CN109758591A - The preparation method and application of planktonic algae containing pseudo- ghost - Google Patents
The preparation method and application of planktonic algae containing pseudo- ghost Download PDFInfo
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Classifications
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- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
- A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
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- 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
- C12N1/02—Separating microorganisms from their culture media
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
Abstract
The present invention provides a kind of preparation method and application of planktonic algae containing pseudo- ghost.The planktonic algae containing pseudo- ghost is in the application for preparing oral acoustic contrast agent.After the planktonic algae for containing pseudo- ghost is used as oral acoustic contrast agent, there is the biological gas bag of pseudo- ghost can effectively play scattering medium effect for it, stomach and intestine wall construction is set clearly to develop under two-dimensional ultrasound and contrast mode, occupying lesion in stomach can be intuitively found under contrast mode, then become image forming medium under two-dimensional ultrasound, it is clear to show swollen object and gastrointestinal tract wall structure and the neighbouring relationship with peripheral organs, and the planktonic algae biological safety containing pseudo- ghost is high.Based on the application of the planktonic algae, the present invention provides a kind of oral acoustic contrast agents containing the planktonic algae.The cultural method of the planktonic algae can effectively improve the multiplication rate of the swim alga, and the more pseudo- ghost airbag structures intracellular of growth in the frustule that enables to swim, and improve its imaging effect.
Description
Technical field
The present invention relates to a kind of medical detection reagent technical fields, and in particular to a kind of planktonic algae containing pseudo- ghost
Preparation method and its application.
Background technique
Enterogastric diseases mainly include acute and chronic gastroenteritis, acute and chronic colitis, peptic ulcer and gastric cancer colorectal cancer
Equal malignant diseases, disease incidence account for about the 20% of national population.Wherein the malignant diseases such as gastrointestinal stromal tumor, gastric cancer not only can be serious
The life for influencing patient and its household, but will threaten to its life.There is data display early stage that the stomach of diffusion transfer does not occur
5 years survival rates can reach 80% or so after the excision of cancer underwent operative.It can be seen that early diagnosis and timely operative treatment are to disease
Lapse to it is most important.
Exist currently used for the gastrointestinal tract barium meal or lipiodol angiography of enterogastric diseases early diagnosis and gastrointestinal tract endoscopy
Clinical diagnosis enterogastric diseases have had wide application, and especially canel barium meal contrast examination is because its method simplicity is even more as preferred inspection
It looks into.But digestive canel barium meal contrast examination is because its radioactive ray allows many patients to have a misgiving the harm of human body, and the inspection can only be seen
Observe whether gastrointestinal tract wall has lesion and its substantially form, cannot but show swollen object to the Infiltrating of gastrointestinal wall and its with surrounding
The neighbouring relationship of organ.Though the inspection that gastrointestinal tract endoscope adds ultrasonic probe inside cavity can solve the above problems, step is checked
Rapid cumbersome, expensive, patient suffering when inspection, this makes many patients hang back, and cannot timely diagnose.
In recent years, with the development of science and technology, medical image has become diagnosis and treats the important auxiliary of disease
Assistant's section.Wherein, contrast-enhanced ultrasound technique monitors after being widely used in medical diagnosis, treatment and treatment with its unique advantage
Equal links.Its advantage includes: safe, noninvasive, cheap；Simple to operate, "dead" pollution, without ionization spoke
It penetrates；Image taking speed is fast, the variation to histoorgan can carry out dynamic image acquisition etc. in real time.Ultrasonic contrast
(ultrasonic contrast) is also known as acoustic contrast (acoustic contrast), be by with tissue acoustic characteristic not
Same substance introduces in vivo, and to improve ultrasonic imaging condition, enhancing tissue echo comparison, raising Doppler signal intensity makes ultrasound
Application range increases and improves the sensitivity and specificity of inspection.With the improvement of instrument performance and going out for novel acoustic contrast agent
Existing, ultrasonic contrast can effectively enhance the two-dimensional ultrasound image and flow Doppler letter of the parenchymatous organs such as cardiac muscle, liver, kidney
Number, the blood perfusion situation of reflection and observation normal tissue and pathological tissues, it has also become one of ultrasound diagnosis is highly important
Developing direction.
The key of contrast-enhanced ultrasound technique is contrast agent, and conventional Ultrasound contrast agent is generally the microvesicle for being enclosed with gas.At present
Clinically common preferably contrast agent is sound Novi (SonoVue) also known as injection sulfur hexafluoride microvesicle, is wrapped in microbubble
Wrap up in sulfur hexafluoride gas, can be used as the reflecting medium of ultrasonic wave with the contact interface of solution medium, under ultrasonography with week
It encloses and forms preferable contrast between tissue.However, due to needing full stomach and enteric cavity when pedestrian's body gastrointestinal barium meal examination
Interference of the stomach and intestine gas to ultrasonic wave is eliminated, gastro intestinal ultrasonic environment is improved with this, so usual examiner needs to drink when checking
There can be more comprehensive display to entire gastrointestinal tract with the contrast agent of 250-500ml, so a large amount of microbubble contrast agent is drunk, and is had
The uncomfortable reaction such as flatulence of patient may be caused after microbubble ruptures, and the current price of sound Novi is still costly, it may
Unnecessary financial burden is brought to patient.Because of above-mentioned many reasons, although sound Novi is widely used in blood pool radiography, but
Application is had no on the ultrasonic contrast of hollow organ.
For the economization and practicability for realizing gastrointestinal contrast agent, it is super that researchers are developed novel edible stomach and intestine
Sound contrast agent, clinically has been widely used, make more fully to detect under the conditions of noninvasive gastrointestinal disease is turned into can
Energy.A kind of instant granular gastrointestinal imaging is disclosed in a patent (application notification number CN1231927) as disclosed at home
Preparation, formula are made of lipid containing plant albumen, rice wheat starch class, Chinese yam, meter Ren, dried orange peel, corrigent.Although its medicine food two
With traditional Chinese medicine ingredients can promoting the circulation of qi dissipate-swelling, dampness elimination invigorating the spleen, but preparation method is needed by repeatedly complicated curing, sieving, grinding
Process, production process are cumbersome.The gastrointestinal tract acoustic contrast agent newly developed occurred in recent years such as exists mostly based on complete traditional Chinese medicine ingredients
In portion patent disclosed in the country (application notification number CN104740657A), researcher used silicon oil foam killer, mannitol and
The grinding of the ten pleasant impression Chinese medicine such as Hericium erinaceus, honeysuckle, turmeric is made, although its contrasting effects is ideal, weighing, preparation process are numerous
It is trivial, pass through the silicon oil foam killer of physical chemistry method combinatorial compound, using dimethicone and defoaming activity agent to exclude in gastrointestinal tract
Gas reduces pseudomorphism, and whether which is not natural origin, and biological safety is poor, have adverse effect still indefinite human body.
