CN102899385A - Application of zebra fish to testing safety of safe substance and method for applying zebra fish to test safety of safe substance - Google Patents
Application of zebra fish to testing safety of safe substance and method for applying zebra fish to test safety of safe substance Download PDFInfo
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- CN102899385A CN102899385A CN2012104553476A CN201210455347A CN102899385A CN 102899385 A CN102899385 A CN 102899385A CN 2012104553476 A CN2012104553476 A CN 2012104553476A CN 201210455347 A CN201210455347 A CN 201210455347A CN 102899385 A CN102899385 A CN 102899385A
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Abstract
The invention relates to an application of zebra fish to testing safety of a safe substance and a method for applying zebra fish to test safety of the safe substance. The method is characterized by comprising the following steps of: preparing an embryo culture solution, then adding the safe substance to be tested to the embryo culture solution, using the embryo culture solution to which the safe substance to be tested is added to culture zebra fish embryos and then observing and recording the developmental aberration rates and mortality of the zebra fish embryos in 4-72 hours to judge the toxicity of the safe substance to be tested toward development of the zebra fish embryos, wherein the addition of the safe substance to be tested ensures that the concentration of the substance to be tested in the culture solution is distributed in echelon; the embryo culture solution is directly prepared from alkalescent water with pH value being 6.7-8.5; and the embryo culture solution contains 5mM of NaCl, 0.17mM of KCl, 0.43mM of CaCl2 and 0.33mM of MgSO4. The method has the following beneficial effect that the embryo culture solution prepared from the alkalescent water ensures that the zebra fish embryos can quickly test the comprehensive toxicity of the safe substance to be tested, thus shortening the detection period.
Description
Technical field
The present invention relates to the toxicity detection field, be specifically related to purposes and method that a kind of spot zebra fish is used for the security of test safety material.
Background technology
Along with the minimizing of natural production mode, industrialization proportion increases gradually, and food raw material is conscious or can introduce unconsciously some chemical substances in the processes such as production, storage, transportation, processing, and this comes increasing unsafe factor for the food securing band.
The use of chemical substance in food is with a long history, has chemical substance together in the production of modern food, processing, storing and the process such as edible, and also these chemical substances being widely used in food just is just so that foodstuffs industry becomes sunrise industry.But the use of chemical substance has brought very important impact also for the security of food, comprising the impact on food safety of the natural poisonous substance matter in drug residue, heavy metal, the food, food-processing, packing, storage.
Especially foodstuff additive, satisfy the index of some aspect in order to reach food, having added can't clear and definite qualitatively material to organism and human-body safety, if these materials are tested in organism, then test period can be long, add human body to the tolerance degree of dangerous material, can't find out in a short time whether certain foodstuff additive have stealthy harm to human body.
The present invention found through experiments, and zebra fish can provide to the material of the unknown safety relatively accurate safety expection.Particularly, the present invention after the adding safe material to be measured, can make the embryo that the untoward reaction of safe material to be measured is showed in the short period of time by using special embryo medium to cultivate zebra fish in nutrient solution, has greatly shortened sense cycle.
Summary of the invention
For solving the problems of the technologies described above, the invention provides purposes and method that a kind of zebra fish is used for the security of test safety material.Particularly, the invention provides the method that a kind of zebra fish is used for the security of test safety material, it is characterized in that the method comprises following steps:
The preparation embryo medium, then in embryo medium, add safe material to be measured, the add-on of safe material to be measured is so that the concentration of test substance in nutrient solution is the echelon distribution, cultivate zebrafish embryo with the embryo medium that has added safe material to be measured, then in abnormal rate and the mortality ratio of 4-72 hour observed and recorded Zebrafish Embryo, judge that safe material to be measured is to the toxicity of Zebrafish Embryo.Wherein embryo medium pH is that the alkalescent water of 6.7-8.5 is directly prepared, and contains 5mM NaCl, 0.17mM KCl, 0.43mM CaCl in the embryo medium
2With 0.33mM MgSO
4
In the aforesaid method, zebrafish embryo is selected by the following method:
Adopt ovum the day before yesterday with sexually matured zebra fish, in female: hero is that 1: 2 ratio is put into and joined cylinder and separate, 9:00 natural crossing in morning next day is also collected zygote, clean the zygote that twice collection obtains with embryo medium, and removal impurity and bad ovum, be placed on afterwards in the embryo medium, control light for 29.3 ℃ and cultivate; When 2hpf, select from same parent, the normal development ratio reaches more than 85% and developmental stage is consistent zebrafish embryo at microscopically.
