CN109329123B - Method for collecting intestinal mucus of aquatic animals - Google Patents
Method for collecting intestinal mucus of aquatic animals Download PDFInfo
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- CN109329123B CN109329123B CN201811190241.1A CN201811190241A CN109329123B CN 109329123 B CN109329123 B CN 109329123B CN 201811190241 A CN201811190241 A CN 201811190241A CN 109329123 B CN109329123 B CN 109329123B
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- 241001465754 Metazoa Species 0.000 title claims abstract description 54
- 230000000968 intestinal effect Effects 0.000 title claims abstract description 37
- 210000003097 mucus Anatomy 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 31
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 51
- 239000002041 carbon nanotube Substances 0.000 claims abstract description 51
- 229910021393 carbon nanotube Inorganic materials 0.000 claims abstract description 51
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 239000002904 solvent Substances 0.000 claims description 6
- 235000003642 hunger Nutrition 0.000 claims description 3
- 230000037351 starvation Effects 0.000 claims description 3
- 239000000243 solution Substances 0.000 description 31
- 241000269368 Xenopus laevis Species 0.000 description 25
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 10
- 239000002048 multi walled nanotube Substances 0.000 description 8
- 239000012528 membrane Substances 0.000 description 7
- 102000011022 Chorionic Gonadotropin Human genes 0.000 description 6
- 108010062540 Chorionic Gonadotropin Proteins 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 229940084986 human chorionic gonadotropin Drugs 0.000 description 6
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- 238000011173 large scale experimental method Methods 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
-
- 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
- Y02A40/81—Aquaculture, e.g. of fish
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Marine Sciences & Fisheries (AREA)
- Animal Behavior & Ethology (AREA)
- Carbon And Carbon Compounds (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of bioscience, and particularly relates to a method for collecting intestinal mucus of aquatic animals. The invention provides a method for collecting intestinal mucus of aquatic animals, which comprises the following steps: step 1, feeding a carbon nanotube solution to aquatic animals; step 2, collecting the excrement of the aquatic animals fed with the carbon nano tube solution; and 3, centrifuging the excrement to obtain the intestinal mucus of the aquatic animal. The collecting method provided by the invention fills the gap that no aquatic animal intestinal mucus collecting method with high biological safety exists at present.
Description
Technical Field
The invention belongs to the technical field of bioscience, and particularly relates to a method for collecting intestinal mucus of aquatic animals.
Background
With the development of modern industrial technology, the influence of new pollutants on the environment is increasingly prominent, such as micro plastics, antibiotics and the like. Many animals or humans die each year from diseases caused by environmental pollution. It has been recently reported that micro-plastics can be detected in marine products such as fish, shrimp, crab, etc., and they are concentrated in the cheek or intestinal tract of animals and cause various injuries and even death. Most animal diseases are caused by the explosive reproduction of pathogens in the gut, eventually leading to death. It has been reported that among stools from 142 infants aged 2-7 in Finland, the macrolide antibiotics, which were analyzed to give the greatest change in the bacterial flora, accounted for 25% of the total Finland antibiotics sold. Therefore, the research on a method for rapidly detecting whether pollutants damage the intestinal tract and the digestive system of an animal or human body is not slow enough.
The aquatic animals are used as important components in the ecological environment, and the influence and the harm degree of the pollutant change of the ecological environment on the intestinal tracts and the digestive systems of animals or human bodies can be directly reflected by utilizing the structural change of the intestinal mucus flora of the aquatic animals, so that the discharge of pollution sources can be controlled, and the human health and the ecological health are better protected. However, no method for collecting intestinal mucus of aquatic animals with high biosafety has been reported at present.
Disclosure of Invention
In view of the above, the present invention provides a method for collecting intestinal mucus of aquatic animals with high biological safety and high efficiency.
The invention provides a method for collecting intestinal mucus of aquatic animals, which comprises the following steps:
and 3, centrifuging the excrement to obtain the intestinal mucus of the aquatic animal.
The invention mainly utilizes the characteristics of high adsorbability, large specific surface area, difficult degradability and no toxic effect on animals.
Preferably, the concentration of the carbon nanotubes in the carbon nanotube solution in a culture system is 1mg/L-10 mg/L.