And when using plant amylum and Chinese medicine etc. as radiography, doctor is still mostly to be observed and diagnoses under two-dimensional imaging mode,
Compared to being difficult to identify gastric content itself and swollen object under contrast mode.Therefore it provides a kind of overall safety or directly edible
Material as contrast medium, while reach the optimization of ultrasonic imaging plot quality and biological safety be always this field research and development
Person attempts to the technical problem solved.
In addition, the tradition culture planktonic algae such as planktonic algae method containing pseudo- ghost mostly uses breathable sealing film, without
Give algae sterile aerobic ventilation, this causes standing swim alga cell proliferation slow and intracellular air bag puppet ghost content is low.And
When standing extraction containing pseudo- ghost preponderant algae, since the frustule that swims has the spy for first sinking and slowly floating again under static condition
Point, the frustule that swims are accumulated at the narrow triangle in separatory funnel bottom of small volume, not only make stand algae be easy conglomeration,
It should not float, and be easy to make the frustule that swims of lower part that can not obtain sufficient air and nutrient, easily lead to the frustule that swims
Decline is rotten.
Summary of the invention
It is an object of the invention to overcome the above-mentioned deficiency of the prior art, a kind of planktonic algae one containing pseudo- ghost is provided
Application method and a kind of oral acoustic contrast agent are planted, existing gastrointestinal contrast agent image quality is undesirable and/or presence is given birth to solve
Poor, the at high cost technical problem of object safety.
It is existing to solve another object of the present invention is to provide a kind of cultural method of planktonic algae containing pseudo- ghost
Planktonic algae cultural method low efficiency, intracellular air bag puppet ghost content are low and the technical issues of easily leading to cell death.
In order to achieve the above-mentioned object of the invention, an aspect of of the present present invention provides a kind of planktonic algae containing pseudo- ghost
Using.The planktonic algae containing pseudo- ghost is in the application for preparing oral acoustic contrast agent.
Another aspect of the present invention provides a kind of oral acoustic contrast agent.The oral acoustic contrast agent, which contains, to be used for
The planktonic algae of ultrasonic development, and the swim alga contains pseudo- ghost structure.
Another aspect of the present invention provides a kind of cultural method of planktonic algae containing pseudo- ghost.It is described to contain puppet
The cultural method of the planktonic algae of ghost includes the following steps:
Swim alga containing pseudo- ghost is seeded in swim alga primary culture medium, and to the swim alga primary culture medium
In be passed through sterile oxygen-containing gas and carry out sterile aerobic culture processing；
After the swim alga is cultured to logarithmic growth phase, the swim alga of logarithmic growth phase is seeded to swim alga
Expand culture medium and carries out sterile aerobic expansion culture processing；
Will through it is described it is sterile it is aerobic expand culture processing swim alga carry out stewing process, after will float on the swim alga
The algal layer for expanding media surface is exported from the swim alga media surface to be collected.
Compared with prior art, the present invention has technical effect below:
After the planktonic algae for containing pseudo- ghost is used as oral acoustic contrast agent, there is the biological gas bag of pseudo- ghost can have
Effect plays the role of scattering medium, so that stomach and intestine wall construction is clearly developed under two-dimensional ultrasound and contrast mode, energy under contrast mode
It intuitively finds occupying lesion in stomach, then becomes image forming medium under two-dimensional ultrasound, it is clear to show swollen object and gastrointestinal tract wall structure
And the neighbouring relationship with peripheral organs.And the planktonic algae biological safety height containing pseudo- ghost.
The present invention takes orally planktonic algae of the acoustic contrast agent due to containing pseudo- ghost, the oral acoustic contrast agent
Can be effectively as gastrointestinal tract acoustic contrast agent, the pseudo- ghost structure-biological air bag contained by the planktonic algae in ultrasonic imaging
Scattering medium effect can be effectively played, so that stomach and intestine wall construction is clearly developed under two-dimensional ultrasound and contrast mode, radiography mould
Occupying lesion in stomach can be intuitively found under formula, then become image forming medium under two-dimensional ultrasound, it is clear to show swollen object and gastrointestinal tract
Wall construction and neighbouring relationship with peripheral organs.And the swim alga cytotostatic containing pseudo- ghost, biological safety are high.
We use optical densitometric method when quantifying to this kind of edible biological contrast agent, instead of traditional cell count
Method, this method is easier, easily operated, is suitable for a large amount of preparations and industrialization.
The present invention contains the cultural method of the planktonic algae of pseudo- ghost using subsection filter, can effectively improve described swim
The multiplication rate of algae, and the more pseudo- ghost airbag structures intracellular of growth in the frustule that enables to swim.In addition, will swim
Algae finally stands to form algal layer and export from swim alga media surface and collect, and the survival rate for the frustule that swims has been effectively ensured,
Improve its imaging effect.
Detailed description of the invention
Present invention will be further explained below with reference to the attached drawings and examples, in attached drawing:
Fig. 1 is the planktonic algae preparation method process flow diagram that the embodiment of the present invention contains pseudo- ghost；
Fig. 2 is Anabaena Flos-aquae deadtime, logarithmic growth phase and resting stage each cultivation stage state in the embodiment of the present invention 1
Photo；
Fig. 3 is to stand the photo for forming floating algae strain algal layer in the embodiment of the present invention 1 under Anabaena Flos-aquae sunlight；
Fig. 4 is 400 times of optical microscopy flowering structure figures of Anabaena Flos-aquae in the embodiment of the present invention 1；
Fig. 5 be by prepared in the embodiment of the present invention 3 control group, value=0.2 OD500,0.5,0.8 oral contrast agent shine
Piece.