In the aforesaid method, the method of cultivating zebrafish embryo is as follows: zebrafish embryo is cleaned twice with embryo medium, then move in the sample hole of 24 orifice plates, 20 pieces in every hole, every hole has added the embryo medium of safe material to be measured, and every hole 4ml adds a cover sealing subsequently, place incubator, cultivate zebrafish embryo at 29.3 ℃.
In the aforesaid method, the time of observed and recorded is to cultivate record abnormal rate and mortality ratio rear the 4th, 12,24,48,72 hour.
In the aforesaid method, pH is 7.0-7.8, and preferably, pH is 7.2.
Deformity refers to that the zebra fish build of the alone embryo medium cultivation of zebra fish build and normal identical incubation time is variant.
Zebra fish was without any reaction when death referred to stirring rod touching zebra fish afterbody.
In the aforesaid method, described safe material to be measured is for needing its any material to biological safety of checking, comprise organism or inorganics, regardless of the solubleness of safe material in water, can be as the safe material to be measured of aforesaid method measurement of the present invention.For the high material of solubleness in the water, directly it is dissolved in the embryo medium according to concentration gradient, for the low solid matter of solubleness in the water, it is crushed to joins the embryo medium high speed behind the 500-1000 order and stir and make suspension.For the low liquid substance of solubleness in the water, will join the stirring of embryo medium high speed and make suspended emulsion.Preferably, safe material comprise think to the poisonous material of human body and it is generally acknowledged but may be poisonous to biological nontoxic safe material.Especially preferably, safe material is such as being the foodstuff additive such as Sodium Glutamate, trimeric cyanamide, Tartrazol yellow, Sodium Benzoate, Sodium Nitrite.
The present invention also provides zebra fish to be used for testing the purposes of safe material toxicity to be measured, it is characterized in that: in embryo medium, add safe material to be measured, the add-on of safe material to be measured is so that the concentration of test substance in nutrient solution is the echelon distribution, cultivate zebrafish embryo with the embryo medium that has added safe material to be measured, then in abnormal rate and the mortality ratio of 4-72 hour observed and recorded Zebrafish Embryo, judge that safe material to be measured is to the toxicity of Zebrafish Embryo.Wherein embryo medium pH is that the alkalescent water of 6.7-8.5 is directly prepared, and contains 5mM NaCl, 0.17mM KCl, 0.43mM CaCl in the embryo medium
2And 0.33mMMgSO
4
The present invention prepares the used sodium-chlor of embryo medium, Repone K, calcium chloride, sal epsom and alkalescent water etc. and is commercial, and wherein inorganic salt are the AR level;
Of particular note, alkalescent water can adopt any alkalescent water that obtains by photoelectrocatalysiwater water electrolysis, the pH of this alkalescent water is 6.7-8.5, can prepare by the alkalescent water preparation facilities on the market, also can be voluntarily mode by electrolysis prepare, it should be noted that alkalescent water of the present invention is not the alkalescent water for preparing by add chemical substance in water, any is not the said alkalescent water of the present invention by adding the alkalescent water that chemical substance prepares in the water.The pH of alkalescent water can regulate in preparation, rather than regulates pH by adding chemical substance.
Beneficial effect of the present invention:
The present invention uses the embryo medium of alkalescent water preparation to be used as testing the nutrient solution of safe material toxicity to be measured, it is not only because the water in the nutrient solution is alkalescent water, also because the specific composition of the inorganic salt in the nutrient solution, so that zebrafish embryo can be tested the comprehensive toxicity of safe material to be measured fast, shortened sense cycle.
Embodiment
In order to understand the present invention, the below further specifies the present invention with embodiment, but the present invention is not restricted to this.
The screening of embodiment 1 best embryo medium
1.1 the preparation of the embryo medium of different ingredients and the effect of cultivating zebra fish
A comprises the embryo medium of following concentration inorganic salt with the ortho-water preparation, regulating pH is 7.2, cultivates zebrafish embryo 72 hours, observes transparency and the survival rate of zebra fish.Concrete outcome sees Table 1:
The embryo medium of table 1 ortho-water preparation is on the impact of zebrafish embryo
It is the embryo medium that 7.2 alkalescent water preparation comprises following concentration inorganic salt that B uses pH by the electrolysis pure water preparation, cultivates zebrafish embryo 72 hours, observes transparency and the survival rate of zebra fish.Concrete outcome sees Table 2:
The embryo medium of table 2 alkalescent water preparation is on the impact of zebrafish embryo
The nutrient solution of the different concns ratio by the preparation of ortho-water and alkalescent water can find out, the nutrient solution of A1 and B1 is best to the culture effect of zebra fish.The inorganic salt concentration that the present invention of this proof adopts is preferred as nutrient solution.
