CN111458467A - Detection method of aflatoxin M1 in milk powder - Google Patents
Detection method of aflatoxin M1 in milk powder Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/02—Food
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Abstract
The invention discloses a method for detecting aflatoxin M1 in milk powder, and relates to the technical field of toxicity detection. The detection method of aflatoxin M1 in the milk powder comprises the following steps: mixing the milk powder extract with water to form a culture solution, culturing zebra fish embryos in the culture solution, and judging the overproof condition of aflatoxin M1 in the milk powder according to the heart rate or the aberration rate of the zebra fish embryos; wherein, the deformity is judged according to the conditions of the liver and yolk sac of the zebra fish embryo. The detection method does not need a large compact instrument, is convenient to operate, and can realize rapid and efficient detection.
Description
Technical Field
The invention relates to the technical field of toxicity detection, and particularly relates to a method for detecting aflatoxin M1 in milk powder.
Background
In recent years, along with the improvement of living standard, the problem of aflatoxin pollution in milk powder gradually becomes the focus of attention. Due to the influence of the process in the processing process, the pollution of aflatoxin and metabolites thereof in milk powder is very common. The aflatoxin M1 is used as a main metabolite of aflatoxin, has strong toxicity and carcinogenicity, seriously threatens the safety and health of people, and has a non-negligible potential risk. However, the existing detection method for aflatoxin M1 mainly focuses on the traditional chemical detection method, has strong dependence on operators and operating environment, and consumes a large amount of manpower and material resources; the result can only determine the concentration level of aflatoxin M1, the actual risk of aflatoxin M1 on the milk powder is difficult to reflect visually, and the safety of the milk powder cannot be evaluated comprehensively.
The existing detection method mainly utilizes a chemical detection method, cannot comprehensively reflect the actual level and potential risk of pollutants from the perspective of individuals, and has high detection cost and low efficiency.
Disclosure of Invention
The invention aims to provide a method for detecting aflatoxin M1 in milk powder, which does not need large-scale precise instruments, has low operation cost and can detect aflatoxin M1 quickly and efficiently.
The technical problem to be solved by the invention is realized by adopting the following technical scheme.
The invention provides a method for detecting aflatoxin M1 in milk powder, which comprises the following steps:
mixing the milk powder extract with water to form a culture solution, culturing zebra fish embryos in the culture solution, and judging the overproof condition of aflatoxin M1 in the milk powder according to the heart rate or the aberration rate of the zebra fish embryos;
wherein, the deformity is judged according to the conditions of the liver and yolk sac of the zebra fish embryo.
The embodiment of the invention provides a method for detecting aflatoxin M1 in milk powder, which has the following beneficial effects: the culture solution for feeding the zebra fish embryos is formed by mixing the milk powder extracting solution and water, and whether the content of aflatoxin M1 in the milk powder exceeds the standard or not is judged by measuring the heart rate of the zebra fish embryos and observing the malformation condition of the zebra fish embryos. The detection method does not need a large compact instrument, is convenient to operate, and can realize rapid and efficient detection.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a comparison of normal zebrafish embryos and developmental malformed zebrafish embryos.
1-liver; 2-yolk sac.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The following describes a method for detecting aflatoxin M1 in milk powder provided by the embodiments of the present invention.
The embodiment of the invention provides a method for detecting aflatoxin M1 in milk powder, which comprises the following steps: mixing the milk powder extracting solution with water to form a culture solution, placing the zebra fish embryo into the culture solution for culture, and judging the overproof condition of aflatoxin M1 in the milk powder according to the heart rate or the aberration rate of the zebra fish embryo; wherein, the deformity is judged according to the conditions of the liver and yolk sac of the zebra fish embryo.
In the embodiment of the invention, the culture solution for feeding the zebra fish embryos is formed by mixing the milk powder extract with water, and whether the content of aflatoxin M1 in the milk powder exceeds the standard or not is judged by measuring the heart rate of the zebra fish embryos and observing the malformation condition of the zebra fish embryos. The detection method does not need a large compact instrument, is convenient to operate, and can realize rapid and efficient detection.
