CN113495125A - Method for evaluating safety of grain crops by using zebra fish - Google Patents

Abstract

The invention provides a method for evaluating the safety of grain crops by using zebra fish, which comprises the steps of constructing a zebra fish toxicity evaluation model, pretreating the grain crops by means of enzymolysis and chemical extraction 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 grain crops. 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

Method for evaluating safety of grain crops by using zebra fish

Technical Field

The invention relates to the technical field of grain safety evaluation, in particular to a method for evaluating the safety of grain crops by using zebra fish.

Background

The grain crops are indispensable consumer food in daily life of people, such as rice, flour, coarse cereals, tuberous root plants, beans, corn extracted starch and other grain crops, and come from cultivation. The safety of the grain crops mainly depends on the planting environment, and whether heavy metals, salts or other pollutants remain in the planting environment is a key factor influencing the food consumption of the grain crops.

According to the national standard of the people's republic of China, namely the inspection part of food sanitation, the current food safety inspection projects are mainly divided into three categories: one is drug residue (including pesticides, veterinary drugs, feed additives, food additives and the like), one is heavy metal, and the other is pathogenic parasite, microorganism and biotoxin. At present, the technology based on the safety detection of grain crops mostly uses chemical analysis means to detect and analyze indexes such as heavy metal, salt and other pollutant residues, and the biological means is rarely used to detect and analyze, 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 food crops 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 food crops. The grain crops have different components and contents, and the safety standard requirements of the grain crops are different from those of medicines and cosmetics, so how to develop a zebra fish model suitable for the safety of the grain crops and a detection method thereof are technical problems which need to be solved urgently by technical personnel in the field.

Disclosure of Invention

The invention aims to solve the technical problem of providing a brand-new method for evaluating the safety of grain crops by using zebra fish, which comprises the steps of carrying out pretreatment of enzymolysis and chemical extraction on the grain crops to obtain an extract of a detection product, and obtaining toxicity evaluation data after the zebra fish is treated, so that the safety of the grain crops is judged and evaluated according to the toxicity evaluation data, and the defects in the prior art are overcome.

Therefore, the invention adopts the following technical scheme:

a method for evaluating the safety of grain crops by using zebra fish comprises the steps of constructing a zebra fish toxicity evaluation model, pretreating the grain crops by means of enzymolysis and chemical extraction 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 grain crops.

Preferably, the method for evaluating the safety of the food crops by using the zebra fish comprises the following steps:

1) preparation of the extract

Mixing grain crops and ultrapure water in a ratio of 1:2, performing ultrasonic treatment, filtering and recovering, and adding enzyme for enzymolysis at 40-50 ℃ for 2-4 hours; after enzymolysis, adding a mixed solution of acetonitrile, DMSO and water in a volume ratio of 1.5:1:1 for extraction for 2 times, recovering and combining the extracting solutions, drying for 2 hours by nitrogen blowing, and performing rotary evaporation for 4 hours; after drying, the mixture is added with 0.5ml of 70% methanol to constant volume and stored at minus 20 ℃;

2) evaluation of safety

Diluting the extract prepared in the step 1) to a zebra fish embryo culture medium to obtain an extract diluent, wherein the dilution concentration is 1.67 mu l/ml, 3.3 mu l/ml, 6.7 mu l/ml, 13.3 mu l/ml and 26.7 mu l/ml;

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 48 hours of exposure, observing the toxicity symptoms of the zebra fish embryos, calculating the death rate of the zebra fish embryos, and judging and evaluating the safety of the food crops.

More preferably, the enzyme added in the step 1) for enzymolysis is one or more selected from amylase, glucosidase, sucrase, levase and protease.

More 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.

More preferably, dosage-effect simulation is carried out on the safety evaluation in the step 2) by adopting a Weibull model, a fitting curve is obtained by carrying out mathematical modeling through MATLAB, the value of a point on the fitting curve with y being 0.1 represents the NOEAL value, and the safety of the grain crops is judged according to the HBGV value; the calculation formula of the HBGV value is as follows: HBGV ═ NOAEL/UFs zebrafish;

wherein, HBGV value represents health guide value, UFs zebra fish represents uncertain coefficient value, and its calculation formula is: UFs Zebra fish UFs mammal ÷ 10^{Average (Log LC50 zebrafish/Log LC50 mammal)}。

The enzymolysis treatment in the invention refers to a process of simulating biodegradation, and has strong biological relevance and specificity.