Fig. 6 is value=0.2 OD500 of the preparation of the embodiment of the present invention 3,0.5,0.8 oral contrast agent and contrast groups are external
Agar imitates body ultrasonic contrast figure under B-mode mode and under Contrast mode under the conditions of 18MHz；
Fig. 7 is value=0.2 OD500 of the preparation of the embodiment of the present invention 3,0.5,0.8 oral contrast agent and contrast groups are external
Agar imitates a of body, u value line chart；
Fig. 8 be the embodiment of the present invention 3 prepare value=0.2 OD500,0.5,0.8 oral contrast agent and contrast groups mouse
Internal ultrasonic contrast imaging figure；Wherein, Fig. 8-A is that oesophagus and stomachus cardiacus, body of stomach, stomach are deep and remote under 40MHZ two dimension high frequency condition
Door portion image；Fig. 8-B is stomach wall image under 40MHZ two dimension high frequency condition；Fig. 8-C is that contrast agent fills stomach under two-dimensional condition
The echogram of chamber and liver, kidney；Fig. 8-D is image under Contrast imaging pattern；
Fig. 9 be the embodiment of the present invention 3 prepare value=0.2 OD500,0.5,0.8 oral contrast agent and contrast groups mouse
Internal a, u value line chart.
Specific embodiment
In order to which technical problems, technical solutions and advantageous effects to be solved by the present invention are more clearly understood, below in conjunction with
Embodiment, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only used to explain
The present invention is not intended to limit the present invention.
The quality of each component noted in the disclosure of the embodiment of the present invention not only may refer to specifically containing for each component
Amount, can also indicate the proportionate relationship of quality between each component, therefore, as long as according to specification each component of the embodiment of the present invention
Content is scaled up or is reduced within specification of embodiment of the present invention scope of disclosure.Specifically, the embodiment of the present invention
Quality described in the specification can be mass unit well known to the chemical fields such as μ g, mg, g, kg.
Inventor's discovery in research and development ultrasonic developer has excellent containing the planktonic algae of pseudo- ghost biological gas bag structure
Development effect.Based on the discovery, the embodiment of the invention provides the planktonic algaes for containing pseudo- ghost hereinafter as ultrasound
The application of contrast agent, based on the application of the planktonic algae containing pseudo- ghost, it is oral that the embodiment of the invention also provides one kind
Acoustic contrast agent, while proposing a kind of cultural method of the planktonic algae containing pseudo- ghost.
On the one hand, the embodiment of the invention provides the planktonic algaes containing pseudo- ghost (gas vesicle) in ultrasonic development
Application in agent field.Inventor has found under study for action, directly using the planktonic algae for containing pseudo- ghost structure as ultrasound
After contrast agent, in ultrasonic imaging, the intracellular existing pseudo- ghost airbag structure of the swim alga surpasses under the action of ultrasonic wave
Sound wave will scatter when meeting the pseudo- ghost air bag formed by glutelin package gas, increase scattering in gastrointestinal tract
By force, there is nebulous echo, this phenomenon can not only become good image forming medium, enable stomach and intestine wall construction in two-dimensional ultrasound item
It is clearly imaged under part, and can intuitively show the profile of gastrointestinal wall under radiography Contrast mode, returned with peripheral organs
Sound comparison is obvious, and can effectively eliminate gas and interfere generated pseudomorphism.When finding filling defect under Photo condition, Ke Yili
Identify the physical property matter that swells in stomach in the 2 d mode, considerably increases the discovery rate for the object that swells in stomach.Therefore, described to contain pseudo- ghost
Planktonic algae can prepare the application in oral acoustic contrast agent directly as oral acoustic contrast agent, therefore.
It that is to say in embodiments of the present invention, the planktonic algae containing pseudo- ghost is to need not move through cell cracking to go purification algae thin
Pseudo- ghost airbag structure intracellular, but directly used using swim alga strain or the frustule that swims as acoustic contrast agent.
Due to being desirable to the planktonic algae containing pseudo- ghost in embodiments of the present invention for super in medical diagnosis clinic
Acoustic imaging is to improve imaging effect, and therefore, the above-mentioned planktonic algae containing pseudo- ghost should be understood that human body and animal
It is safe planktonic algae, the non-toxic or low-toxic planktonic algae that can be such as received by medicine.In one embodiment, described to contain puppet
The planktonic algae of ghost is cyanobacteria.In a particular embodiment, the cyanobacteria includes in anabena, synnema algae, Microcystis aeruginosa and the algae that quivers
At least one.Those planktonic algaes selected not only have the airbag structure of abundant pseudo- ghost, but also are safe and non-toxic, biological
The high algae strain of compatibility.
On the other hand, based on application of the planktonic algae of pseudo- ghost as oral acoustic contrast agent is contained above, originally
Inventive embodiments additionally provide a kind of oral acoustic contrast agent and (are referred to as edible contrast medium for stomach and intestine ultrasonic imaging, or oral
Biological acoustic contrast agent).The oral acoustic contrast agent contains the planktonic algae for ultrasonic development, and the swim alga contains
Pseudo- ghost structure.It that is to say that the oral acoustic contrast agent contains the planktonic algae containing pseudo- ghost described above.In this way, base
Described in the planktonic algae application above containing pseudo- ghost containing pseudo- ghost planktonic algae have high quality ultrasonic imaging and
High biological safety special efficacy, therefore, the oral acoustic contrast agent is effectively as gastrointestinal tract acoustic contrast agent, in ultrasonic imaging
Pseudo- ghost biological gas bag structure contained by the planktonic algae can effectively play scattering medium effect, and stomach and intestine wall construction is enable to exist
Clearly develop under two-dimensional ultrasound and contrast mode, can intuitively find occupying lesion in stomach under contrast mode, under two-dimensional ultrasound
Then become image forming medium, it is clear to show swollen object and gastrointestinal tract wall structure and the neighbouring relationship with peripheral organs.And described contain
The swim alga cytotostatic of pseudo- ghost, can directly take orally, easy to use, safe and non-toxic.In addition, by the institute containing pseudo- ghost
It is similar to the seaweed of people's daily consumption, kelp etc. to state planktonic algae such as cyanobacteria, and without special odor, is easy to be accepted by patients, takes
It can be discharged by eubolism approach after, biological safety is high.
