CN113495129A - Method for evaluating safety of dairy product by using zebra fish - Google Patents
Method for evaluating safety of dairy product by using zebra fish Download PDFInfo
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Abstract
The invention provides a method for evaluating the safety of a dairy product by using zebra fish, which comprises the steps of constructing a zebra fish toxicity evaluation model, pretreating the dairy product in an extraction mode to obtain an extract of a detection product, obtaining toxicity evaluation data after the zebra fish is treated, and analyzing and evaluating the safety of the dairy product. The method provided by the invention has the advantages of high detection accuracy, scientific and reasonable detection method and short molding period, greatly reduces the detection cost, and provides practical feasibility for popularizing the detection method for the masses of people, enterprises and governments.
Description
Technical Field
The invention relates to the technical field of dairy product safety evaluation, in particular to a method for evaluating the safety of a dairy product by using zebra fish.
Background
Dairy products are popular because of their easy absorption, high nutritional value. With the development of economy in China, the consumption level of residents is improved, and the consumption of dairy products is also rapidly increased. However, the detection means cannot keep pace with market development, so that the supervision has a leak, and some illegal manufacturers do not participate in counterfeiting while the illegal manufacturers do, so that the safety problem of the dairy products is high in popularity. Although the supervision of the dairy industry is increased in recent years and related safety problems are alleviated to a certain extent, the detection technology has limitations.
At present, the technologies based on dairy product safety detection mostly use chemical analysis means for detection and analysis, and the biological means for detection and analysis are rarely used, so that the following technical defects exist:
1) for historical reasons, most detection indexes in the existing national standard are established by referring to foreign standards, but whether the toxicity of chemicals presents toxicity differences due to ethnic differences or not is not supported by a large amount of sample data verification at present, which means that whether chemicals which are harmless to Chinese people are harmless under the standard dosage of caucasians or not is uncertain.
2) The synergistic toxicity of different chemicals to the human body cannot be detected. Because the physicochemical properties of different chemicals are unknown, whether synergistic effect or synergistic effect can be generated or not during interaction exists, and uncertainty exists; the difficulty of judging the detection of the synergistic toxicity of the sample to be detected is more than that of a single toxic substance.
3) Unknown additives cannot be detected, and only known chemicals can be detected. Given the presence of analogs, derivatives, and metabolites of known chemical species in the human body, etc., as unidentified additives, detection is difficult.
4) The toxic substances are various in types, and cannot be comprehensively detected due to the cost factors such as time, manpower and material resources.
The homology of zebra fish and human genes is as high as 85%, because the genome and the proteome have high similarity with human, the aspects of disease pathogenesis, signal transduction pathway and the like are basically similar to human, and the biological structure and physiological function are highly similar to those of mammals. Compared with other experimental animals, zebrafish also has the characteristics of transparent embryos and young fish bodies (the development process of each organ can be directly observed in vivo by naked eyes and a dissecting microscope), small volume (the development process can be analyzed by a micropore plate), short development period, in vitro fertilization, strong reproductive capacity, higher single egg laying number and the like (Zon LI, Peterson RT. in vivo Drug Discovery in the zebrafish [ J ]. Drug Discovery,2005,4: 35-44.). The model organism zebra fish has become one of the human disease models in recent years, has the advantages of quick in vitro experiment, high efficiency, low cost, small dosage and the like, has the advantages of strong mammal experiment predictability, high contrast, capability of observing a plurality of organs and the like, and is widely applied to compound safety evaluation (Barros T P, Alderton W K, et al. Zebraphish: An experimental technology for in vivo pharmacological assessment to identification potential safety availability traits in early drug discovery [ J ]. British Journal of pharmacy, 2008,154: 1400: 1413.).
Therefore, the applicant considers that the zebra fish model is adopted to carry out overall biological evaluation, and the safety of the dairy product is judged by observing the in-vivo state of the zebra fish, so that the zebra fish model is used as supplement and improvement of the traditional chemical detection means.
Through the search of documents, the safety evaluation of the existing zebra fish model is found, and the evaluation objects are all medicines and cosmetics, and are not applied to the field of dairy products. The dairy products have different components and contents, and the safety standard requirements of the dairy products are different from those of medicines and cosmetics, so how to develop a zebra fish model suitable for the safety of the dairy products and a detection method thereof is a technical problem which needs to be solved urgently by a person skilled in the art.
Disclosure of Invention
The invention aims to solve the technical problem of providing a brand-new method for evaluating the safety of a dairy product by using zebra fish.