In the present invention, the extraction of the sample of the enzymatic hydrolysate is preferably performed by organic and inorganic solvents (for example, extraction in which acetonitrile, DMSO, and water are mixed in a certain ratio).

The data analysis in the present invention includes simulation of the toxic dose response of the samples using the Weibull model, calculation of NOAEL and further health guidance value calculation (HBGV) to provide the maximum daily intake from a food safety perspective.

Mathematical modeling

Dose-effect simulation was performed on food safety using the Weibull model, a fitted curve was obtained by mathematical modeling with MATLAB, the Weibull model formula: theta1-theta2 Exp [ theta3+ theta4 Log [ x ].

The safety evaluation formula of the grain crops is as follows: HBGV ═ NOAEL/UFs zebrafish, where HBGV represents health guidance values, NOAEL represents no-effect dose values, and UFs represents uncertainty coefficient values.

1) Calculation of NOEAL:

the definition of OECD for normal mortality of zebrafish was met by finding a point value for y ═ 0.1 on the fitted curve.

2) UFs calculation:

uncertainty coefficients generally use default coefficients to account for uncertainty and variability. The uncertainty factor of 100 has been commonly used when converting NOAEL from a previous experimental animal study to a health guideline value for human exposure (see IPCS standard, 1987). But with data deficits, such as lack of efficacy in chronic disease studies, no NOAEL value could be determined for each experimental dose. Thus, LOAEL can also be used to formulate health guidance values (see IPCS Standard, 1994). The default 100-fold uncertainty factor represents the product of two independent 10-fold coefficients (i.e., 100 ═ 10 × 10), and the 2 "10-fold" coefficients mean: firstly, the difference between the average response of experimental animals for POD and the response of general population is deduced; ② variability of the response of highly sensitive people to the general population (IPCS, 1987).

The research value of the health guidance value of 100-10 x 10 is that different uncertainty coefficients are used by the NOAEL obtained based on the population experimental research and the NOAEL obtained based on the traditional model animal (such as rat, dog and the like) experimental research, so that the research of the health guidance value is more flexible.

However, the 10-fold difference between the average response of experimental animals deriving POD and the response of general population is not suitable for zebra fish, because most POD experimental animals are fed or injected, and the toxicity test of zebra fish is carried out by absorption, which are different from each other in experimental principle. Therefore, the applicant creatively converts zebra fish and LC50 of mammalian chemical toxicity evaluation to calculate UFs suitable for the zebra fish model, and the conversion formula is as follows:

UFs Zebra fish UFs mammal ÷ 10^{Average (Log LC50 zebrafish/Log LC50 mammal)}。

And calculating to obtain different types of food crops, wherein the range value of UFs zebra fish is 5-20.

Compared with the prior art, the method for evaluating the safety of the grain crops by using the zebra fish has the following beneficial effects:

the method selects proper homogenizing proportion, enzymolysis temperature and time and extraction solvent to carry out pretreatment of enzymolysis and chemical extraction and zebra fish treatment on the grain crops to obtain toxicity evaluation data, judges and evaluates the safety of the grain crops based on mathematical modeling analysis, has high detection accuracy, scientific and reasonable detection method and short modeling period, greatly reduces the detection cost, and provides practical feasibility for popularizing the detection method to the masses of people, enterprises and governments.

Drawings

FIG. 1 is a curve fitted to farm C rice safety assessment using the Weibull model of example 1 of the present invention.

Detailed Description

In order to make the technical solution of the present invention better understood by those skilled in the art, the technical solution in the present embodiment will be specifically described below with reference to the accompanying drawings in the present application. It should be noted that the following examples are only for illustrating the present invention and are not to be construed as limiting the present invention, and any modifications and changes made to the present invention within the spirit and scope of the claims are included in the scope of the present invention.

Example 1 examination of pretreatment method

This example compares the advantages and disadvantages of the enzymatic hydrolysis method, the chemical extraction method and the enzymatic hydrolysis-chemical extraction method in the pretreatment of grain crops, and the applicability in zebra fish experiments. Because the grain contains a large amount of polysaccharide substances, substances such as starch and the like can have a large influence on the living environment of the zebra fish and need to be removed. The amylase can effectively degrade starch and other substances in the grain, destroy the tissue structure of the grain and not influence toxic and harmful substances in the grain. Chemical extraction can also remove nutrient substances such as starch and the like, but can also remove a part of other harmful substances at the same time, and conflicts with the concept of omnibearing detection of zebra fish.

