condition of ice bath, is obtained
Bacterial fermentation object.
The third is inoculum supernatant, preparation method:
Inoculum living obtained above is centrifuged 20 minutes in 12000rpm, makes bacterial precipitation, the supernatant of collection
For inoculum supernatant.
Frustule method of counting
1) Tissue Culture Flask is carefully shaken, adherent Gambia algae is made to be detached from and be uniformly distributed.
2) 100 μ L algae solutions are drawn with pipettor rapidly, be laid on self-control circular groove glass slide.
3) it takes pictures under stereoscopic dissecting microscope to algae solution, obtains jpeg format photo, repeat three times.
4) all photos are converted into no compressed version tiff format using picture format switching software.
5) Bio-Rad Quantity one software is opened, tiff format photo is loaded into.
6) Analysis-Colony counting is clicked, sets 8.0 for Sensitivity, count is clicked and is counted
Number.
7) it calculates and records every milliliter of frustule concentration, repeat three times.
8) growth curve of Gambia algae is drawn.
Bacterium mid-term stimulation test result is as shown in Figure 5, it can be seen that (the ridge ratio at this time at Gambia's algae growth regulation 15 days
Sub- algae has just enter into the growth platform phase) stimulant is added, growth curve breaks up in two days, and different stimulants has not
Same effect.
For adding inoculum living, control group (refers to the ridge that any bacterium, bacterium solution or extra nutritional is not added
Than sub- algae) algae cell density concentration maintains 3000cells/mL, add high concentration bacterium (5 × 105Algae is thin when cells/mL)
Born of the same parents' density can be improved 6500cells/mL, and double high concentration (1 × 106Cells/mL) and four times of high concentrations (2 ×
106Cells/mL) there is certain inhibiting effect to the growth of frustule, algae cell density is down to 800~1200cells/mL's
It is horizontal.This explanation needs to control the additive amount of bacterium, no more than 5 × 10 to obtain a large amount of Gambia algaes5cells/mL。
For adding inoculum supernatant, addition concentration is respectively 5 × 105Cells/mL and 2 × 106cells/mL
When, after culture 32 days, algae cell density rises to 8200 and 7500cells/mL level respectively, with control group
(3000cells/mL) is compared and 2.07 and 1.5 times (Fig. 5) has been respectively increased.
For adding bacterial fermentation object, it will also be seen that the stimulation of frustule, addition concentration is respectively 5 ×
105Cells/mL and 2 × 106When cells/mL, algae cell density is twice of control group up to 6000cells/mL or more；Most
For it is apparent that addition 1 × 106Cells/mL dosage group, when cultivating 32 days, frustule concentration can rise to 11000cell/mL,
It is 3.67 times of control group.
In order to eliminate interference of the nutrients to experiment, the present invention is provided with nutritive salt group, that is, adds the K of 100 times of concentration
Medium algae culturing liquid is in algae solution.Nutritive salt group frustule quantity after culture in 25 days reaches 4100cells/mL, for control
Twice of group.However, nutritive salt group is still limited to the growth promotion of frustule, only bacterium supernatant compared with bacterium additive
(2×106) and bacterium tunning (1 × 10 cells/mL6Cells/mL 40-50%).It is main to show that the growth of frustule promotes
It is derived from the effect of bacterium and its product.
Embodiment 3, bacillus thuringiensis (Bacillus thuringiensis serovar konkukian) GDMCC
No.60074 increases the detection of ciguatoxin secretion
1. the extraction of ciguatoxin
1) algae solution is filtered using 0.22 μm of water phase miillpore filter, filter membrane is transferred in 50ml centrifuge tube.
2) 25ml anhydrous methanol is added into centrifuge tube, rocks so that filter membrane sufficiently immerses.
3) centrifuge tube is placed in ultrasonication instrument under condition of ice bath, preparatory rinse probe.
4) setting Pulse is to open for 5 seconds, is closed within 1 second, power 40%, every pipe is ultrasonically treated 5 minutes.
5) sample after ultrasonic treatment is transferred in ultracentrifugation pipe, 12000rpm is centrifuged 10 minutes.
6) 25ml anhydrous methanol piping and druming precipitating is added into centrifuge tube into 250ml round-bottomed flask for transfer supernatant.