More preferably, the concentration of the carbon nano tubes in the carbon nano tube solution in a culture system is 1mg/L-5 mg/L.
Wherein, the concentration of the carbon nano tube solution is too high to directly cause damage and death of gastrointestinal barrier of aquatic animals, and the concentration is too low to collect enough intestinal mucus.
Preferably, the solvent of the carbon nanotube solution is water.
More preferably, the solvent of the carbon nanotube solution is an aquatic animal acceptable solution.
Preferably, the pore diameter of the carbon nanotube is 0 to 50 μm.
Preferably, the preparation method of the carbon nanotube solution is as follows: and ultrasonically mixing the carbon nano tube with the solvent to prepare a carbon nano tube solution.
Preferably, the frequency of the ultrasonic wave is 40HZ, and the ultrasonic time is 30-50 min.
Wherein, the ultrasonic mixing makes the obtained carbon nano tube uniformly dispersed in the solvent to form a uniform carbon nano tube solution which is used as a subsequent feeding material.
Preferably, the aquatic animal is an aquatic animal in a starvation state.
Preferably, the aquatic animals in a starved state have a starvation time of 24 hours.
Preferably, in step 1, the feeding time is 60-120 min.
Wherein the feeding time is not too long, otherwise the feed is enriched in aquatic animals and difficult to excrete.
Preferably, the intestinal mucus of the aquatic animal is preserved at 4-8 ℃.
It is to be noted that the aquatic animal of the present invention is an aquatic animal capable of feeding carbon nanotubes, such as fish or early tadpoles.
Preferably, the aquatic animals are tadpoles of xenopus laevis, specifically xenopus laevis Stage49-50 tadpoles.
Specifically, the method for obtaining xenopus laevis Stage49-50 tadpoles comprises the following steps: a pair of sexual mature tropical xenopus laevis are selected and put into a self-made propagation culture box, and the method of manually injecting Human Chorionic Gonadotropin (HCG) is adopted to induce spawning. Performing subcutaneous injection for 2 times, performing interval of 24-28h after the first injection, performing the second injection, placing into an incubator, and collecting embryo after the Xenopus laevis naturally embraces and spawns for about 3-5 h. Treating with 3% L-cysteine with pH of 7.9-8.1 for 2-3min, removing membrane, rinsing with 7.3-7.5 embryo culture solution for 6-7 times, culturing with 1-1.5% agarose culture dish, and culturing for 12-14 days to obtain experimental Xenopus laevis stage49-50 tadpoles.
The xenopus laevis is a classical model organism, the genomics of the xenopus laevis is similar to that of human beings by 87%, and the intestinal tract of xenopus laevis tadpoles can be compared with that of human beings. In addition, the tropical xenopus laevis has the characteristics of short culture period, large egg laying amount and easy survival, and can be used for large-scale experiments. The invention adopts the xenopus laevis Stage49-50 tadpoles, because the tadpole body shape permeability in the period is good, the whole process of ingestion, digestion and excretion of substances is easy to observe.
Wherein the injection interval time between the first injection and the second injection is 24-28h, preferably 25 h.
Wherein the pH of the L-cysteine is 7.9-8.1, preferably 8.0.
Wherein the embryo membrane eliminating time of L-cysteine is 2-3min, preferably 2.5 min.
Wherein the pH value of the embryo culture solution is 7.3-7.5, preferably 7.4.
Wherein, the mass percent of the agarose is between 1 percent and 1.5 percent, and is preferably 1.2 percent.
Wherein, the concentration range of the carbon nano tube in the culture system is 1-10mg/l, and preferably 5 mg/l.
More preferably, the culture system specifically comprises: aerating distilled water for 24h to make the dissolved oxygen in the water reach more than 90%, and then filtering by a 0.45-micron microporous filter membrane to obtain a culture system.
Preferably, step 2 specifically comprises: adding the carbon nanotube solution into a simulated culture system, keeping the concentration of the carbon nanotubes in the culture system at 1-10mg/L, continuously exposing, stopping feeding after the aquatic animals eat the carbon nanotubes for 1-2h and are in a half-full state, and standing; the step 3 specifically comprises the following steps: and collecting excrement by using a suction pipe after the aquatic animals excrete so as to be convenient for a centrifugal pipe, oscillating and uniformly mixing the excrement and the excrement to fully dissolve, and centrifuging to obtain supernatant liquid to obtain the intestinal mucus of the aquatic animals.