The enzymolysis-chemical extraction method combines the enzymolysis method with the chemical extraction method, and after nutrient substances are removed, the chemical extraction method is used for concentration, so that the sample adding amount is reduced, and the influence on the growth environment of the zebra fish is reduced.

Aiming at most of grains, the starch content is relatively high, and amylase degradation is the method for removing main nutrient substances firstly. For grains with higher cellulose, protein and sugar contents, the degradation by using corresponding enzymes can be considered. For chemical extractions, comparing 10% to 100% concentration of methanol, acetonitrile, DMSO and tetrahydrofuran, acetonitrile at 70% to 80% concentration was found: DMSO, DMSO: water 1: 1.5: the extraction rate is optimal when the extraction rate is 0.5, and the extraction rate of various substances can reach 75-80%. The degreasing step may also be applied to some grain samples, and the ratio of cyclohexane to polar organic phase is found to be 1: the degreasing effect is best when 2.

The results of the comparison of the three pretreatment methods are shown in table 1:

TABLE 1 comparison of three grain pretreatment methods

	Recovery of toxic substances	Nutrient removal profile	Zebra fish living environment influence
				Chemical extraction method	+	++	+
Enzymolysis method	+++	+++	+++
				Enzymolysis-chemical extraction method	+++	+++	+

Comprehensively considering the recovery rate of toxic substances, the removal rate of nutrient substances and the influence on the growth environment of the zebra fish, the extraction rate of the toxic substances is more than ' +++, the removal condition of the nutrient substances is more than ' ++, and the shadow response to the growth environment of the zebra fish is less than ' ++.

In conclusion, it was determined that the food crops of the present invention were pretreated by enzymatic hydrolysis-chemical extraction (i.e., enzymatic hydrolysis followed by chemical extraction).

Example 2 examination of the homogeneity ratio

Mixing grain crops and ultrapure water in a ratio of 1: 2(w/v) in the ratio comparative 1: 1. 1: 2. 1: 4 and 1: 8, taking the homogenizing effect and the volume of the chemical extraction water phase into consideration. For 1:1, the sample is difficult to be completely homogenized due to small liquid content, and the obtained homogenized liquid is very turbid and cannot be filtered smoothly. For 1: 4. and 1: the mixing ratio of 8 influences subsequent chemical extraction due to the large water content of the homogeneous liquid, so that the drying step of the chemical extraction is very complicated and time-consuming.

After comprehensive consideration, the homogenizing ratio of the grain crops to the ultrapure water is determined to be 1:2 (w/v).

EXAMPLE 3 examination of enzymatic hydrolysis temperature and time

Depending on the nature of the nutrient content in the sample, it is contemplated to add different amounts of enzymes, amylase, glucosidase, sucrase, levase, protease, etc. The enzyme mixture was added to the pre-configured nutrient solution and the optimal reaction conditions for the mixed enzymes were tested. The results are shown in Table 2.

TABLE 2 relationship of enzymolysis efficiency with temperature and time

In conclusion, the enzymolysis temperature and time are obtained by comparing different results, and the enzymolysis efficiency is highest when the temperature is 40-50 ℃ and the time is 2-4 h.

Example 4 examination of extraction solvent

Comparing the extraction rates of 10-100% methanol, acetonitrile, DMSO, acetone and tetrahydrofuran and a mixture of the methanol, the acetonitrile, the DMSO, the acetone and the tetrahydrofuran to grain samples, carrying out extraction rate analysis on a self-made known residual positive sample, wherein the positive additive mainly comprises pesticide, pollutant, food additive and heavy metal salt, and the comparison result shows that the acetonitrile: DMSO, DMSO: water 1.5:1: the extraction rate of 1 repeated extraction for three times is optimal, and the extraction rate of various substances can reach 75-80%. Drying the extractive solution by mixing at 55 deg.C and 55 deg.C under nitrogen blowing. The optimal volume fixing solution after drying is 70% methanol, and the consideration factors are the toxicity of the organic solvent to the zebra fish and the solubility of the organic solvent aqueous solution to the extract. The results are shown in Table 3.

