RU2604802C1 - Method of determining safety of food ingredients with help of cell test systems - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, namely to hygienic safety facilities of food purposes. Method of determining safety of food ingredients is disclosed, in which test systems are cell cultures of mammals and human. Method involves three stages: preparation of analyzed object, preparation of test system and determination of safety of object for test system. Safety of food ingredients is estimated on basis of determination of cell culture viability using Goryaev's chamber and cytopathic activity of analyzed objects.

EFFECT: invention provides higher accuracy and reliability of results of determination of safety of food ingredients for humans.

1 cl, 4 tbl, 2 ex

Description

The invention relates to the field of hygienic safety of food objects and can be used in biotechnology for preclinical assessment of toxicity of biologically active substances and products of microbial synthesis, in the food industry for routine quality control of raw materials, food ingredients, additives and products, as well as in research works.

Ensuring food safety is an important part of the sanitary and epidemiological well-being of the population, since poor-quality food products can lead to diseases of various etiologies. To prevent this, it is necessary to carefully conduct toxicity testing of food items, including at the preclinical stage.

A biological safety assessment of food products and ingredients is carried out using unicellular protists, insects, small crustaceans, algae and seeds of higher plants.

Known methods of biotesting food using Tetrahymena pyriformis infusoria as a test organism, including a method for determining the safety of vegetable oils [6] and the express method of toxicological biological assessment of meat, meat products and milk [2]. In both methods, toxicity is determined by the number of ciliates that multiplied in test samples during 72-96 hours of cultivation, the nature of the movement of ciliates, the presence of altered forms and dead cells in the culture. The number of protozoa is counted under a small magnification of the microscope in the Goryaev’s chamber. The data obtained are compared with the number of ciliates in the control, and the result - safety (B) - is expressed as a percentage according to formula (1) as follows:

where K _and - the number of microorganisms in the test sample of oil;

To _to - the number of microorganisms in the control sample of oil.

Express methods are known for determining the general toxicity of feed and raw materials for their production, including microbiological synthesis products, milk powder, concentrated feed additives according to GOST 31674-2012 [1]. Biotesting is carried out at the ciliates, stilonychia (Stylonychia mytilus) or colodia (Colpoda steinii). The method is based on the extraction of various fractions of toxic substances from the studied feeds in parallel with acetone and water, followed by the action of these extracts on stylohychy or on colpodes. Assessment of the biotest result is given by the reaction of the death of ciliates. Safe food is considered to be defined as non-toxic (the survival rate of stylonichia is more than 70%; about 90% of colpods remained mobile), while a simultaneous study of both acetone and water extract. The survival rate of stylonychia N,% is calculated by the formula (2) as follows:

where N ₂ is the arithmetic mean (of five tests) of the number of stylonichia at the end of the experiment after 1 h of exposure, pcs .;

N ₁ is the arithmetic mean (of five tests) of the number of stylonichia at the beginning of the experiment, pcs.

A known method for a comprehensive assessment of the toxicity of feed and food products, which consists in the use of ciliates Paramecium caudatum, cultivated on grains of long-grain rice. Toxicity is assessed by the percentage of surviving ciliates in an aqueous solution of the acetone extract of the test product, calculated by a formula similar to formula (2). The degree of toxicity of the product when testing its aqueous solution is determined by the time during which the death of the ciliates occurs [5].

The disadvantage of these methods is that test organisms belong to the kingdom of Protozoa and have a much simpler cellular organization than higher vertebrates, whose organisms consist of various types of tissues and cells.

When determining the safety of food additives and ingredients, higher animals (young individuals of rats, mice, rabbits, cats, etc.), including agricultural ones (for example, chickens), are usually used as a model system of the human body.

Known methods for determining the total toxicity of feed, feed and feed additives by biotesting in parallel on rabbits (skin test) and mice (acute experience) according to GOST 31674-2012 [1].