7) centrifuge tube is placed in ultrasonic water bath pot, is ultrasonically treated 30 minutes.
8) step 5-7 is repeated twice.
9) by the supernatant rotary evaporation collected in round-bottomed flask processing, revolving temperature setting is 45 DEG C, revolving speed 120rpm.
10) 30ml anhydrous methanol is added after being evaporated to re-dissolve.
11) solution is transferred in the 250ml separatory funnel of preparatory rinse, 20ml distilled water is added, rocks mixing.
12) 50ml methylene chloride is added into separatory funnel, rocks and is vented 2 minutes repeatedly, stands liquid separation.
13) after layering occurs in liquid, the methylene chloride organic phase of lower layer is collected, 30ml methylene chloride is added and extracts again.
14) after collecting lower layer's methylene chloride organic phase, 20ml methylene chloride third time is added and extracts.
15) lower layer's methylene chloride phase, rotary evaporation processing are collected.Rotating temperature setting is 40 DEG C, revolving speed 120rpm.
16) sample being evaporated is collected using anhydrous methanol, is collected in three times, each 2ml rinse, total 6ml.
17) by 6ml sample loaded in 15ml sterile centrifugation tube, number is recorded.
18) -20 DEG C of cryo-conservations are placed in.
2. ciguatoxin measuring method
Ciguatoxin quantitative determination is carried out based on liquid chromatogram and cell experiment.
1) cell line and basic maintenance
The cell strain system used under study for action is the neuroblastoma cells of mouse, Neuro-2a.It is in U.S.'s allusion quotation
A kind of standard cell system with the number of CCL131 is compiled in type Cell Culture Center (ATCC).By Hong Kong University (Pokfulam
Road, HKSAR, China) Department of Zoology Billy doctor Chow give.Cell routine uses the culture of C-RPMI culture medium, and
Ensure not exceeding the 80% of its totality under monitoring.Contain 5%CO at 37 DEG C2Wet air in cultivate cell.Again
Culture need to use trypsin digestion.Cell monolayer first is rinsed with 10ml PBS buffer solution, adds 3ml trypsase-EDTA, so
It is cultivated at 37 DEG C afterwards.Period detects cell using inverted microscope, until discovery cell detachment and assembles.By to culture bottle
Middle addition 10mlC-RPMI can block trypsin digestion, can add into the new tissue culture flasks for be added 10ml C-RPMI
Enter 1ml resuspension cell.The survival ability of cell is often more than 90% in the incompatible experiment of trypan blue.
2) cell density measures
Cell density can be measured with hemocytometer.PBS is added in ingredient after dilution, cell suspension is made.Mixed liquor is added again
Enter into two rooms of the haemocytometer with the coverslip suitably slided.Calculate the cell number in five grid, then with
Following formula determines last cell concentration:
Average × extension rate × 10 in every ml cell number=each grid4。
3) cell viability measures
The survival ability of cell is detected by the incompatible experiment of trypan blue, and principle is that living cells cannot be through as trypan blue one kind
Dyestuff, and dead cell can pass through.Experiment is by the way that cell suspension (50 μ L) directly (to be added with trypan blue solution in 50 μ L
0.4%PBS buffer) it mixes, then mixed liquor is added in hemocytometer in 30 minutes using optical microphotograph under the microscope.
In more or less a hundred cell, the cell for being dyed to blue can be identified.Cell survival is calculated using following formula:
Total cell number × 100 survivaling cell number (undyed) ÷ of survival rate (%)=total
4) cell viability measurement is assessed by MTT experiment
Cell viability measurement can by Mosmann T and Hansen describe in 96 orifice plates with 3- (4,5- dimethyl
Thiazole -2-y) assessment of -2,5- diphenyltetrazolium bromide bromide (MTT) decoration method.This method, which is based on living cells, to be converted into MTT
The principle of the first a ceremonial jade-ladle, used in libation of blue.MTT solution (5mg/ml) is prepared using PBS.With 96 orifice plate culture cells, using C-RPMI solution as
Control.In culture latter stage, culture medium is sucked out from 96 orifice plates, then 50 μ L are added (by itself and C-RMPI with 1:10 into each hole
It is diluted) MTT solution.After having cultivated 1 hour at 37 DEG C, 100 μ L are added thereto and contain 1M for dark Crystallization
HCl isopropanol with terminate reaction.The OD value (OD) in each hole can be with 550 microplate reader of Bio-Rad with 655nm
It is read at 595nm as reference wavelength.Viable count obtains and with the conversion of the wavelength ratio of control group.Various toxin
Virulence is standardized by the ratio with non-toxic controls group.