TABLE 3 comparison of extraction rates of organic solvent mixtures in different proportions

Comprehensively considering the influence of the organic solvent on the toxicity of the zebra fish, the extraction rate of harmful substances and the single extraction rate of the harmful substances, determining the selection of the extraction solvent: acetonitrile: DMSO, DMSO: water 1.5:1: 1.

example 5 Zebra fish toxicity evaluation test

(1) Acute toxic effect

Selecting zebra fish with 4-128 cell stages as a test living body, contacting the extract of the embryonated fish sample, and selecting a static or semi-static system to test according to the stability of a tested object, wherein the indexes are embryo coagulation and non-developmental somite. The lack of heartbeats and the absence of tail separation from the yolk sac, these endpoints were taken as markers for the determination of zebrafish death, and were observed at 24h and 48h recordings and used to calculate the median lethal concentration LC50 and NOAEL values for the sample extracts. And 24h and 48h are proper observation time for screening, and the consideration factor is the timeliness and accuracy of the zebra fish experiment.

After 72h, the influence of the zebra fish growth environment on the zebra fish growth environment begins to increase, and in addition, the toxic effect is extremely high in the period from 48h to 72h, so the evaluation of the safety of the sample by considering the result after 48h is not accurate. Other non-death toxic effects are recorded and used as toxicity judgment indexes, such as cardiovascular toxicity, liver toxicity and renal toxicity.

(2) Specific toxic effects

Phenotype specific toxicity: the sample extracts were tested for their specific effects on zebrafish embryos for their specific tissue, organ or hormonal system effects. Including cardiotoxicity, hepatotoxicity, muscle toxicity, neurotoxicity, nephrotoxicity, ototoxicity, susceptibility to epilepsy, endocrine disruption, intestinal motility, changes in skin pigmentation, pancreatic toxicity, cancer induction.

Hepatotoxicity test: zebrafish have been studied as a model of drug-induced hepatotoxicity. The transparency of zebrafish after several days post fertilization enables in vivo visual observation of internal organs including the liver. Zebrafish complete primary liver morphogenesis after 48h fertilization (HPF). When exposed to liver toxicants, changes in liver morphology can be observed under a microscope. Endpoints for liver toxicity were assessed as: liver degeneration, changes in liver size and shape, and delayed yolk sac absorption.

Cardiotoxicity test: the zebrafish heart is composed of ventricles and atria, develops rapidly, and cardiovascular and heartbeat are observed 24 hours after fertilization, and cardiovascular circulation, cavity formation and blood circulation are completed 72 hours ago. The cardiac toxicity index is evaluated as follows: heart rate, heart rhythm, pericardial edema, hemorrhage, slowed blood flow, venous sinus congestion, and the like.

Application example 1 evaluation of safety of rice

Purpose of the experiment: the rice produced in the three places of the farm A, B, C is taken for acute toxicity test of zebra fish, and safety is evaluated.

The experimental method comprises the following steps:

(1) preparation of the extract

15g of rice from different origins were mixed with 30ml of ultrapure water and homogenized mechanically. Filtering the extractive solution with filter paper, recovering, adding amylase, levulose enzyme, glucolase and sucrase 0.15g each, placing in shaking table, mixing at 45 deg.C, and performing enzymolysis for 2 hr. A mixture of acetonitrile, DMSO and water (v: v: v ═ 1.5:1: 1) was added to the mixture to conduct extraction. Recovering liquid phase, extracting twice, mixing extractive solutions, drying the extractive solution at 55 deg.C under nitrogen flow for 2 hr, and rotary evaporating at 55 deg.C for 4 hr. The dried material was made up to volume with 0.5ml 70% methanol and stored at-20 ℃ until tested.

(2) Evaluation of safety

Taking the rice extract prepared in the step (1), and diluting the rice extract to the concentration of 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) of 1.67. mu.l/ml, 3.3. mu.l/ml, 6.7. mu.l/ml, 13.3. mu.l/ml and 26.7. mu.l/ml. Zebrafish embryos at 4-128 cell stages were exposed to six well plates containing dilutions of the extract, each well plate holding 30 roe eggs. The culture medium and 3.7mg/L dichloroaniline were set as negative and positive controls. After 48 hours of exposure at 26 ℃, the zebrafish embryos were observed under a stereomicroscope for signs of toxicity. Mortality was calculated at each concentration as the acute toxicity endpoint. The test results are shown in Table 4.