Therefore, the invention adopts the following technical scheme:
a method for evaluating the safety of a dairy product by using zebra fish comprises the steps of constructing a zebra fish toxicity evaluation model, pretreating the dairy product in an extraction mode to obtain an extract of a detection product, processing the zebra fish to obtain toxicity evaluation data, and analyzing and evaluating the safety of the dairy product.
Preferably, the method for evaluating the safety of the dairy product by using the zebra fish comprises the following steps:
1) preparation of the extract
Mixing dairy product with acetonitrile, homogenizing, extracting, adding anhydrous magnesium sulfate and sodium chloride, oscillating, centrifuging at high speed, collecting supernatant, extracting residue with acetonitrile for 1 time, mixing supernatants, rotary evaporating in 50 deg.C water bath, drying, diluting with 1% methanol to desired volume, and storing at-20 deg.C; the volume ratio of acetonitrile used in the 1 st extraction to the 2 nd extraction is (2:1) - (3: 1);
2) evaluation of safety
Diluting the extract prepared in the step 1) to a zebra fish embryo culture medium, wherein the dilution concentration is 0.31 mu L/mL, 0.63 mu L/mL, 1.25 mu L/mL, 2.5 mu L/mL and 5.0 mu L/mL, so as to obtain an extract diluent;
exposing 4-128 cell-stage zebra fish embryos to a microplate containing extract diluent, and setting zebra fish embryo culture media and 3.7mg/L dichloroaniline as a negative control group and a positive control group respectively; and after 48h of exposure, observing toxicity symptoms of the zebra fish embryos, calculating the death rate of the zebra fish embryos, and judging and evaluating the safety of the dairy product.
Preferably, the volume ratio of acetonitrile used in the 1 st extraction to the 2 nd extraction in step 1) is 2: 1.
Preferably, the zebra fish embryo culture medium in the step 2) comprises the following components in percentage by weight: 294.0mg/L anhydrous sodium chloride, 123.3mg/L magnesium sulfate heptahydrate, 63.0mg/L sodium bicarbonate, 5.5mg/L potassium chloride in deionized water.
Preferably, the dairy product in step 1) is selected from one of liquid milk, yogurt, milk powder and cheese.
Compared with the prior art, the method for evaluating the safety of the dairy product by using the zebra fish has the following beneficial effects:
according to the method, the appropriate extraction solvent, the amount of methanol and the dilution concentration of the extracting solution are selected, the dairy product is subjected to extraction pretreatment and zebra fish treatment, toxicity evaluation data are obtained and used for judging and evaluating the safety of the dairy product, the detection accuracy is high, the detection method is scientific and reasonable, the detection cost is greatly reduced due to the short molding period, and practical feasibility is provided for popularizing the detection method to the masses of people, enterprises and governments.
Detailed Description
The following are specific examples of the present invention and further describe the technical solutions of the present invention, but the scope of the present invention is not limited to these examples. All changes, modifications and equivalents that do not depart from the spirit of the invention are intended to be included within the scope thereof.
Example 1 examination of the composition of extraction solvent in the pretreatment stage
This example explores and identifies suitable extraction solvents by comparing the extraction rates of methanol, acetonitrile, acetone, ethanol, propanol. The examination method is as follows:
firstly, representative antibiotics according to GB standard are added into dairy products, and the addition amount is shown in Table 1:
TABLE 1 milk antibiotic addition
| Name (R) | The addition amount is mu g/L | Name (R) | The addition amount is mu g/L |
| Cefalexin | 100 | Fenvalerate | 100 |
| Clavulanic acid | 200 | Tetracycline derivatives | 100 |
| Chlorohydroxypyridine | 20 | Sulfonamides | 100 |
| Enrofloxacin | 100 | Thiamphenicol | 50 |
| Erythromycin | 40 | Trichlorfon | 50 |
Secondly, weighing 10g of dairy product sample (accurate to 0.01g) into a 50mL centrifuge tube, adding 20mL of extraction solvent (methanol, acetonitrile, acetone, ethanol and propanol), homogenizing and extracting for 1min, adding 12g of anhydrous magnesium sulfate and 4g of sodium chloride, oscillating, and centrifuging at a high speed of 4000r/min for 5 min. The supernatant was aspirated, the residue was extracted again with 10mL acetonitrile for 1 time, the supernatants were combined, concentrated by rotary evaporation in a 50 ℃ water bath to near dryness, and dissolved in 1mL methanol.
Finally, the concentration of the antibiotic (Table 1) remaining in the extract was measured by HPLC using a 0.45 μm membrane, and the recovery rate was calculated, and the results are shown in Table 2.