5) cellular morphology is observed
Cellular morphology is observed using microscope (20 × object lens, 10 × eyepiece) and is taken pictures.Cellular morphology variation can be detected
It such as cell shrinkage and swells, out film bubble etc..
6) prepared by drug
G-Strophanthin and black false hellebore pyridine are dissolved in the stock solution for being made into 10mM and 1mM in 0.01M HCl respectively.By short brown dinoflagellate poison
Plain titer is dissolved in the stock solution that 10 μ g/ml are made into methanol.
7) mouse neuroblastoma cells test (MNA)
It is 2.5 × 10 that mouse neuroblastoma cells suspension, which is resuspended into concentration,5Cell/ml.Suspension cell (200 μ L,
5×104Cell) it can be redistributed into 96 orifice plates (flat-bottom hole, tissue cultures grade), and with 5%CO2Wet environment
In in 37 DEG C cultivate 24 hours.After hatching, it is replaced with the C-RPMI of 200 μ L G-Strophanthins containing 0.2mM and 0.02mM black false hellebore pyridine.
The C-RPMI for being free of G-Strophanthin and black false hellebore pyridine is added in control wells.10 μ L toxin samples or poison is added into control combination experimental group
Plain standard specimen.Sample and standard specimen do 3-4 repetition.Edge effect generates in order to prevent, in the hole of the row and column outside 96 orifice plates
Cell will be not used for testing, and 300ul PBS can be added thereto.96 orifice plates are cultivated 18 hours at 37 DEG C.Again will
Culture medium is sucked out, and 50ul MTT solution (with C-RPMI with the dilution proportion of 1:10) is added into each hole and detects cell activity.
After 37 DEG C have been cultivated 45-60 minutes, then the isopropanol of 100 HCls of the μ L containing 0.1M is added thereto.OD value is with Bio-
550 microplate reader of Rad is to read at 595nm referring to wavelength in 655nm.
Testing result observes different addition concentration as shown in fig. 6, in Gambia algae growth initial period addition bacterial fermentation object
Under (high dose group concentration be 5 × 106Cells/mL, middle dose group are 5 × 105Cells/mL, low dose group be 5 ×
104Cells/mL) the production poison situation of Gambia algae different times.After can be seen from the chart addition bacillus thuringiensis, energy
The Toxin producing C of different degrees of increase Gambia algae.Toxicity can be increased up 15 × 10 in maximum dose level group when to latter stage- 4pg P-CTX-1Eq/cell or more is 2.2 times of control group toxicity；And this toxin stimulation is with the increase of bacterial concentration
And increase.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples
It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, modifies, replacement and variant.
Claims (14)
1. a kind of microorganism is bacillus thuringiensis (Bacillus thuringiensis) GQS09, in September 5 in 2016
It is preserved in Guangdong Province's Culture Collection day, deposit number is GDMCC No.60074.
2. a kind of preparation, which is characterized in that the work inoculum in the preparation containing microorganism described in claim 1, institute
Inoculum living is stated to obtain in the following manner:
Expand culture after microorganism described in claim 1 is activated, obtain inoculum living, wherein bacterial concentration living is
5×105。
3. a kind of preparation, which is characterized in that the fermentation material containing microorganism described in claim 1 in the preparation, the fermentation
Object obtains in the following manner:
Expand culture after microorganism described in claim 1 is activated, obtain inoculum living, wherein bacterial concentration living is
5×104-5×106；
The inoculum living is ultrasonically treated 5 minutes in cell crushing instrument under condition of ice bath, obtains bacterial fermentation object.
4. a kind of preparation, which is characterized in that the fermentation material containing microorganism described in claim 1 in the preparation, the fermentation
Object obtains in the following manner:
Expand culture after microorganism described in claim 1 is activated, obtain inoculum living, wherein bacterial concentration living is
5×105-1×106；
The inoculum living is ultrasonically treated 5 minutes in cell crushing instrument under condition of ice bath, obtains bacterial fermentation object.