In one embodiment, the content of the contained planktonic algae that is to say that concentration can be in the oral acoustic contrast agent
Control is optical densitometric method OD500=0.5 or more, because the type difference radiography prevailing concentrations of swim alga used have different.
By controlling the concentration of the planktonic algae, to improve the ultrasonic imaging effect of the oral acoustic contrast agent.Wherein, the mouth
The solvent for taking acoustic contrast agent can be the isotonic solution for meeting medical requirement, in physiological saline, PBS (phosphate buffer)
At least one.
In another aspect, the embodiment of the invention also provides the planktonic algaes containing pseudo- ghost in each embodiment above
A kind of preparation method.The screening of the planktonic algae containing pseudo- ghost expands cultural method process flow as shown in Figure 1, it is wrapped
Include following steps:
Step S01: the swim alga containing pseudo- ghost is seeded in swim alga primary culture medium, and to it is described swim it is primary
It is passed through sterile oxygen-containing gas in algae culture medium and carries out sterile aerobic culture processing；
Step S02: after the swim alga is cultured to logarithmic growth phase, the swim alga of logarithmic growth phase is inoculated with
Expand culture medium to swim alga and carries out sterile aerobic expansion culture processing；
Step S03: will carry out stewing process through the sterile aerobic swim alga for expanding culture, after will float on it is described floating
The algal layer for swimming algae expansion media surface is exported from the swim alga media surface to be collected.
Wherein, in the step S01, the sterile aerobic culture processing preferably carries out in accordance with the following steps:
Step S011: it after the swim alga is seeded in the swim alga primary culture medium, is placed in illumination and is protected from light friendship
For carried out in the environment of transformation it is sterile it is aerobic culture until the swim alga primary culture medium light green color is become from light green when, then
Fresh swim alga originally culture base fluid is added and continues sterile aerobic culture；
Step S012: when deepening green to green apple by light green color to the swim alga primary culture medium wait cultivate, again plus
Enter fresh swim alga primary culture medium or former swim alga be subjected to sub-bottle, the fresh primary algae culture medium that swims is added, continue into
The sterile aerobic culture of row is until the swim alga primary culture medium color burn is green to green apple.
Led to by above-mentioned preferred sterile aerobic culture processing with shortening the time of the swim alga deadtime of inoculation
It crosses sub-bottle and expands culture to filter out floating advantage algae strain more containing a large amount of pseudo- ghosts within the same time.Wherein, the step
The swim alga that the swim alga in rapid S011 can be the freeze-dried powder of the swim alga containing pseudo- ghost or cultivate in advance.
If it is freeze-dried powder, then needs first to carry out at room temperature to place one day rewarming under gnotobasis, be then seeded to described float
Swim algae primary culture medium.In one embodiment, the primary culture medium can be but not just for BG11 culture medium.
The swim alga is seeded to the amount in the swim alga primary culture medium can be according to the inoculation of conventional swim alga
Amount be inoculated with, such as in a particular embodiment, can with but not only according to the swim alga and the swim alga primary culture medium
Volume ratio is that the ratio of 1:50 is inoculated with.In the illumination in step S011 and the environment for being protected from light checker, light
It can be 12-16h according to the time, the specific can be that 14h；Be protected from light the time be can be 8-12h, the specific can be that 10h.
In addition, swim alga primary culture medium described in step S011 becomes the jade-green time about 6 days from light green,
The swim alga primary culture medium in step S012 is deepened by light green color to the green apple green time about 4 days.In other words
The time of the sterile aerobic culture processing of step S01 has altogether about 10 days.Certainly due to culture scale and stirring and ventilatory capacity
Difference, the transformation period for the swim alga primary culture medium color that the sterile aerobic culture handles each stage also has one
Fixed difference.
When the swim alga primary culture medium color light green color of step S012 deepens green to green apple, the swim alga
The swim alga in primary culture medium enters logarithmic growth phase, that is to say after cultivating to step S01, changes culture medium, into
Enter the advantage swim alga for expanding and cultivating and can producing largely containing more pseudo- ghost.Therefore, the sterile aerobic expansion in the step S02
It, can the amount of acquisition be big and advantage swim alga containing more pseudo- ghost after big culture processing.
In one embodiment, the sterile aerobic method for expanding culture processing in the step S02 can be according to such as
Under two methods cultivated.
The sterile aerobic expansion cultural method one includes the following steps:
After contain after the swim alga of aerobic culture processing sterile in step S01 is cultured to logarithmic growth phase
The swim alga primary culture medium of logarithmic growth phase swim alga is divided at least two parts, and is separately added into swim alga and expands culture medium
Sterile aerobic expansion culture processing is carried out, until when swim alga expansion culture medium is presented emerald green, it again will be emerald
The swim alga expands culture medium and is divided at least two parts, and fresh swim alga is added again and expands culture medium and carries out sterile aerobic
Culture, until the algae culture medium presentation of swimming is emerald green.
The sterile aerobic expansion cultural method two includes the following steps:
After the swim alga is cultured to logarithmic growth phase, the originally culture containing the swim alga is based on quiet at sunlight
Set processing, the swim alga for floating on the originally culture primary surface is taken out and be seeded to swim alga expand culture medium in carry out nothing
The aerobic culture processing of bacterium.
Certainly, the sterile aerobic expansion cultural method two can also comprise the step of: to it is sterile it is aerobic culture until
When the algae culture medium that swims is presented emerald green, the emerald swim alga is divided at least two parts again, is added again new
Fresh swim alga expands culture medium and carries out sterile aerobic culture, until the algae culture medium presentation of swimming is emerald green.
The sterile aerobic expansion cultural method one and the sterile aerobic expansion cultural method two may be implemented containing
The raised growth of the swim alga of pseudo- ghost, in contrast, the sterile aerobic expansion cultural method is second is that first contain puppet to primary
The swim alga of ghost is screened, and expanding culture containing pseudo- ghost swim alga for screening, and therefore, described sterile have
It is relatively high that oxygen expands efficiency of the acquisition of cultural method two containing pseudo- ghost swim alga.Wherein, the sterile aerobic expansion culture side
In method one and the sterile aerobic expansion cultural method two, expand from swim alga expansion culture medium to the swim alga is added
The emerald time about 4-7 days time is presented in culture medium.In addition, the number of sub-bottle culture can be it is multiple, such as at least two
It is secondary, it is specific such as 2-3 times.Finally, becoming presenting when the swim alga expands culture medium color after sterile aerobic culture processing
When emerald green, and when continuing culture color obviously not deepened in two days, illustrate that the swim alga has come into growth retardation
Phase, the quality of algae may be will affect by continuing culture, should be terminated in time culture and be collected, be may be implemented at this time to through sterile aerobic
The swim alga of culture processing is collected processing, the step S03 processing as described in carrying out.