The inventor finds that the detection accuracy of the aflatoxin M1 by using the zebra fish is higher, which is probably because the zebra fish has high similarity with human genes and has similar tissue organs and systems with human.
The three aspects of the preparation of the culture solution, the feeding of the zebra fish, embryo selection and culture test method are introduced below.
(1) Preparation of the culture solution
The culture solution is formed by mixing milk powder extracting solution and water, the volume ratio of the milk powder extracting solution to the water is 0.8-1.2:1000, preferably 0.9-1.1:1000, and the detection accuracy is improved by controlling the proportion of the milk powder extracting solution to the water.
The preparation process of the milk powder extracting solution comprises the following steps: mixing and dissolving milk powder and water, mixing and extracting with an extractant solution, then mixing with chloride, and separating supernatant; the resulting supernatant was evaporated to dryness and redissolved.
Preferably, the extractant is selected from at least one of 0.5-1.5% acetonitrile acetate, 0.5-1.5% acetonitrile formate, methanol and acetonitrile, more preferably 0.5-1.5% acetonitrile acetate, the dosage of the extractant solution corresponding to each gram of milk powder is 5-10M L, the dosage of the water corresponding to each gram of milk powder is 3-8M L, the chloride is sodium chloride, and the dosage of the sodium chloride corresponding to each gram of milk powder is 1.5-2.5 g.
Further, the process of separating the supernatant is to swirl for 0.5-1.5min, then centrifuge for 3-8min at the rotation speed of 4000-. The salting-out process can be accelerated by vortexing and centrifuging to separate acetonitrile acetate and water.
Further, the solvent used in the redissolution process is selected from at least one of dimethyl sulfoxide and acetone; more preferably dimethyl sulfoxide; the volume ratio of the supernatant to the solvent used in the reconstitution process is 70-80: 1. And by evaporation and redissolution and by selecting a solvent with lower toxicity for redissolution, the solvent is prevented from being used as an interference factor to influence the test result.
(2) Zebra fish breeding and embryo selection
The zebra fish is cultured for a long time before mating and spawning, the zebra fish is cultured under the conditions of water temperature of 27-29 ℃, dissolved oxygen of 6-10 mg/L and pH value of 7-7.3, and a light cycle comprises 12-16h of light condition and 8-12h of dark condition in the culturing process.
Preferably, the water for cultivation is aerated for 4 to 6 days; the feeding bait is at least one of tropical small fish feed and fairy shrimp, and is fed for 3 times per day; feeding bait at 10:00-11:00, 15:00-16:00 and 20:00-21:00 each day.
The zebra fish embryo is formed by mating adult AB-series zebra fish according to the male-female ratio of 1:1, collecting fertilized eggs and selecting the normally developed zebra fish embryo of 3-4hpf, and the inventor finds that the fertilized zebra fish embryo of about 3h is more suitable for detection, so that the detection accuracy can be further improved.
Further, one night before mating and egg taking day, the selected adult male and female zebra fish are transferred into an egg laying container, and the male and female zebra fish are separately cultured for 10-13h while keeping the noise value less than 40-50dB and the temperature at 26-28 ℃. Specifically, the male and female zebra fish are separately cultured 20-21 nights before the egg taking day, the male and female zebra fish are mixed at 8:00-10:00 of the egg taking day for free mating and egg laying, and fertilized eggs in an egg laying container are collected at 12:00-1:00 of the egg taking day; after collecting the fertilized eggs, cleaning the impurities in the fertilized eggs, flushing the fertilized eggs, and then transferring the fertilized eggs into a culture solution for culture.
(3) Culture test method
Controlling the temperature at 27-28 deg.C during culture in culture solution, culturing for 90-100h (preferably 95-98h), processing in dark place during culture, and measuring heart rate or aberration rate after culture.