The result is determined by the totality of reactions in both methods: the feed is non-toxic (non-toxic in both tests), the feed is toxic (toxic in at least one test).

The first method is based on the dermonecrotic effect on the skin of rabbit of toxic substances, mainly of mycogenic origin, extracted from feed with acetone. The toxicity of the studied feed is determined by the presence of an inflammatory process on the skin area with the applied extract: the feed is non-toxic if there is no inflammation.

The second method is based on the introduction of an aqueous or acetone feed extract once into the stomach of white mice. The reaction is taken into account on the basis of an analysis of the state of internal organs (gastrointestinal tract, liver, spleen, and kidneys) at opening of mice. The food is nontoxic if all the mice are alive, and there were no pathological changes detected during the autopsy of the killed mice.

Known methods for determining the safety and effectiveness of dietary supplements to food [4] and assess the harmlessness of probiotic microorganisms [3] on models of conventional linear laboratory animals of both sexes - mice, rats, guinea pigs, rabbits, with oral administration of standard and aggravated doses of substances.

When determining the harmlessness of strains of probiotic microorganisms, colony of a 24-hour culture of bifidobacteria or lactobacilli grown on agarized MPC medium is removed from the agar loop, suspended in 0.5 ml of 0.9% sodium chloride solution to a concentration of 10 ⁸ cells / ml and incubated 30 min at a temperature of (37 ± 1) ° С. 0.5 ml of the resulting suspension is introduced using a special nozzle on the syringe into the stomach of 5 white mice weighing (15 ± 1) g. Observation of the mice is carried out for 5 days. In the case of the death of at least one mouse, the experiment is repeated on twice the number of animals. If in a repeated experiment none of the mice dies, the strain is considered harmless, otherwise the strain is rejected [3].

The disadvantages of methods using higher mammals are the high cost, cumbersomeness, length of analysis and ethical issues. Therefore, a simple, affordable and reliable method of assessing the impact of various food ingredients on a living organism and its system is needed.

The above disadvantages are deprived of the proposed method of biological assessment using animal and human cell cultures as test systems. In vitro cells respond to chemical and biological factors adequately to the original organism.

The objective of the invention is to develop an effective method for determining the safety of food ingredients for humans.

The technical result of the proposed method is to expand the technological capabilities of the method, which include determining not only toxicity, but also the overall safety and functional potential of food ingredients, as well as improving the accuracy and reliability of the results and their quantitative determination.

The advantages of the proposed method using cell cultures over methods using higher animals and protozoa in the following:

- simultaneous formulation of a large number of experiments is possible;

- the ability to assess the dynamics of the behavior of the cellular system depending on time, i.e. in addition to the acute toxicity of the object, evaluate chronic toxicity;

- the opportunity within 24 hours to make a preliminary conclusion about the safety of the product;

- the use of several types of human tissues and cells provides the basis for extrapolating the results to the human body as a whole;

- the method of cultivation in a monolayer and intravital observation of human cells using a microscope allows us to determine their viability and, accordingly, the degree of impact of the test object with greater accuracy.

The proposed method for determining the safety of food ingredients, in which mammalian and human cell cultures are used as test systems, includes three stages:

1) preparation of the test object: a series of double serial dilutions of decreasing concentrations are prepared from the initial concentration of the drug; for the preparation of dilutions from each sample of an ingredient or product by a weight or volumetric method, a sample is taken; the weight (volume) of the sample is individual for each specific ingredient; for the preparation of dilutions of water-soluble objects, saline is used, for soluble in organic solvents - ethyl alcohol, oils, if necessary - solvents (DMSO, etc.) that do not have cytotoxic activity;

2) preparation of the test system: grow a monolayer of cells in a culture vial with an area of 25 cm ² on the appropriate nutrient medium EMEM, DMEM or 199, including 10% serum of fetal bovine or cattle, 0.05% of the antibiotic gentamicin or penicillin-streptomycin; remove the nutrient medium from the formed monolayer; washed with a monolayer of cells twice with phosphate-buffered saline, or Versen's solution, or saline;