The time that the swim alga of entire S02 pairs of step carries out sterile aerobic expansion culture processing may swim according to dividing
The number that algae expansion culture medium expands culture is different and different, and swim alga is divided to expand what culture medium expanded culture every time
Time about 4 days, certainly due to culture scale and stirring and the difference of ventilatory capacity, the sterile aerobic culture handled each stage
The transformation period of the algae culture medium color of swimming also have certain difference.The swim alga, which expands culture medium, can be but not
Only G625 culture medium.
In addition, specific aerobic culture sterile as described in step S011 and S012 and step S02 in above-mentioned steps S01
In sterile aerobic expansion culture during preferably with stir process and be passed through sterile oxygen-containing gas such as air in described
It swims in algae culture medium, to enhance the uniformity of the content of oxygen and illumination in culture medium, in a particular embodiment, the stirring
The revolving speed of processing can be with but not just for 100rpm, wherein the stirring can be realized with shaking table.When rigid inoculated and cultured, revolving speed
Do not answer it is excessively high, illumination do not answer it is too strong, otherwise may cause algae recover bad, growth restriction.In above-mentioned steps S01 specifically such as
Temperature can during the sterile aerobic culture in step S011 and S012 and the sterile aerobic expansion culture in step S02
To be room temperature, if temperature is 25 DEG C.
Stewing process in the step S03 is to utilize the swim alga after the culture of step S01 and step S02,
Therefore generating more pseudo- ghost airbag structure into the cell can be good at floatation characteristic, and during stewing process
It cracking can float on the algal layer that the surface of the algae culture medium that swims is formed.It is real one in order to improve stewing process efficiency
It applies in example, the method for the swim alga stewing process includes the following steps: will be through the aerobic swim alga for expanding culture
Aerobic stewing process is carried out in sunlight irradiation environment.In a particular embodiment, can be placed in it is normal it is sunlit at room temperature
Stand 4 hours.
It will be formed by what the algal layer was collected from swim alga media surface export through stewing process in step S03
Method preferably includes following steps: directly exporting the algal layer from the swim alga media surface by the way of absorption and receives
Collection.In this way, since the algal layer directly exported and being collected by the way of absorption, the frustule that can adequately guarantee to swim fills
It point is contacted with oxygen, and ensure that nutrient, therefore, the method for the export algal layer can guarantee to swim the bioactivity of frustule, shape
State more preferably, the airbag structure of puppet ghost intracellular save it is more preferable, thus when the swim alga is used for acoustic contrast agent, imaging
Effect is also more preferable.
The swim alga that collection is exported in step S03 wouldn't such as be needed for radiography, the swim alga can be added enough
Culture solution is placed in 4 DEG C of environment and is saved, and concentration should be less than 1:100, at this temperature save can save about 1 year when
Between.
Due to the swim alga that the cultural method culture of the planktonic algae containing pseudo- ghost contains pseudo- ghost be for
It is used directly as acoustic contrast agent, therefore, into S03, links should be protected to above-mentioned steps S01 during the cultivation process
Demonstrate,prove it is sterile, as described in step S01 sterile aerobic culture processing, the expansion of step S02 culture processing and step S03 in
The export collection of stewing process and the algal layer should be ensured that gnotobasis is handled.The planktonic algae for inoculation
Also should be ensured that it is sterile with the algae culture medium that swims for cultivating the swim alga.Preferably it is passed through during the cultivation process
It should also be sterile containing gas such as air in culture medium.By those sterile culture processing, guarantee that is obtained swims
Algae safety can be directly used for acoustic contrast agent use.
In addition, in each embodiment of cultural method described above the planktonic algae of used inoculation it is as described above can
It is safe planktonic algae to be to human body and animal, the non-toxic or low-toxic planktonic algae that can be such as received by medicine.It is real one
It applies in example, the planktonic algae containing pseudo- ghost is cyanobacteria.In a particular embodiment, the cyanobacteria includes anabena, synnema
Algae, Microcystis aeruginosa and at least one of the algae that quivers.Those planktonic algaes selected not only have the airbag structure of abundant pseudo- ghost, and
And it is safe and non-toxic.It is, of course, also possible to be other contain pseudo- ghost structure other be suitable for field of medicaments other planktonic algaes.
The cultural method of the planktonic algae containing pseudo- ghost by mentioned earlier uses subsection filter, can effectively improve institute
The multiplication rate of swim alga is stated, and the more pseudo- ghost airbag structures intracellular of growth in the frustule that enables to swim, such as Fig. 4
It is shown.And finally stand to form swim alga algal layer and export from swim alga media surface and collect, it has been effectively ensured and has swum
The survival rate of frustule improves its imaging effect.In addition, the planktonic algae containing pseudo- ghost used in the cultural method
For the strain of nontoxic algae, and cultivate in an aseptic environment, and biological safety is high.Secondly, the cultural method condition is easily-controllable, training
Feeding technique is effectively simplified, and the planktonic algae reproduction speed is fast, is effectively increased the increment efficiency of planktonic algae, is effectively reduced
The cost of culture, may be implemented enterprise scale culture.
Answering for the planktonic algae that embodiment contains pseudo- ghost is illustrated the present invention below by way of multiple specific embodiments
With and cultural method.
Embodiment 1: the cultural method of the planktonic algae containing pseudo- ghost
The cultural method for present embodiments providing a kind of planktonic algae containing pseudo- ghost, includes the following steps:
S11: it the culture (about 10 days) of Anabaena Flos-aquae deadtime: opens Anabaena Flos-aquae freeze-dried powder and (it is raw to be purchased from the U.S.