Generally, the criteria for determining deformity include: the heart (pericardial edema, no atrium or ventricle), brain (degeneration, reduction, irregularity), ear (reduction), mandible (loss, deformity), eye (reduction, deformity, hypopigmentation), liver (loss, degeneration, size abnormality), yolk sac absorption (delay), intestine (viscera loss, fold loss, size abnormality, color abnormality), and the abnormality of the index such as trunk, tail fin, spinal cord deformity, muscle, body segment, circulatory system, body color. The inventor creatively finds that only observing the condition of the liver or yolk sac can judge whether the zebra fish embryo exposed to the aflatoxin M1 is malformed. As shown in FIG. 1, A shows normal zebrafish, B and C show malformed zebrafish, and liver 1 and yolk sac 2 have obvious malformed changes, respectively.
Preferably, the heart rate of the zebrafish embryo is measured when the culture time reaches 20-30h, 40-55h and 65-75h respectively, and the malformation condition of the zebrafish embryo is observed to observe the change of the zebrafish during the culture process.
In other embodiments, a constant temperature water control group (which only uses constant temperature water with the same amount as that in the embodiment for culture), a solvent control group (which only uses a solvent in a re-dissolving process to be mixed with water and uses the same amount as that in the embodiment), and a process blank control group (which is different from the embodiment in that milk powder extract is not included) can be set for simultaneous culture, so as to improve the detection accuracy.
After the culture in the culture solution is finished, if the heart rate is less than or equal to 126 times/min, the content of the aflatoxin M1 in the milk powder exceeds 0.5 mug/Kg; when the heart rate is less than or equal to 121 times/min, the content of the aflatoxin M1 in the milk powder exceeds 5 mug/Kg; when the heart rate is less than or equal to 110 times/min, the content of the aflatoxin M1 in the milk powder exceeds 50 mug/Kg. The inventor finds that whether the content of aflatoxin M1 in milk powder exceeds the standard or not by continuously exploring and controlling each index of the culture solution and setting the specific numerical value of the heart rate. The limit standard of aflatoxin M1 in milk and dairy products stipulated in China and America is 0.5 mug/Kg, and the standard of European Union is more strict and is 0.05 mug/Kg.
After the culture in the culture solution is completed, the distortion rate is greater than or equal to 30%, and the content of the aflatoxin M1 in the milk powder is more than 0.5 mu g/Kg. The inventor finds that when the content of the aflatoxin M1 reaches 5 mug/Kg, even 50 mug/Kg, the distortion rate is not obviously increased.
Preferably, when the zebra fish embryos are observed, a microscope is adopted for observation, and the magnification is adjusted to be 3-4 times; the zebrafish embryos were fixed in lateral position using methyl fiber gel during observation.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment of the invention provides a method for detecting aflatoxin M1 in milk powder, which comprises the following steps:
(1) the preparation of the culture solution comprises the steps of accurately weighing 2.0g of milk powder sample (the content of aflatoxin M1 is detected to be about 0.55 mu g/kg), adding 10M of L of water for dissolving, adding 15M of L1% of acetic acid acetonitrile for vortex mixing, weighing 4.0g of NaCl, carrying out vortex mixing for 1min and 4500rpm/min for centrifugation for 5min to obtain supernatant, transferring the obtained 15M of L supernatant into a heart-shaped flask, carrying out vortex evaporation to dryness, adding 200 mu of L dimethyl sulfoxide for redissolving to obtain milk powder extracting solution, and mixing the milk powder extracting solution with water to form the culture solution, wherein the volume ratio of the milk powder extracting solution to the water is 1: 1000.
(2) The method comprises the steps of obtaining and selecting zebra fish embryos, wherein the zebra fish products are wild AB zebra fish, the zebra fish are bred in fish tanks with constant-temperature circulating water systems (the male and female zebra fish are bred in different fish tanks respectively), water for breeding is aerated for five days, a heating rod is used in a water tank for heating to maintain the water temperature, the temperature is 27-29 ℃, the dissolved oxygen in the water is more than 6.0 mg/L, the pH value is about 7.0, illumination is alternately carried out (illumination is carried out for 14 h/dark 10h), breeding bait is tropical small fish feed and fairy shrimp, the breeding bait is fed for 3 times every day, and the breeding bait is fed at 10:00-11:00, 15:00-16:00 and 20:00-21:00 respectively.