According to the regulation of GB/T27101-; when the content of the tested components is 0.1-1mg/kg, the recovery rate is required to be 80-110%; when the content of the tested components is 1-100mg/kg, the recovery rate is required to be in the range of 90-110%.
TABLE 2 Effect of different organic solvents on recovery
As is clear from table 2, only acetonitrile was extracted from the 5 organic solvents in a recovery ratio that meets the GB requirement, and therefore acetonitrile was selected as the solvent for extraction.
Example 2 examination of the amount of extraction solvent used in the pretreatment stage
On the basis of example 1, this example explores and determines the ratio of acetonitrile amounts used in the previous and next 2 extractions by comparing the volume amounts of acetonitrile. The examination method is as follows:
weighing 10g of sample (accurate to 0.01g) into a 50mL centrifuge tube, adding 15, 20, 30 and 40mL of acetonitrile, homogenizing and extracting for 1min, adding 12g of anhydrous magnesium sulfate and 4g of sodium chloride, oscillating, and centrifuging at a high speed of 4000r/min for 5 min. The supernatant was aspirated, the residue was extracted again with 10mL acetonitrile for 1 time, the supernatants were combined, concentrated by rotary evaporation in a 50 ℃ water bath to near dryness, and dissolved in 1mL methanol.
After extraction, the extract was passed through a 0.45 μm membrane, and the concentration of the antibiotic (Table 1) remaining in the extract was measured by HPLC mass spectrometry, and the recovery rate was calculated, and the results are shown in Table 3.
TABLE 3 influence of acetonitrile dosage ratio of front and back 2 extractions on recovery
As can be seen from Table 3, the recovery rate of extraction meets GB requirements when the ratio of the amount of acetonitrile used in the 1 st extraction to that used in the 2 nd extraction is 2:1 and 3: 1. And comprehensively considering the solvent loss, determining the dosage ratio of acetonitrile used in the 1 st extraction and the 2 nd extraction as the optimal dosage ratio of 2: 1.
Example 3 examination of the amount of methanol used in the pretreatment stage
Considering that the solvent methanol of the extracting solution has certain toxicity, in order to eliminate death of the zebra fish embryos caused by inappropriate methanol concentration and avoid influencing experimental results, the zebra fish embryos are exposed to methanol with different concentrations, and the tolerance condition of the zebra fish embryos is observed.
In the present example, only the amount of methanol (concentration: 20%, 10%, 5%, 2.5%, 1.25%, 1%, 0.5%, 0.25%) was changed, and the remaining experimental conditions were unchanged, and the mortality of zebra fish was calculated by observing 120 hours and recording every 24 hours, and the results are shown in table 4.
Generally, the death rate of zebrafish does not exceed 10%, and the concentration is considered safe.
TABLE 4 influence of methanol at different concentrations on the mortality rate of zebrafish embryos
As can be seen from Table 4, when the concentration of methanol is not more than 1%, the test requirements are met, so that methanol with a concentration of 1% is selected as the maximum concentration that the zebrafish embryo can tolerate.
Example 4 examination of dilution concentration of extract at Zebra fish treatment stage
Based on example 3, this example explores and determines the appropriate dilution concentration of the extract by comparing the mortality rate of zebrafish embryos after treating different dilution concentrations of extract in 24h, 48h, 72h, 96h and 120 h. The examination method is as follows:
1) preparation of the extract
Weighing 10g of dairy product sample (accurate to 0.01g) into a 50mL centrifuge tube, adding 20mL of acetonitrile, homogenizing and extracting for 1min, adding 12g of anhydrous magnesium sulfate and 4g of sodium chloride, oscillating, and centrifuging at 4000r/min at a high speed for 5 min. Sucking supernatant, extracting residue with 10mL acetonitrile for 1 time, mixing supernatants, rotary evaporating in 50 deg.C water bath for concentrating to near dry, dissolving with 1% methanol, and storing at-20 deg.C;
2) evaluation of safety
Diluting the extract prepared in the step 1) to zebrafish embryo culture medium (deionized water containing 294.0mg/L anhydrous sodium chloride, 123.3mg/L magnesium sulfate heptahydrate, 63.0mg/L sodium bicarbonate and 5.5mg/L potassium chloride) with dilution concentration of 0.08 μ L/mL, 0.15 μ L/mL, 0.31 μ L/mL, 0.63 μ L/mL, 1.25 μ L/mL, 2.5 μ L/mL and 5.0 μ L/mL to obtain extract diluent;
zebrafish embryos at 4-128 cell stages were exposed to six-well plates containing dilutions of extracts, each plate containing 30 roes, and the following 4 experimental groups were set up: blank sample group, add antibiotic sample group A, B and C; after a period of exposure (set at 24h, 48h, 72h, 96h and 120h), toxicity symptoms of the zebrafish embryos were observed with a stereomicroscope, and the mortality of the zebrafish embryos was calculated. The results are shown in Table 5.