TABLE 4 acute toxicity of Rice extracts of different origins

(3) Data analysis

The data of farm C were simulated using the Weibull model, and the data simulated using the Weibull model were compared with experimental values to find that the model fitted well, and four parameter values in the model were calculated, theta 1-1.0261, theta 2-1.0369, theta 3-1.3155, and theta 4-1.5207. The fitted coherence curve was plotted (see fig. 1).

From the fitted curve, UFs of the rice was 16.2, and based on this, the HBGV value was calculated to be 0.00617 g/ml-6.17 g/kg, and the health guidance value for safely eating farm C rice was 370 g/day, based on the weight of the human being as 60 kg.

Application example 2 safety evaluation of flour

Purpose of the experiment: flour produced by the three farms A, B, C is taken for acute toxicity test of zebra fish, and safety is evaluated.

The experimental method comprises the following steps:

(1) preparation of the extract

Mixing flour 15g from different producing areas with ultrapure water 30ml, and ultrasonic treating for 10 min. Filtering the extractive solution with filter paper, recovering, adding amylase, levulose enzyme, glucolase and sucrase 0.2g each, placing in shaking table, and performing enzymolysis at 40 deg.C for 2 hr. A mixture of acetonitrile, DMSO and water (v: v: v ═ 1.5:1: 1) was added to the mixture to conduct extraction. Recovering liquid phase, extracting twice, mixing extractive solutions, drying the extractive solution at 55 deg.C under nitrogen flow for 2 hr, and rotary evaporating at 55 deg.C for 4 hr. The dried material was made up to volume with 0.5ml 70% methanol and stored at-20 ℃ until tested.

(2) Evaluation of safety

Taking the flour extract prepared in the step (1), and diluting the flour extract to the concentration of 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) of 1.67 mul/ml, 3.3 mul/ml, 6.7 mul/ml, 13.3 mul/ml and 26.7 mul/ml. Zebrafish embryos at 4-128 cell stages were exposed to six well plates containing dilutions of the extract, each well plate holding 30 roe eggs. The culture medium and 3.7mg/L dichloroaniline were set as negative and positive controls. After 48 hours of exposure at 26 ℃, the zebrafish embryos were observed under a stereomicroscope for signs of toxicity. Mortality was calculated at each concentration as the acute toxicity endpoint. The test results are shown in Table 5.

TABLE 5 acute toxicity of flour extracts of different sources

Application example 3 evaluation of safety of potatoes

Purpose of the experiment: potatoes produced in three places of a farm A, B, C are taken and subjected to an acute toxicity test of zebra fish, and safety is evaluated.

The experimental method comprises the following steps:

(1) preparation of the extract

Potatoes were first peeled off and 15g were mixed with 30ml of ultrapure water and homogenized mechanically. Filtering the extractive solution with filter paper, recovering, adding amylase, levulose enzyme, glucolase and sucrase 0.15g each, placing in shaking table, mixing at 50 deg.C, and performing enzymolysis for 4 hr. A mixture of acetonitrile, DMSO and water (v: v: v ═ 1.5:1: 1) was added to the mixture to conduct extraction. Recovering liquid phase, extracting twice, mixing extractive solutions, drying the extractive solution at 55 deg.C under nitrogen flow for 2 hr, and rotary evaporating at 55 deg.C for 4 hr. The dried material was made up to volume with 0.5ml 70% methanol and stored at-20 ℃ until tested.

(2) Evaluation of safety

The potato extract prepared in step (1) is diluted to a concentration of 1.67. mu.l/ml, 3.3. mu.l/ml, 6.7. mu.l/ml, 13.3. mu.l/ml and 26.7. mu.l/ml in zebrafish embryo culture medium (deionized water containing 294.0mg/L of anhydrous sodium chloride, 123.3mg/L of magnesium sulfate heptahydrate, 63.0mg/L of sodium bicarbonate and 5.5mg/L of potassium chloride). Zebrafish embryos at 4-128 cell stages were exposed to six well plates containing dilutions of the extract, each well plate holding 30 roe eggs. The culture medium and 3.7mg/L dichloroaniline were set as negative and positive controls. After 48 hours of exposure at 26 ℃, the zebrafish embryos were observed under a stereomicroscope for signs of toxicity. Mortality was calculated at each concentration as the acute toxicity endpoint. The test results are shown in Table 6.