5. a kind of preparation, which is characterized in that the fermentation material containing microorganism described in claim 1 in the preparation, the fermentation
Object obtains in the following manner:
Expand culture after microorganism described in claim 1 is activated, obtain inoculum living, wherein bacterial concentration living is
1-5×106；
The inoculum living is ultrasonically treated 5 minutes in cell crushing instrument under condition of ice bath, obtains bacterial fermentation object.
6. a kind of preparation, which is characterized in that the inoculum supernatant in the preparation containing microorganism described in claim 1,
The inoculum supernatant obtains in the following manner:
Expand culture after microorganism described in claim 1 is activated, obtain inoculum living, wherein bacterial concentration living is
5×105-2×106；
The inoculum living is centrifuged 20 minutes in 12000rpm, makes bacterial precipitation, the supernatant of collection is Bacteria Culture
Liquid supernatant.
7. a kind of promotion Gambia algae growth or photosynthetic method characterized by comprising
The Gambia algae is cultivated to logarithmic phase, preparation described in claim 5 is added in Gambia's algae culturing liquid,
Continue follow-up cultivation.
8. intensity of illumination is the method according to the description of claim 7 is characterized in that the temperature of the culture is 26 DEG C
3800Lux, 12h:12h Dark-light cycle.
9. a kind of method for promoting the growth of Gambia algae characterized by comprising
The Gambia algae was cultivated to plateau, the Gambia is added in the described in any item preparations of claim 2,4,6
In algae culturing liquid, continue follow-up cultivation.
10. according to the method described in claim 9, intensity of illumination is it is characterized in that, the temperature of the culture is 26 DEG C
3800Lux, 12h:12h Dark-light cycle.
11. a kind of method for promoting Ya Biya algae secretion ciguatoxin characterized by comprising
Preparation as claimed in claim 3 is added in Gambia algae growth initial period, continues follow-up cultivation.
12. according to the method for claim 11, which is characterized in that the temperature of the culture is 26 DEG C, and intensity of illumination is
3800Lux, 12h:12h Dark-light cycle.
13. a kind of kit, which is characterized in that the kit includes that microorganism described in claim 1 or claim 2-6 appoint
One preparation.
14. kit described in any one of microorganism described in claim 1 or claim the 2-6 preparation or claim 13
Promoting the purposes in the growth of Gambia algae, photosynthesis or secretion ciguatoxin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610920840.9A CN106544296B (en) | 2016-10-20 | 2016-10-20 | Promote the growth of Gambia algae, photosynthesis and microorganism, method and the kit of secreting ciguatoxin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610920840.9A CN106544296B (en) | 2016-10-20 | 2016-10-20 | Promote the growth of Gambia algae, photosynthesis and microorganism, method and the kit of secreting ciguatoxin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106544296A CN106544296A (en) | 2017-03-29 |
| CN106544296B true CN106544296B (en) | 2019-07-16 |
Family
ID=58392060
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610920840.9A Active CN106544296B (en) | 2016-10-20 | 2016-10-20 | Promote the growth of Gambia algae, photosynthesis and microorganism, method and the kit of secreting ciguatoxin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106544296B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108977477B (en) * | 2018-08-01 | 2021-07-13 | 浙江海洋大学 | Method for preparing ciguatoxin by fermenting marine toxigenic dinoflagellate commensal bacteria |
| CN115992104B (en) * | 2022-07-22 | 2024-01-30 | 浙江大学 | Schizochy polysaccharide monooxygenase from bacillus subtilis and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008140988A1 (en) * | 2007-05-10 | 2008-11-20 | Danisco Us Inc., Genencor Division | Stable enzymatic peracid generating systems |
| CN102253023A (en) * | 2011-06-03 | 2011-11-23 | 深圳市疾病预防控制中心 | Sodium fluorescence probe detection method of ciguatoxin |
| CN102788850A (en) * | 2012-07-31 | 2012-11-21 | 舟山出入境检验检疫局综合技术服务中心 | Detection method for ciguatoxin in aquatic products |