The toxicity of embodiment 2 zebra fish test safety materials
Embryo medium with above-mentioned numbering A1 and B1 adds safe material to be measured respectively, the content of safe material to be measured (mass content) is prepared according to echelon, and described solution carried out the zebra fish toxicity test, wherein each is processed and repeats 5 times, average, contrast as not adding embryo medium A1 and the B1 of safe material.Mainly measure by following steps:
1) cultivation of zebra fish:
Getting sexual maturity AB is that common zebra fish male and female are divided cylinder, raises in the flowing water recirculation system, and the dark 10h of illumination 14h/ hockets, and water temperature remains on 28 ℃, regularly feeds the shrimps with artificial incubation 24h;
2) embryo selects:
Adopt ovum the day before yesterday with sexually matured zebra fish, in female: hero is that 1: 2 ratio is put as joining in the cylinder and separating, 9:00 natural crossing in morning next day is also collected zygote, clean the zygote that twice collection obtains with embryo medium B1, and removal impurity and bad ovum, be placed on afterwards among the embryo medium B1, control light for 29.3 ℃ and cultivate; When 2hpf, select from same parent, the normal development ratio reaches more than 85% and developmental stage is consistent embryo at microscopically, clean in the sample hole of twice rear immigration 24 orifice plate 20 pieces in every hole with embryo medium B1;
3) process:
Siphon away the embryo medium B1 in the sample hole, and in the embryo medium that contains safe material to be measured that will prepare and the nutrient solution A1 that does not contain safe material to be measured and the rapid adding of the B1 difference sample hole, every hole 4ml adds a cover sealing subsequently, places 29.3 ℃ of cultivations of incubator;
4) observe embryo and record:
Regularly examine under a microscope and record embryo's abnormal rate (getting the mean value of 5 parallel tests) and mortality ratio (getting the mean value of 5 parallel tests).
As a result, see Table 3:
Table 3 contains the embryo medium result of safe material to be measured
Sodium Glutamate is as the main component of monosodium glutamate, in routine consciousness, be useful to human body, but how much actually edible is safe, generally not accurately definition, measuring Sodium Glutamate by method Quick Measuring of the present invention is that the situation of 0.05% weight can cause the hyperplasia of zebra fish to increase in concentration, and to cause mortality ratio be 35.2%, and to during the concentration of 0.1% weight, it can cause the most of deformity of zebra fish and dead gradually.
Use zebra fish and measure the security of the materials such as trimeric cyanamide, Tartrazol yellow, Sodium Benzoate, Sodium Nitrite, the same application method of the present invention of finding can measure described safe material to the safe dose of zebra fish by Quick Measuring.Trimeric cyanamide just can cause zebra fish at 24 hours deformity to occur in 0.1%, and the zebra fish unusual death occurs by 72 hours.In 0.2%, use method of the present invention and measure, the zebra fish hyperplasia occured at 4 hours, the mortality ratio of the moon 8.7% appearred at 12 hours.The concentration of Sodium Nitrite the zebra fish hyperplasia occurred in 48 hours in 0.001% weight, in the time of 0.004% weight, the zebra fish hyperplasia occurred in 4 hours, zebra fish death occurred in 12 hours, and mortality ratio is 4.8%.
Use thus 5mM NaCl, 0.17mM KCl, the 0.43mM CaCl of alkalescent water preparation
2With 0.33mM MgSO
4Nutrient solution can be so that the zebrafish embryo rapid detection goes out the toxicity of safe material to be measured.
Claims (8)
1. a zebra fish is used for the method for the security of test safety material, it is characterized in that comprising following steps:
The preparation embryo medium, then in embryo medium, add safe material to be measured, the add-on of safe material to be measured is so that the concentration of test substance in nutrient solution is the echelon distribution, cultivate zebrafish embryo with the embryo medium that has added safe material to be measured, then in abnormal rate and the mortality ratio of 4-72 hour observed and recorded Zebrafish Embryo, judge that safe material to be measured is to the toxicity of Zebrafish Embryo, wherein embryo medium pH is that the alkalescent water of 6.7-8.5 is directly prepared, and contains 5mM NaCl in the embryo medium, 0.17mM KCl, 0.43mM CaCl
2With 0.33mM MgSO
4
2. method according to claim 1, wherein zebrafish embryo is selected by the following method:
Adopt ovum the day before yesterday with sexually matured zebra fish, in female: hero is that 1: 2 ratio is put into and joined cylinder and separate, 9:00 natural crossing in morning next day is also collected zygote, clean the zygote that twice collection obtains with embryo medium, and removal impurity and bad ovum, be placed on afterwards in the embryo medium, control light for 29.3 ℃ and cultivate; When 2hpf, select from same parent, the normal development ratio reaches more than 85% and developmental stage is consistent zebrafish embryo at microscopically.