TABLE 6 acute toxicity of potato extracts of different origins

Application example 4 evaluation of safety of Soybean

Purpose of the experiment: soybeans produced from three farms A, B, C were taken and subjected to acute toxicity test of zebra fish to evaluate safety.

The experimental method comprises the following steps:

(1) preparation of the extract

15g of soybeans were mixed with 30ml of ultrapure water and homogenized mechanically. Filtering the extractive solution with filter paper, recovering, adding cellulase, levulose enzyme, glucolase and sucrase 0.1g each, placing in shaking table, mixing at 45 deg.C, and performing enzymolysis for 2 hr. A mixture of acetonitrile, DMSO and water (v: v: v ═ 1.5:1: 1) was added to the mixture to conduct extraction. Recovering the liquid phase, repeatedly extracting for two times, combining the extracting solutions, and mixing the extracting solutions according to the volume ratio of 1:2, adding cyclohexane for washing and degreasing, and repeating the washing twice. The extract was dried at 55 ℃ for 2h under a nitrogen stream and rotary evaporated at 55 ℃ for 4 h. The dried material was made up to volume with 0.5ml 70% methanol and stored at-20 ℃ until tested.

(2) Evaluation of safety

Taking the soybean extract prepared in the step (1), and diluting the soybean extract to the concentration of 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) of 1.67 mul/ml, 3.3 mul/ml, 6.7 mul/ml, 13.3 mul/ml and 26.7 mul/ml. Zebrafish embryos at 4-128 cell stages were exposed to six well plates containing dilutions of the extract, each well plate holding 30 roe eggs. The culture medium and 3.7mg/L dichloroaniline were set as negative and positive controls. After 48 hours of exposure at 26 ℃, the zebrafish embryos were observed under a stereomicroscope for signs of toxicity. Mortality was calculated at each concentration as the acute toxicity endpoint. The test results are shown in Table 7.

TABLE 7 acute toxicity of Soybean extracts from different sources

Claims (5)

1. A method for evaluating the safety of grain crops by using zebra fish is characterized by constructing a zebra fish toxicity evaluation model, pretreating the grain crops by means of enzymolysis and chemical extraction 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 grain crops.

2. The method of claim 1, comprising the steps of:

1) preparation of the extract

Mixing grain crops and ultrapure water in a ratio of 1:2, performing ultrasonic treatment, filtering and recovering, and adding enzyme for enzymolysis at 40-50 ℃ for 2-4 hours; after enzymolysis, adding a mixed solution of acetonitrile, DMSO and water in a volume ratio of 1.5:1:1 for extraction for 2 times, recovering and combining the extracting solutions, drying for 2 hours by nitrogen blowing, and performing rotary evaporation for 4 hours; after drying, the mixture is added with 0.5ml of 70% methanol to constant volume and stored at minus 20 ℃;

2) evaluation of safety

Diluting the extract prepared in the step 1) to a zebra fish embryo culture medium to obtain an extract diluent, wherein the dilution concentration is 1.67 mu l/ml, 3.3 mu l/ml, 6.7 mu l/ml, 13.3 mu l/ml and 26.7 mu l/ml;

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 48 hours of exposure, observing the toxicity symptoms of the zebra fish embryos, calculating the death rate of the zebra fish embryos, and judging and evaluating the safety of the food crops.

3. The method as claimed in claim 2, wherein the enzyme added in step 1) is selected from one or more of amylase, glucosidase, sucrase, levulose enzyme and protease.

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 claim 2, wherein the safety evaluation in step 2) is performed by using a Weibull model for dose-effect simulation, a fitting curve is obtained by performing mathematical modeling through MATLAB, a point value of y-0.1 on the fitting curve represents a NOEAL value, and the safety of the food crop is judged according to the HBGV value; the calculation formula of the HBGV value is as follows: HBGV ═ NOAEL/UFs zebrafish;

wherein, HBGV value represents health guide value, UFs zebra fish represents uncertain coefficient value, and its calculation formula is:

UFs Zebra fish UFs mammal ÷ 10^{Average (Log LC50 zebrafish/Log LC50 mammal)}。

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