The invention discloses a method for collecting intestinal mucus of aquatic animals, which is mainly characterized in that a carbon nano tube solution is used for feeding the aquatic animals, and the carbon nano tube has the characteristics of difficult degradability, no biotoxicity, high adsorbability, large specific surface area and the like, so that the intestinal mucus can be better attached to the carbon nano tube solution. Due to the difficult degradability, the carbon nano tubes can be finally excreted in a way that the animals swallow and vomit by themselves, the aquatic animals cannot be damaged, and the carbon nano tubes can be recycled on the premise of not damaging and killing the aquatic animals. The collecting method provided by the invention collects the feces pulled out after the carbon nano tubes are ingested in the fasting state, and centrifuges to obtain the intestinal mucus of the aquatic animals, so that a large amount of intestinal mucus can be rapidly collected and detected and analyzed, and the method can be used in the fields of medical detection, environmental monitoring and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows a scanning electron micrograph of a multiwalled carbon nanotube of an embodiment of the invention;
fig. 2 shows an excretion diagram of xenopus laevis Stage49-50 tadpoles of example 1 of the present invention;
fig. 3 shows a micrograph of stool from xenopus laevis Stage49-50 tadpoles according to example 1 of the present invention, wherein 1 is multiwall carbon nanotubes, and 2 is intestinal mucus;
fig. 4 shows intestinal mucus supernatant after centrifugation of feces according to example 1 of the present invention, wherein 3 is intestinal mucus supernatant.
Detailed Description
The invention provides a method for collecting intestinal mucus of an aquatic animal, which fills the gap that no method for collecting intestinal mucus of an aquatic animal with high biological safety exists at present.
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The raw materials used in the following examples are all commercially available or self-made.
Example 1
The embodiment provides a method for collecting intestinal mucus of an aquatic animal, comprising the following steps:
(1) obtaining xenopus laevis Stage49-50 tadpoles: a pair of bred and sexually mature tropical xenopus laevis are selected and put into a self-made propagation culture box, and the method of manually injecting Human Chorionic Gonadotropin (HCG) is adopted to induce spawning. Each Xenopus laevis is injected subcutaneously for 2 times at intervals of 24h, and then placed in an incubator until the Xenopus laevis embraces the eggs, and embryos can be collected after 3 h. Treating with 3% L-cysteine (pH 8.7.9) for 2.5min to remove membrane, rinsing with 7.3% embryo culture solution for 6 times, culturing with 1.2% agarose culture dish, and culturing for 13 days to obtain tadpoles of stage 49-50.
(2) Establishing a simulated culture system: aerating the contained distilled water for more than 24 hours to ensure that the dissolved oxygen in the water is more than 90 percent, filtering the water by using a 0.45um microporous filter membrane to obtain experimental water, and placing the experimental water in an experimental tank for later use to form a simulated culture system.
(3) Preparing a carbon nanotube solution: weighing 0.01g of multi-walled carbon nanotube in a beaker, uniformly mixing with 100ml of distilled water, putting the mixture into an ultrasonic cleaner, and carrying out ultrasonic treatment for half an hour under the condition of 40HZ to uniformly disperse the mixture to obtain a carbon nanotube solution.
(4) Obtaining intestinal mucus: and (3) feeding the carbon nanotube solution obtained in the step (3) and the tadpoles starved for 24 hours into a simulated culture system, so that the concentration of the carbon nanotubes in the carbon nanotube solution in the simulated culture system is 5mg/L, continuously exposing, stopping feeding the tadpoles after the tadpoles are in a half-full state after the tadpoles eat the carbon nanotube solution, standing, collecting excrement by using a suction pipe after the tadpoles are excreted so as to be convenient for a centrifugal tube, oscillating and uniformly mixing the excrement, fully dissolving the excrement, and centrifuging to obtain supernatant liquid of the excrement, thereby obtaining the intestinal mucus of the Stage49-50 tadpoles.