Object standard items collecting center ATCC) lid, one day rewarming is placed under room-temperature sterile environment, and freeze-dried powder is poured into 50ml taper
In bottle, cyanobacteria BG11 culture medium is added according to the volume ratio of 1:50, shakes up, insertion airway connects air pump in conical flask, uses
It sterilizes after cotton filling taper bottleneck, is sealed with sealing strip (paraflim), keep being gnotobasis in bottle；Cultivate light application time
Dark for 14h illumination/10h, 25 DEG C of temperature, shaking speed 100rpm, culture solution is from initial such as Fig. 2-A institute after culture about 6 days
When the light green shown becomes light green color, equivalent fresh medium is added and continues to cultivate, deepens to culture solution green to such as Fig. 2-B institute
The green apple shown is changed to 250ml conical flask culture when green, and fresh culture is added to final volume about 150ml, continues culture about 4
It is green that it to liquid in bottle becomes green apple again；
S12: logarithmic growth phase culture (about 4 days): being divided into 2 parts for culture to the green culture medium of green apple in step S11,
It pours into addition G625 culture medium to 250ml final volume in 500ml conical flask respectively to continue to cultivate, therefore phase cell proliferation is accelerated,
I.e. visible culture solution color becomes emerald green within about 4 days, can continue mass propgation after sub-bottle at this time, when such as Fig. 2-is presented in algae in bottle
When kingfisher shown in C (ink) is green, illustrates to be again introduced into growth retardation phase (resting stage), following step S13 separation can be carried out
Processing；
S13: in the 500ml conical flask after the Anabaena Flos-aquae culture solution that step S12 is cultivated to be poured into high-temp steam sterilizing,
Bottleneck covers bacteriological filtration film, and standing about 4 hours in the case where normal sunlight irradiates room temperature (about 23 DEG C), i.e. visible level floats bottle green algae
Layer, as shown in Figure 3；Using 1ml liquid-transfering gun, pipette tips close to floating algal layer surface slowly draws float cyanobacteria, be collected in 15ml from
Heart pipe, standing are layered to algae with culture solution completely, and liquid-transfering gun exhausts culture supernatants as far as possible.It wouldn't such as need for radiography, it can
Algae solution is placed in 4 DEG C of environment.It is learnt through experiment, the time saved at this temperature about 1 year.
For the Anabaena Flos-aquae that culture is obtained in carrying out 400 times of optical microphotographs under the microscope, photo is as shown in Figure 4.By
Fig. 4 is it is found that shown Anabaena Flos-aquae airbag structure rich in.
Embodiment 2: the cultural method of the planktonic algae containing pseudo- ghost
The cultural method for present embodiments providing a kind of planktonic algae containing pseudo- ghost, includes the following steps:
S11: the culture (about 10 days) of Microcystis aeruginosa deadtime: Microcystis aeruginosa is poured into 50ml conical flask, according to the body of 1:50
Product shakes up, insertion airway connects air pump in conical flask, clogs conical flask with sterilizing cotton than cyanobacteria BG11 culture medium is added
It after mouthful, is sealed with sealing strip (paraflim), keeps being gnotobasis in bottle；Culture light application time is 14h illumination/10h dark,
25 DEG C of temperature, shaking speed 100rpm, after culture about 6 days culture solution from it is initial it is colorless and transparent become light green color when, be added etc.
It measures fresh BG11 culture solution to continue to cultivate, 250ml conical flask culture is changed to when culture solution green deepens green to green apple, and add
Enter fresh culture to final volume about 150ml, continues culture about 4 days to liquid in bottle and become green apple again and is green；
S12: the green culture medium of green apple extremely logarithmic growth phase culture (about 6 days): is divided into 2 by culture in step S11
Part, the fresh BG11 culture medium of addition to 250ml final volume in 500ml conical flask is poured into respectively to be continued to expand culture until green apple
When green, illustrate to be again introduced into growth retardation phase (resting stage), following step S13 separating treatment can be carried out；
S13: in the 500ml conical flask after the micro-capsule algae culturing liquid that step S12 is cultivated to be poured into high-temp steam sterilizing, bottleneck
Bacteriological filtration film is covered, standing about 4 hours in the case where normal sunlight irradiates room temperature (about 23 DEG C), i.e. visible level floats bottle green algal layer, such as
Shown in Fig. 3；Using 1ml liquid-transfering gun, pipette tips slowly draw floating cyanobacteria close to floating algal layer surface, are collected in 15ml centrifuge tube,
Standing is layered to algae with culture solution completely, and liquid-transfering gun exhausts culture supernatants as far as possible.It wouldn't such as need for radiography, it can be by algae solution
It is placed in 4 DEG C of environment.It is learnt through experiment, the time saved at this temperature about 1 year.
Embodiment 3: the cultural method of the planktonic algae containing pseudo- ghost
The cultural method for present embodiments providing a kind of planktonic algae containing pseudo- ghost, includes the following steps:
S11: the culture (about 10 days) for algae deadtime of quivering: the algae that will quiver pours into 50ml conical flask, according to the volume ratio of 1:50
Cyanobacteria BG11 culture medium is added, shakes up, insertion airway connects air pump in conical flask, clogs taper bottleneck with sterilizing cotton
Afterwards, it is sealed with sealing strip (paraflim), keeps being gnotobasis in bottle；Culture light application time is 14h illumination/10h dark, temperature
25 DEG C, shaking speed 100rpm of degree obtains upper layer swim alga until to become green apple again green for liquid in bottle；
S12: logarithmic growth phase culture (about 4 days): 3 parts are divided by swim alga is obtained in step S11, is poured into respectively
Fresh BG11 culture medium to 250ml final volume is added in 500ml conical flask to continue to cultivate, therefore phase cell proliferation is accelerated, about 4 days
I.e. visible culture solution color becomes emerald green, can continue mass propgation after sub-bottle at this time, when algae is presented as shown in Fig. 2-C in bottle
Kingfisher (ink) it is green when, illustrate to be again introduced into growth retardation phase (resting stage), following step S13 separating treatment can be carried out；
S13: step S12 is cultivated in the 500ml conical flask after the algae culturing liquid that quivers pours into high-temp steam sterilizing, bottleneck covering
Bacteriological filtration film, normal sunlight irradiate room temperature (about 23 DEG C) under stand about 4 hours i.e. visible level float bottle green algal layer, such as Fig. 3
It is shown；Using 1ml liquid-transfering gun, pipette tips slowly draw floating cyanobacteria close to floating algal layer surface, are collected in 15ml centrifuge tube, stand
It is layered completely to algae with culture solution, liquid-transfering gun exhausts culture supernatants as far as possible.It wouldn't such as need algae solution can be placed for radiography
In 4 DEG C of environment.It is learnt through experiment, the time saved at this temperature about 1 year.