And (3) selecting well-developed adult male and female zebra fish (male-female ratio is 1:1) to move into a spawning tank at 8: 00-9: 00 night before egg taking, carrying out isolated culture on the male and female zebra fish by using a partition plate, closing all noise sources, and keeping the noise value below 40dB and the constant temperature condition of about 27 ℃ in the process. And (3) taking the partition plate in the spawning tank out 9:00 the next morning to ensure that the male and female zebra fishes freely mate and spawn in the spawning tank, keeping the noise value and the temperature under the original conditions, and collecting fertilized eggs in the spawning tank at 12:00-1:00 noon. And (3) cleaning impurities from the taken fertilized eggs, washing the fertilized eggs with constant-temperature culture water for 2 times, transferring the fertilized eggs into a culture dish, and selecting the embryos which are normally developed and are at 3hpf under a microscope.
(3) And (3) exposing the zebra fish embryos, namely taking a twelve-hole plate, adding 1m L of the culture solution prepared in the step (1) and 10 selected embryos into each hole, covering the infected embryos, sealing and wrapping the covered embryos by tinfoil, and putting the covered embryos into a constant-temperature incubator for culturing for 96 hours at the constant-temperature culture temperature of about 27 ℃.
And (3) fixing the zebra fish embryo on a cover of a twelve-well plate by using methylcellulose gel in a side body position during observation and recording at 96h, and observing and photographing under a microscope to obtain a zebra fish embryo lateral position image, wherein the microscope magnification is 3.5 times. The heart rate measuring method comprises the following steps: and randomly selecting 5 zebra fish embryos from each group, observing the zebra fish embryos under a microscope, manually counting the heart rate within 10 seconds, repeatedly measuring for three times, averaging, and multiplying the obtained result by 6 to obtain the heart rate per minute (times/minute). The method for measuring the distortion rate comprises the following steps: and (3) observing all 10 zebra fish embryos in each group under a microscope, counting the number of the zebra fish embryos with deformities, and dividing by 10 times 100% to obtain the deformity rate of each group.
Comparative example 1
This comparative example was cultured using only water as the culture medium, and the other reference example 1 was labeled CK (constant temperature culture water).
Comparative example 2
In this comparative example, only a mixed solution of dimethyl sulfoxide and water was used as a culture medium, and the mass fraction of dimethyl sulfoxide was controlled to 0.1%, in other words, see example 1.
Comparative example 3
The culture solution used in this comparative example is different from the culture solution in example 1 in that: no milk powder extract was added, called process blank, labeled SCK.
Comparative example 4
The culture solution used in this comparative example is different from the culture solution in example 1 in that: the milk powder sample used was aflatoxin M1 blank.
Comparative example 5
This comparative example differs from the culture solution in example 1 in that: the aflatoxin M1 standard solution extract was mixed with water to form a culture solution, using a culture solution containing aflatoxin M1 standard solution extract, the aflatoxin M1 concentration in the culture solution was 0.5. mu.g/kg, and other parameters and culture methods were referred to example 1.
Comparative example 6
The comparative example is different from the culture solution in comparative example 4 in that: the culture solution containing the aflatoxin M1 standard solution extract and the milk powder extract is adopted, the milk powder extract (extracted from milk powder with trace aflatoxin M1 content and can be ignored) is added on the basis of the comparative example 4, the volumes of the milk powder extract and the aflatoxin M1 standard solution extract are the same, and the volume ratio of the aflatoxin M1 standard solution extract to water is 1: 1000.
Comparative example 7
This comparative example differs from the culture solution in example 1 in that: the aflatoxin M1 standard solution extract was mixed with water to form a culture solution, using a culture solution containing aflatoxin M1 standard solution extract, the aflatoxin M1 concentration in the culture solution was 5. mu.g/kg, and other parameters and culture methods were referred to example 1.