Example 2
The embodiment provides a second method for collecting intestinal mucus of an aquatic animal, comprising the following steps:
(1) obtaining xenopus laevis Stage49-50 tadpoles: a pair of bred and sexually mature tropical xenopus laevis are selected and put into a self-made propagation culture box, and the method of manually injecting Human Chorionic Gonadotropin (HCG) is adopted to induce spawning. Each Xenopus laevis is injected subcutaneously for 2 times at intervals of 28h, and then placed in an incubator until the Xenopus laevis embraces the eggs, and embryos can be collected after 5 h. Treating with 3% L-cysteine with pH of 8.1 for 2.5min for membrane removal, rinsing with 7.5% embryo culture solution for 7 times, culturing with 1.2% agarose culture dish, and culturing for 13 days to obtain tadpoles of stage 49-50.
(2) Establishing a simulated culture system: aerating the contained distilled water for more than 24 hours to ensure that the dissolved oxygen in the water is more than 90 percent, filtering the water by using a 0.45um microporous filter membrane to obtain experimental water, and placing the experimental water in an experimental tank for later use to form a simulated culture system.
(3) Preparing a carbon nanotube solution: weighing 0.01g of multi-walled carbon nanotube in a beaker, uniformly mixing with 100ml of distilled water, putting the mixture into an ultrasonic cleaner, and carrying out ultrasonic treatment for half an hour under the condition of 40HZ to uniformly disperse the mixture to obtain a carbon nanotube solution.
(4) Obtaining intestinal mucus: and (3) feeding the carbon nanotube solution obtained in the step (3) and the tadpoles starved for 24 hours into a simulated culture system, so that the concentration of the carbon nanotubes in the carbon nanotube solution in the simulated culture system is 1mg/L, continuously exposing, stopping feeding the tadpoles after the tadpoles are in a half-full state after the tadpoles eat the carbon nanotube solution, standing, collecting excrement by using a suction pipe after the tadpoles are excreted so as to be convenient for a centrifugal tube, oscillating and uniformly mixing the excrement, fully dissolving the excrement, and centrifuging to obtain supernatant liquid of the excrement, thereby obtaining the intestinal mucus of the Stage49-50 tadpoles.
FIG. 1 is a scanning electron microscope image of a multi-walled carbon nanotube, which shows that the multi-walled carbon nanotube has a tubular structure; fig. 2 is a diagram showing excretion of xenopus laevis Stage49-50 tadpoles in example 1, wherein it can be seen that feces of xenopus laevis Stage49-50 tadpoles contain intestinal mucus and multi-walled carbon nanotubes; fig. 3 is a micrograph of stool from xenopus laevis Stage49-50 tadpoles of example 1, and it can be seen from fig. 3 that intestinal mucus contains a large amount of black multi-walled carbon nanotubes; fig. 4 shows the supernatant of intestinal mucus after centrifugation of stool in example 1, which shows that intestinal mucus can be obtained only by centrifugation after collecting stool from xenopus laevis Stage49-50 tadpoles, and the separation process is very simple.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. A method for collecting intestinal mucus of aquatic animals is characterized by comprising the following steps:
step 1, feeding aquatic animals and a carbon nano tube solution into a culture system, and feeding the aquatic animals with the carbon nano tube solution; the concentration of the carbon nano tubes in the carbon nano tube solution in a culture system is 1mg/L-10mg/L, and the aperture of the carbon nano tubes is 0-50 mu m;
step 2, collecting the excrement of the aquatic animals fed with the carbon nano tube solution;
and 3, centrifuging the excrement to obtain the intestinal mucus of the aquatic animal.
2. The method of claim 1, wherein the solvent of the carbon nanotube solution is water.
3. The method of claim 2, wherein the carbon nanotube solution is prepared by: and ultrasonically mixing the carbon nano tube with the solvent to prepare a carbon nano tube solution.
4. The method as set forth in claim 3, wherein the ultrasonic frequency is 40Hz and the ultrasonic time is 30-50 min.
5. The method of claim 1, wherein the aquatic animal is starved.
6. The method as set forth in claim 5, wherein the starvation time of the starved aquatic animals is 24 hours.
7. The method as set forth in claim 1, wherein the feeding time is 60-120min in step 1.
8. The method of claim 1, wherein the intestinal mucus of the aquatic animal is stored at 4-8 ℃.
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| Title |
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