TABLE 5 Effect of extracts of different dilution concentrations, different time on the mortality of zebra fish embryos
Note: comparison with blank sample set<0.05; compared with the group A with the antibiotic sample added,+p<0.05。
from table 5, it can be seen that:
the death rate of the zebra fish embryos in the four experimental groups is unchanged after 48 hours, and is different from the data of 24 hours;
compared with the blank group, the antibiotic sample group A, B and the antibiotic sample group C have obvious difference (p is less than 0.05) when the dilution concentration is 0.31-10 mu L/mL in 48 h;
comparing with the antibiotic sample group A, when the dilution concentration is 0.31-5 mu L/mL in 48h, the antibiotic sample group B and the antibiotic sample group C have obvious difference (p is less than 0.05), which shows that whether the addition of the antibiotics in the three groups of the sample A, B, C exceeds the standard or not can be distinguished;
in conclusion, the dilutions of the extracts were determined to be 0.31, 0.63, 1.25, 2.5, 5.0. mu.L/mL.
Application example 1 safety evaluation of pure milk
1) Preparation of the extract
Weighing 10g (accurate to 0.01g) of pure milk sample of brand A, B, C into a 50mL centrifuge tube, adding 20mL acetonitrile, homogenizing and extracting for 1min, adding 12g anhydrous magnesium sulfate and 4g sodium chloride, shaking, and centrifuging at 4000r/min for 5 min. Sucking supernatant, extracting residue with 10mL acetonitrile for 1 time, mixing supernatants, rotary evaporating in 50 deg.C water bath for concentrating to near dry, dissolving with 1% methanol, and storing at-20 deg.C;
2) evaluation of safety
Diluting the extract prepared in the step 1) to zebra fish embryo culture medium (deionized water containing 294.0mg/L anhydrous sodium chloride, 123.3mg/L magnesium sulfate heptahydrate, 63.0mg/L sodium bicarbonate and 5.5mg/L potassium chloride) with dilution concentration of 0.31 μ L/mL, 0.63 μ L/mL, 1.25 μ L/mL, 2.5 μ L/mL and 5.0 μ L/mL to obtain extract diluent;
zebrafish embryos at 4-128 cell stages were exposed to six well plates containing dilutions of the extract, 30 roe were placed in each plate, and medium and 3.7mg/L dichloroaniline were set as negative and positive controls. After exposure at 26 ℃ for 48h, toxicity symptoms of the zebrafish embryos were observed with a stereomicroscope, and the mortality of the zebrafish embryos was calculated. The results are shown in Table 6.
TABLE 6 acute toxicity of pure milk extracts of different brands
Application example 2 safety evaluation of milk powder
1) Preparation of the extract
Weighing 10g (accurate to 0.01g) of a milk powder sample of brand A, B, C in a 50mL centrifuge tube, adding 10mL of water, oscillating in a water bath at 30 ℃ for 10min, adding 20mL of acetonitrile, homogenizing and extracting for 1min, adding 12g of anhydrous magnesium sulfate and 4g of sodium chloride, oscillating, and centrifuging at a high speed of 4000r/min for 5 min. Sucking supernatant, extracting residue with 10mL acetonitrile for 1 time, mixing supernatants, rotary evaporating in 50 deg.C water bath for concentrating to near dry, dissolving with 1% methanol, and storing at-20 deg.C;
2) evaluation of safety
Diluting the extract prepared in the step 1) to zebra fish embryo culture medium (deionized water containing 294.0mg/L anhydrous sodium chloride, 123.3mg/L magnesium sulfate heptahydrate, 63.0mg/L sodium bicarbonate and 5.5mg/L potassium chloride) with dilution concentration of 0.31 μ L/mL, 0.63 μ L/mL, 1.25 μ L/mL, 2.5 μ L/mL and 5.0 μ L/mL to obtain extract diluent;
zebrafish embryos at 4-128 cell stages were exposed to six well plates containing dilutions of the extract, 30 roe were placed in each plate, and medium and 3.7mg/L dichloroaniline were set as negative and positive controls. After exposure at 26 ℃ for 48h, toxicity symptoms of the zebrafish embryos were observed with a stereomicroscope, and the mortality of the zebrafish embryos was calculated. The results are shown in Table 7.