Comparative example 8
The comparative example is different from the culture solution in comparative example 6 in that: the culture solution containing the aflatoxin M1 standard solution extract and the milk powder extract is adopted, the milk powder extract (extracted from milk powder with trace aflatoxin M1 content and can be ignored) is added on the basis of the comparative example 6, the volumes of the milk powder extract and the aflatoxin M1 standard solution extract are the same, and the volume ratio of the aflatoxin M1 standard solution extract to water is 1: 1000.
Comparative example 9
The difference between the comparative example and the culture solution in example 1 is that the culture solution containing aflatoxin M1 standard solution extract is adopted, aflatoxin M1 standard solution extract is mixed with water to form the culture solution, the concentration of aflatoxin M1 in the culture solution is 50 mug/L, and other parameters and culture methods refer to example 1.
Comparative example 10
The culture solution of this comparative example is different from that of comparative example 8 in that: the culture solution containing the aflatoxin M1 standard solution extract and the milk powder extract is adopted, the milk powder extract (extracted from milk powder with trace aflatoxin M1 content and can be ignored) is added on the basis of the comparative example 8, the volumes of the milk powder extract and the aflatoxin M1 standard solution extract are the same, and the volume ratio of the aflatoxin M1 standard solution extract to water is 1: 1000.
Test example 1
The zebrafish embryos of example 1, comparative examples 1-10 were tested for heart rate or teratogenesis after 96 hours of culture as shown in table 1.
TABLE 1 Zebra fish embryo Heart Rate or teratogenesis Rate test results
As can be seen from the test data in Table 1, the heart rate of the comparative examples 1-4, namely the normal control, the solvent control, the process blank group and the substrate blank group, is 130-150 times/min, the aberration rate is 0-20%, and the indexes are indexes of the zebra fish embryos under normal conditions; comparative examples 5 and 6, namely 0.5 mug/kg standard solution group and 0.5 mug/kg matrix standard addition group, the heart rate is between 121 and 126 times/min, and the aberration rate is higher than 30 percent; comparative examples 7 and 8, namely 5 mug/kg standard solution group and 5 mug/kg matrix standard adding group, the heart rate is between 110 and 121 times/min, and the aberration rate is higher than 30%; the heart rate of the comparative examples 9 and 10, namely the standard solution group of 50 mug/kg and the matrix adding standard group of 50 mug/kg, is between 102 and 110 times/min, and the aberration rate is higher than 30%. Example 1 (the content of aflatoxin M1 is about 0.55 mu g/kg), the heart rate is 124-127, and the aberration rate is higher than 30%. In conclusion, when the embryo heart rate is lower than 126 times/minute and the aberration rate exceeds 30%, the aflatoxin M1 in the milk powder is judged to be overproof (the aflatoxin M1 is higher than 0.5 mu g/kg); when the heart rate is lower than 110 times/minute and the aberration rate exceeds 30 percent, the aflatoxin M1 in the milk powder is judged to be seriously out of standard (the aflatoxin M1 is higher than 50 mu g/kg).
In summary, according to the method for detecting aflatoxin M1 in milk powder, provided by the invention, a culture solution for feeding zebra fish embryos is formed by mixing a milk powder extracting solution and water, and whether the content of aflatoxin M1 in the milk powder exceeds the standard or not is judged by measuring the heart rate of the zebra fish embryos and observing the malformation condition of the zebra fish embryos. The detection method does not need a large compact instrument, is convenient to operate, and can realize rapid and efficient detection.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. 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.
Claims (10)
1. A method for detecting aflatoxin M1 in milk powder is characterized by comprising the following steps:
mixing the milk powder extract with water to form a culture solution, placing the zebra fish embryo in the culture solution for culture, and judging the overproof condition of aflatoxin M1 in the milk powder according to the heart rate or the aberration rate of the zebra fish embryo;
wherein, the deformity is judged according to the conditions of the liver and yolk sac of the zebra fish embryo.