Embodiment 4: the preferred cultural method of the planktonic algae containing pseudo- ghost:
The cultural method for present embodiments providing a kind of planktonic algae containing pseudo- ghost, includes the following steps:
S11: it the culture (about 10 days) of Anabaena Flos-aquae deadtime: opens Anabaena Flos-aquae freeze-dried powder and (it is raw to be purchased from the U.S.
Object standard items collecting center ATCC) lid, one day rewarming is placed under room-temperature sterile environment, and freeze-dried powder is poured into 50ml taper
In bottle, cyanobacteria BG11 culture medium is added according to the volume ratio of 1:50, shakes up, insertion airway connects air pump in conical flask, uses
It sterilizes after cotton filling taper bottleneck, is sealed with sealing strip (paraflim), keep being gnotobasis in bottle；Cultivate light application time
Dark for 14h illumination/10h, 25 DEG C of temperature, shaking speed 100rpm, culture solution is from initial such as Fig. 2-A institute after culture about 6 days
When the light green shown becomes light green color, equivalent fresh medium is added and continues to cultivate, deepens to culture solution green to such as Fig. 2-B institute
The green apple shown is changed to 250ml conical flask culture when green, and fresh culture is added to final volume about 150ml, continues culture about 4
It is green that it to liquid in bottle becomes green apple again；
S12: after the swim alga is cultured to logarithmic growth phase, the swim alga expansion culture of logarithmic phase: will be contained
BG11 culture based on stewing process at sunlight, the swim alga for floating on the BG11 media surface is taken out and is seeded to floating
Sterile aerobic culture processing is carried out in trip algae G625 culture medium, cultivates again to logarithmic phase, G625 culture medium is divided into 2
Part, addition G625 culture medium to 250ml final volume in 500ml conical flask is poured into respectively and continues to cultivate, therefore phase cell proliferation adds
Fastly, about 4 days i.e. visible culture solution colors become emerald green, can continue mass propgation after sub-bottle at this time, when algae is presented such as in bottle
When kingfisher shown in Fig. 2-C (ink) is green, illustrates to be again introduced into growth retardation phase (resting stage), following step S13 can be carried out
Separating treatment；
S13: in the 500ml conical flask after the Anabaena Flos-aquae culture solution that step S12 is cultivated to be poured into high-temp steam sterilizing,
Bottleneck covers bacteriological filtration film, and standing about 4 hours in the case where normal sunlight irradiates room temperature (about 23 DEG C), i.e. visible level floats bottle green algae
Layer, as shown in Figure 3；Using 1ml liquid-transfering gun, pipette tips close to floating algal layer surface slowly draws float cyanobacteria, be collected in 15ml from
Heart pipe, standing are layered to algae with culture solution completely, and liquid-transfering gun exhausts culture supernatants as far as possible.It wouldn't such as need for radiography, it can
Algae solution is placed in 4 DEG C of environment.It is learnt through experiment, the time saved at this temperature about 1 year.
Embodiment 5: oral acoustic contrast agent embodiment
The present embodiment provides a kind of oral acoustic contrast agents, specifically provide by the following method:
Embodiment is applied to the Anabaena Flos-aquae collected in 1 and appropriate PBS isotonic solution mixed processing, sets up concentration, setting 3
The oral acoustic contrast agent of a concentration gradient.
(because not diluted is made after the oral acoustic contrast agent isotonic solution 1:10 of 3 concentration gradients of acquisition is diluted
Shadow agent excessive concentration, microplate reader can not measure its concentration), using microplate reader (wavelength sets 500nm) measurement OD value, and use PBS
Isotonic solution is as control.OD value through measuring the oral acoustic contrast agent of 3 concentration gradients after diluting is respectively 0.2/
0.5/0.8, the photo of each concentration is as shown in Figure 5.
Certain oral acoustic contrast agent concentration can also use the side such as counting or granular cell calculating instrument under microscope
Formula quantifies.
Embodiment 6: the oral in-vitro simulated ultrasonic imaging experiment of acoustic contrast agent
The oral acoustic contrast agent that the OD500 value provided in above-described embodiment 3 is respectively 0.2/0.5/0.8 is carried out respectively
The experiment of external supersonic radiography, specific experiment method are as follows: body opening is imitated to the agar for filling algae using Vevo2100 ultrasonic imaging device
Carry out radiography, alignment probe well.200 μ L algae samples are added in 4 holes respectively, PBS is added in the 1st hole as blank
It compares, the contrast agent suspension that the OD value prepared in embodiment 3 is 0.2,0.5,0.8 is separately added into the hole 2-4, is filled before sample-adding
Divide oscillation to shake up, and observes its radiography situation under two-dimensional ultrasound mode and under contrast mode and measure its echo signal intensity
(probe setting: two-dimensional model 40MHZ, imaging pattern 18HMZ).
Experimental result: the contrast agent of OD500=0.2,0.5,0.8 imitates body 18MHz ultrasonic imaging such as Fig. 6 in vitro.It measures
A, u value broken line are as shown in Figure 7.By Fig. 6 and it is shown in Fig. 7 the result shows that, in PBS control hole two dimension and contrast mode under all without
Obvious echo, average echo intensity a, u 192.6.The contrast agent of OD500=0.2,0.5,0.8 can be clear under two modes
Clear imaging, average echo intensity a, u is improved, respectively 641.5,1262.8,1732.8 with the increase of concentration, wherein with OD
The imaging effect of value=0.5 and 0.8 is more satisfactory.