-
2016
- 2016-10-20 CN CN201610920840.9A patent/CN106544296B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008140988A1 (en) * | 2007-05-10 | 2008-11-20 | Danisco Us Inc., Genencor Division | Stable enzymatic peracid generating systems |
| CN102253023A (en) * | 2011-06-03 | 2011-11-23 | 深圳市疾病预防控制中心 | Sodium fluorescence probe detection method of ciguatoxin |
| CN102788850A (en) * | 2012-07-31 | 2012-11-21 | 舟山出入境检验检疫局综合技术服务中心 | Detection method for ciguatoxin in aquatic products |
Non-Patent Citations (3)
| Title |
|---|
| Detailed LC-MS/MS Analysis of Ciguatoxins Revealing Distinct;Kentaro Yogi等;《Analytical chemistry》;20111019;第8886–8891页 * |
| Large Outbreaks of Ciguatera after Consumption of Brown Marbled Grouper;Thomas Y. K. Chan等;《Toxins》;20140711;第2041-2049页 * |
| Toxins: Bacterial and Marine Toxins;Matthew Salzman等;《Clinics in laboratory medicine》;20060630;第397–419页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106544296A (en) | 2017-03-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Shah et al. | Isolation and characterization of plant growth-promoting endophytic fungi from the roots of Dendrobium moniliforme | |
| CN103923841B (en) | One strain has Strain of Beauveria bassiana and the application thereof of highly pathogenicity to silkworm | |
| CN105886405B (en) | Dendrobium candidum endogenetic fungus and its application | |
| CN106167776A (en) | A kind of can bacillus cereus (Bacillus cereus) TH 35 of heavy metal cadmium and application thereof in activating soil | |
| CN109722388A (en) | Microalgae commensalism bacterium isolation medium, separation method and the crucial bacterium high-throughput screening method for influencing micro algae growth | |
| CN107460133A (en) | Dark color has every endogenetic fungus HS40 and its application in dendrobium candidum production | |
| CN106544296B (en) | Promote the growth of Gambia algae, photosynthesis and microorganism, method and the kit of secreting ciguatoxin | |
| Lakshmi et al. | Anticancer potentials of secondary metabolites from endophytes of Barringtonia acutangula and its molecular characterization | |
| Yu et al. | Diversity and annual variation of phytoplankton community in Yesso scallop (Patinopecten yessoensis) farming waters of North Yellow Sea of China | |
| CN101629136A (en) | High-efficient culture and sorting method and culture device of microorganisms | |
| CN108913796A (en) | For detecting Green Fluorescent Protein gene egfp specific primer, kit and the method for green muscardine fungus plant endogenesis | |
| CN102747005B (en) | Bacillus atrophaeus for prevention and control of cotton boll blight, and microbial agent thereof | |
| CN102586143B (en) | Bacillus mojavensis and microorganism bacterium agent thereof for controlling cucumber downy mildew | |
| US7011957B2 (en) | Isolation and cultivation of microorganisms from natural environments and drug discovery based thereon | |
| AU2002335757B2 (en) | Isolation and cultivation of microorganisms from natural environments and drug discovery based thereon | |
| CN106047956B (en) | A method of the Penicillium citrinum Asc2-4 that grown nonparasitically upon another plant altogether using ascidian fermentation preparation is rich in the grease of gamma-Linolenic acid | |
| CN108531415A (en) | The pale air bacillus Jh-7 of one plant of antagonism rice leaf spot bacteria | |
| CN109810920A (en) | One thuringiensis strain bacillus and its application | |
| AU2002335757A1 (en) | Isolation and cultivation of microorganisms from natural environments and drug discovery based thereon | |
| CN110295132A (en) | One plant has the active bacillus CCPM7645 of powerful anticancer and its application | |
| CN106591173A (en) | Bacillus flexus HL-37 capable of activating soil heavy metal cadmium, and applications thereof | |
| CN103333816A (en) | Screening method for seawater fermentation-type bacterial strains producing biosurfactants | |
| CN102533610B (en) | Bacillus subtilis for preventing and treating cucumber ampho disease, and microbial agent thereof | |
| CN101182478A (en) | Root nodule azotobacter strain BDYD1 and uses thereof | |
| CN110184226A (en) | Bacillus Mangrove-2008, microbial inoculum and its purposes and method in inhibition Aspergillus Niger Growth |
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 |