2. The method for detecting aflatoxin M1 in milk powder as claimed in claim 1, wherein the milk powder extract is in the form of milk powder extract, the volume ratio of the milk powder extract to water is 0.8-1.2:1000, and the heart rate or the teratocardiology rate is measured after culturing in the culture solution for 90-100 h;
preferably, malformation refers to degeneration of the liver or delayed yolk sac absorption.
3. The method for detecting aflatoxin M1 in milk powder as claimed in claim 2, wherein the volume ratio of the milk powder extract to water is 0.9-1.1: 1000;
preferably, the temperature is controlled to be 27-28 ℃ during the culture process in the culture solution;
preferably, the culture time in the culture solution is 95-98h, and light-shielding treatment is carried out during the culture process.
4. The method for detecting aflatoxin M1 in milk powder as claimed in claim 3, wherein the preparation process of the milk powder extract comprises: mixing and dissolving milk powder and water, mixing and extracting with an extractant solution, and separating supernatant after salting out; evaporating the obtained supernatant to dryness and redissolving;
preferably, the extractant is selected from at least one of 0.5-1.5% acetonitrile acetate, 0.5-1.5% acetonitrile formate, methanol and acetonitrile; more preferably 0.5-1.5% acetonitrile acetate.
5. The method for detecting aflatoxin M1 in milk powder as claimed in claim 4, wherein the mass fraction of the extractant solution is 0.5-1.5%, the dosage of the extractant solution per gram of milk powder is 5-10M L, and the dosage of water per gram of milk powder is 3-8M L;
preferably, the agent used in the salting-out process is chloride; more preferably, the chloride is sodium chloride, and the amount of the sodium chloride per gram of the milk powder is 1.5-2.5 g.
6. The method for detecting aflatoxin M1 in milk powder according to claim 4, wherein the solvent used in the reconstitution process is at least one selected from dimethyl sulfoxide and acetone; more preferably dimethyl sulfoxide;
preferably, the volume ratio of the supernatant to the solvent used in the reconstitution process is 70-80: 1;
preferably, the process of separating the supernatant is to vortex for 0.5-1.5min, then centrifuge for 3-8min at the rotation speed of 4000-.
7. The method for detecting aflatoxin M1 in milk powder of any one of claims 2-6, wherein after the culture in the culture solution is completed, the heart rate is less than or equal to 126 times/min, and the content of aflatoxin M1 in the milk powder is more than 0.5 μ g/Kg;
preferably, if the heart rate is less than or equal to 121 times/min, the content of the aflatoxin M1 in the milk powder is more than 5 mug/Kg;
preferably, if the heart rate is less than or equal to 110 times/min, the content of aflatoxin M1 in the milk powder is more than 50 mug/Kg.
8. The method for detecting aflatoxin M1 in milk powder of any one of claims 2-6, wherein after the culture in the culture solution is completed, the distortion rate is greater than or equal to 30%, and the content of aflatoxin M1 in the milk powder is more than 0.5 μ g/Kg.
9. The method for detecting aflatoxin M1 in milk powder according to claim 2, wherein the heart rate of the zebrafish embryos is measured and the malformation condition of the zebrafish embryos is observed when the culture time reaches 20-30h, 40-55h and 65-75h respectively;
preferably, when the zebra fish embryos are observed, a microscope is adopted for observation, and the magnification is adjusted to be 3-4 times; more preferably, the zebrafish embryos are fixed in lateral position using methyl-fibre gel during observation.
10. The method for detecting aflatoxin M1 in milk powder according to claim 1, wherein the zebrafish embryos are selected from the group consisting of normally developing 3-4hpf embryos;
preferably, the zebra fish embryo is a 3-4hpf zebra fish embryo which is normally developed and is obtained by mating adult AB-line zebra fish according to the male-female ratio of 1:1, collecting fertilized eggs and selecting.
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