TABLE 7 acute toxicity of different brands of powdered milk extracts
Application example 3 safety evaluation of cheese
1) Preparation of the extract
Weighing 10g (accurate to 0.01g) of a cheese sample of brand A, B, C in a 50mL centrifuge tube, adding 10mL of water, oscillating in a water bath at 30 ℃ for 10min, adding 20mL of acetonitrile, homogenizing and extracting for 1min, adding 12g of anhydrous magnesium sulfate and 4g of sodium chloride, oscillating, and centrifuging at a high speed of 4000r/min for 5 min. Sucking supernatant, extracting residue with 10mL acetonitrile for 1 time, mixing supernatants, rotary evaporating in 50 deg.C water bath for concentrating to near dry, dissolving with 1% methanol, and storing at-20 deg.C;
2) evaluation of safety
Diluting the extract prepared in the step 1) to zebra fish embryo culture medium (deionized water containing 294.0mg/L anhydrous sodium chloride, 123.3mg/L magnesium sulfate heptahydrate, 63.0mg/L sodium bicarbonate and 5.5mg/L potassium chloride) with dilution concentration of 0.31 μ L/mL, 0.63 μ L/mL, 1.25 μ L/mL, 2.5 μ L/mL and 5.0 μ L/mL to obtain extract diluent;
zebrafish embryos at 4-128 cell stages were exposed to six well plates containing dilutions of the extract, 30 roe were placed in each plate, and medium and 3.7mg/L dichloroaniline were set as negative and positive controls. After exposure at 26 ℃ for 48h, toxicity symptoms of the zebrafish embryos were observed with a stereomicroscope, and the mortality of the zebrafish embryos was calculated. The results are shown in Table 7.
TABLE 7 acute toxicity of cheese extracts of different brands
Claims (5)
1. A method for evaluating the safety of dairy products by using zebra fish is characterized by comprising the following steps: constructing a zebra fish toxicity evaluation model, pretreating the dairy product in an extraction mode to obtain an extract of a detection product, treating the zebra fish to obtain toxicity evaluation data, and analyzing and evaluating the safety of the dairy product.
2. The method of claim 1, comprising the steps of:
1) preparation of the extract
Mixing dairy product with acetonitrile, homogenizing, extracting, adding anhydrous magnesium sulfate and sodium chloride, oscillating, centrifuging at high speed, collecting supernatant, extracting residue with acetonitrile for 1 time, mixing supernatants, rotary evaporating in 50 deg.C water bath, drying, diluting with 1% methanol to desired volume, and storing at-20 deg.C; the volume ratio of acetonitrile used in the 1 st extraction to the 2 nd extraction is (2:1) - (3: 1);
2) evaluation of safety
Diluting the extract prepared in the step 1) to a zebra fish embryo culture medium, wherein the dilution concentration is 0.31 mu L/mL, 0.63 mu L/mL, 1.25 mu L/mL, 2.5 mu L/mL and 5.0 mu L/mL, so as to obtain an extract diluent;
exposing 4-128 cell-stage zebra fish embryos to a microplate containing extract diluent, and setting zebra fish embryo culture media and 3.7mg/L dichloroaniline as a negative control group and a positive control group respectively; and after 48h of exposure, observing toxicity symptoms of the zebra fish embryos, calculating the death rate of the zebra fish embryos, and judging and evaluating the safety of the dairy product.
3. The method according to claim 2, wherein the volume ratio of acetonitrile used in the 1 st extraction to the 2 nd extraction in the step 1) is 2: 1.
4. The method as claimed in claim 2, wherein the zebrafish embryo culture medium in the step 2) comprises the following components in percentage by weight: 294.0mg/L anhydrous sodium chloride, 123.3mg/L magnesium sulfate heptahydrate, 63.0mg/L sodium bicarbonate, 5.5mg/L potassium chloride in deionized water.
5. The method according to any one of claims 1 to 4, wherein the dairy product is selected from one of liquid milk, yoghurt, milk powder and cheese.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116411050A (en) * | 2023-02-01 | 2023-07-11 | 山东省科学院生物研究所 | Method for controlling Xinkeshu quality by using differential gene screened by zebra fish gene expression profile |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116411050A (en) * | 2023-02-01 | 2023-07-11 | 山东省科学院生物研究所 | Method for controlling Xinkeshu quality by using differential gene screened by zebra fish gene expression profile |
| CN116411050B (en) * | 2023-02-01 | 2024-03-19 | 山东省科学院生物研究所 | Method for controlling Xinkeshu quality by using differential gene screened by zebra fish gene expression profile |
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