3. method according to claim 1 and 2, the method of wherein cultivating zebrafish embryo is as follows: zebrafish embryo is cleaned twice with embryo medium, then move in the sample hole of 24 orifice plates, 20 pieces in every hole, every hole has added the embryo medium of safe material to be measured, and every hole 4ml adds a cover sealing subsequently, place incubator, cultivate zebrafish embryo at 29.3 ℃.
4. method according to claim 1, wherein the time of observed and recorded is after cultivating the 4th, 12,24,48,72 hour, record abnormal rate and mortality ratio.
5. each described method according to claim 1-4, wherein pH is 7.0-7.8.
6. method according to claim 5, wherein pH is 7.2.
7. each described method according to claim 1-6, wherein said safe material to be measured is Sodium Glutamate, trimeric cyanamide, Tartrazol yellow, Sodium Benzoate or Sodium Nitrite.
8. a zebra fish is for the purposes of testing safe material toxicity to be measured, it is characterized in that: in embryo medium, add safe material to be measured, the add-on of safe material to be measured is so that the concentration of test substance in nutrient solution is the echelon distribution, cultivate zebrafish embryo with the embryo medium that has added safe material to be measured, then in abnormal rate and the mortality ratio of 4-72 hour observed and recorded Zebrafish Embryo, judge that safe material to be measured is to the toxicity of Zebrafish Embryo, wherein embryo medium pH is that the alkalescent water of 6.7-8.5 is directly prepared, and contains 5mM NaCl in the embryo medium, 0.17mM KCl, 0.43mM CaCl
2With 0.33mM MgSO
4
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Cited By (7)
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| CN107893099A (en) * | 2017-12-14 | 2018-04-10 | 广东省保化检测中心有限公司 | The method for evaluating safety of sunscreen product |
| CN109321451A (en) * | 2018-10-11 | 2019-02-12 | 常州市环境监测中心 | It is a kind of can multiple groups experiment zebrafish embryo acute toxicity detection kit |
| CN111551679A (en) * | 2020-03-22 | 2020-08-18 | 南京新环检测科技有限公司 | Method for rapidly detecting safety of on-site catering |
| CN111766354A (en) * | 2020-03-22 | 2020-10-13 | 杭州环特生物科技股份有限公司 | Method for evaluating safety of meat product by using zebra fish |
| CN112858592A (en) * | 2021-02-01 | 2021-05-28 | 广州鲁比生物科技有限公司 | Method for evaluating mild irritation of cosmetics by using zebra fish embryos |
| CN112857539A (en) * | 2021-01-08 | 2021-05-28 | 新乡医学院 | Method for weighing zebra fish embryos and juvenile fish |
| CN113447104A (en) * | 2021-06-25 | 2021-09-28 | 成都理工大学 | Method for measuring fresh weight of miniature aquatic organisms, miniature device and application |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107893099A (en) * | 2017-12-14 | 2018-04-10 | 广东省保化检测中心有限公司 | The method for evaluating safety of sunscreen product |
| CN109321451A (en) * | 2018-10-11 | 2019-02-12 | 常州市环境监测中心 | It is a kind of can multiple groups experiment zebrafish embryo acute toxicity detection kit |
| CN109321451B (en) * | 2018-10-11 | 2021-08-10 | 常州市环境监测中心 | Zebra fish embryo acute toxicity detection kit for multiple groups of experiments |
| CN111551679A (en) * | 2020-03-22 | 2020-08-18 | 南京新环检测科技有限公司 | Method for rapidly detecting safety of on-site catering |
| CN111766354A (en) * | 2020-03-22 | 2020-10-13 | 杭州环特生物科技股份有限公司 | Method for evaluating safety of meat product by using zebra fish |
| CN112857539A (en) * | 2021-01-08 | 2021-05-28 | 新乡医学院 | Method for weighing zebra fish embryos and juvenile fish |
| CN112858592A (en) * | 2021-02-01 | 2021-05-28 | 广州鲁比生物科技有限公司 | Method for evaluating mild irritation of cosmetics by using zebra fish embryos |
| CN113447104A (en) * | 2021-06-25 | 2021-09-28 | 成都理工大学 | Method for measuring fresh weight of miniature aquatic organisms, miniature device and application |
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