Embodiment 7: ultrasonic imaging experiment in oral acoustic contrast agent body
The oral acoustic contrast agent that the OD value provided in above-described embodiment 3 is respectively 0.2/0.5/0.8 is carried out respectively small
Mouse gastrointestinal series experiment in vivo, specific experiment method are as follows: randomly select the BALB/c mouse of 48 week old of health, about 23g, SPF
Grade is deprived of food but not water processing 12 hours before experiment, Baoding carries out shaving to abdomen on mouse plate after isoflurane gas anesthetized mice
Preserved skin.It is detected using vevo 2100 in mouse left upper abdomen, the ultrasonic imaging situation before observation experiment.Sufficiently shake up 3 concentration
After the contrast agent suspension of gradient, extract 0.2ml sample and give the processing of 3 intragastric administration on mice respectively, carried out at once after stomach-filling ultrasound at
Picture observes oesophagus and stomachus cardiacus, body of stomach, stomachus pyloricus in two-dimentional high frequency probe respectively, compares gastral cavity signal and liver kidney letter
Number, its radiography situation is observed under contrast mode and measures its echo intensity.Blank control group mouse uses same amount of normal saline
Stomach-filling.
Experimental result: blank control group and experimental mice oesophagus and stomachus cardiacus, stomach under 40MHZ two dimension high frequency condition
As shown in Fig. 8-A, blank control group and the experimental mice stomach wall under two-dimentional high frequency condition are imaged for body portion, stomachus pyloricus image
Figure is as shown in Fig. 8-B, time of blank control group and experimental mice contrast agent filling gastral cavity and liver, kidney under two-dimensional condition
Acoustic imaging figure is as shown in Fig. 8-C, under Contrast imaging pattern shown in imaging contrast Fig. 8-D, a measured, u value broken line such as Fig. 9
It is shown,.By Fig. 8 and it is shown in Fig. 9 the result shows that, it is more that PBS control group is imaged pneumatosis in visible magenblase, gastral cavity and stomach wall structure
It can not clearly show, average a under Photo condition, u value is 161.2.Pour into the oral algae contrast agent for ultrasonic imaging of various concentration
Mouse, air in the visible contrast agent uniform pack gastral cavity of two-dimensional imaging, emptying stomach, comparison is obvious, and stomach wall structure echo can
Clear display.See the strong echo uniform pack gastral cavity of contrast agent under contrast mode, the clear-cut display echo intensity of gastral cavity is averaged a, u
Value improves, respectively 641.5,4731.7,5559.8 with the increase of concentration.It is suspended with the contrast agent of OD500=0.8 concentration
Liquid imaging effect is best, and gastral cavity filling display is preferable under contrast mode.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (10)
1. the planktonic algae containing pseudo- ghost is preparing the application in oral acoustic contrast agent.
2. application according to claim 1, it is characterised in that: the planktonic algae containing pseudo- ghost is the cyanobacteria that swims.
3. application according to claim 2, it is characterised in that: the cyanobacteria that swims includes anabena, synnema algae, Microcystis aeruginosa
With at least one of the algae that quivers.
4. a kind of oral acoustic contrast agent, it is characterised in that: containing the planktonic algae for ultrasonic development, and the swim alga contains
There is pseudo- ghost structure.
5. oral acoustic contrast agent according to claim 4, it is characterised in that: the content of the planktonic algae is optical density
Method detects OD500=0.5 or more.
6. a kind of cultural method of the planktonic algae containing pseudo- ghost, includes the following steps:
Swim alga containing pseudo- ghost is seeded in swim alga primary culture medium, and is led into the swim alga primary culture medium
Enter sterile oxygen-containing gas and carries out sterile aerobic culture processing；
After the swim alga is cultured to logarithmic growth phase, the swim alga of logarithmic growth phase is seeded to swim alga and is expanded
Culture medium carries out sterile aerobic expansion culture processing；
To carry out stewing process through the sterile aerobic swim alga for expanding culture processing, after will float on the swim alga and expand
The algal layer of media surface is exported from the swim alga media surface and is collected.
7. cultural method according to claim 6, which is characterized in that by the algal layer from the swim alga media surface
The method that export is collected includes the following steps:
The algal layer is directly expanded media surface export from the swim alga by the way of absorption to collect.
8. cultural method according to claim 6 or 7, which is characterized in that the method for the swim alga stewing process includes
Following steps: aerobic stewing process will be carried out in sunlight irradiation environment through the aerobic swim alga for expanding culture.
9. cultural method according to claim 6 or 7, which is characterized in that the sterile aerobic originally culture processing method
Include the following steps:
After the swim alga is seeded in the swim alga primary culture medium, it is placed in illumination and is protected from light in the environment of checker
Sterile aerobic culture is carried out until adding fresh swim when the swim alga primary culture medium becomes light green color from light green
Algae originally culture base fluid continues sterile aerobic culture；
When deepening green to green apple by light green color to the swim alga primary culture medium wait cultivate, fresh swim alga is added again
Primary culture medium carries out sub-bottle to former algae solution of swimming and supplements fresh swim alga primary culture medium, continues sterile aerobic
Culture is until the algae culture medium color burn that swims is green to green apple.
10. cultural method according to claim 6 or 7, which is characterized in that the sterile aerobic side for expanding culture processing
Method includes the following steps:
After the swim alga is cultured to logarithmic growth phase, by the primary training of the swim alga containing logarithmic growth phase swim alga
Feeding base is divided at least two parts, and is separately added into fresh swim alga and expands the sterile aerobic culture processing of culture medium progress, until described
When swim alga expansion culture medium is presented emerald green, the emerald swim alga is expanded into culture medium again and is divided at least two parts,
Fresh swim alga is added again to expand culture medium and carry out sterile aerobic culture, until the algae culture medium presentation of swimming is emerald green
Color；
Or
After the swim alga is cultured to logarithmic growth phase, the originally culture containing the swim alga is based at sunlight at standing
Reason, the swim alga for floating on the originally culture primary surface is taken out and is seeded in swim alga expansion culture medium sterile have
Oxygen culture processing.
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| CN111035771A (en) * | 2019-12-13 | 2020-04-21 | 深圳先进技术研究院 | Nano ultrasonic contrast agent and preparation method thereof |
| CN111166897A (en) * | 2019-11-25 | 2020-05-19 | 彭利 | Sonoweiwei compound contrast agent and application |
| CN113186134A (en) * | 2021-05-17 | 2021-07-30 | 中国科学院水生生物研究所 | Blue algae air sac extraction method and application thereof |
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