3) determining the safety of the object for the test system: 0.5 ml of the solution of the test object and 10 ml of EMEM, DMEM or 199 medium containing 10% bovine or cattle serum are added to the vial with the formed cell monolayer; cultured for 1 h in a CO ₂ incubator at a temperature of 37 ° C and a gas concentration of 5.0%; remove the nutrient medium and wash the cell monolayer twice with phosphate-buffered saline, or Versen's solution, or saline; make 10 ml of fresh nutrient medium EMEM, DMEM or 199, including 10% serum of fetal bovine or cattle; the bottle is placed for 24 hours in a CO ₂ incubator at a temperature of 37 ° C and a gas concentration of 5.0%; remove the nutrient medium; monolayer removal is carried out with 1 ml of a mixture of Versen solutions 0.02% and trypsin 0.25% in a ratio of 3: 1; cells are stained with trypan blue dye solution; they count living and dead cells using a Goryaev camera and determine viability and cytopathic activity.

As a control, use a bottle with cells grown on a nutrient medium without adding a test substance. The tests are repeated at least three times. The data obtained are compared with the control, and the result - viability (G) - is expressed as a percentage according to formula (3) as the ratio of the number of living cells grown on the medium with the addition of the studied ingredient to the total number of cells:

where KZ is the arithmetic mean of the number of living cells at the end of the experiment, pcs .;

To - the arithmetic mean of the total number of cells at the beginning of the experiment, pcs .;

100 - conversion factor in percent.

For the concentration range of the test ingredient, TC ₅₀ is determined - an indicator that characterizes the concentration of the test object in a nutrient medium, which leads to the destruction of the cell population by 50%, i.e. corresponds to a viability of 50%.

The object has low cytotoxicity, and is safe for use in food products, if based on the content of pure substance, its concentration, which results in a 50% destruction of the cellular system is greater than 0.03% (TC _50/10).

Otherwise, the object has a high cytotoxicity (as pure substance based on its concentration, which results in a 50% destruction of the cell system does not exceed 0.03% (TC _50/10)) is safe as a food ingredient and not recommended for use in food production.

Determining the safety of food ingredients is carried out on monolayer permanent (transplantable) cell lines of various origins, morphology, tissue type, for example HEK 293 - kidney of a human embryo; FLECH (FLECH) - fibroblasts of the human lung embryo; Ner-2 - epidermoid carcinoma of the human larynx; CaCO-2 - adenocarcinoma of the human colon; Girardi Heart - human atrial cells; VERO - African green monkey kidney cell line.

To maintain cell cultures, standard synthetic liquid nutrient media are used: MEM needle (minimal essential medium), DMEM (Dulbecco's modified Eagle's medium), 199, alpha MEM, F12, which are based on Earl and Hanks saline solutions. As a source of growth factors, blood serum of cattle or fetal bovine in the amount of 10-15% is used. The nutrient medium is prepared according to the passports of cell lines of the Russian collection of vertebrate cell cultures (RKKK P). To prevent contamination, use antibiotics penicillin-streptomycin or gentamicin. To avoid inactivation during storage, an antibiotic solution is prepared immediately before changing the medium: under sterile conditions, a weighed portion of the antibiotic is dissolved in saline and introduced into the cell culture medium at the rate of 100-200 UNITS per 1 ml of medium or 0.05%. As essential substances are depleted and the pH of the system changes, the medium is replaced in culture bottles every 2-3 days.

The preparation of the monolayer (removal and sieving) is as follows. The nutrient medium is removed from the formed monolayer by a laboratory aspirator; washed with a monolayer of cells twice with phosphate-buffered saline, or Versen's solution, or saline; cells are disaggregated into a suspension: 1 ml of Versene solution 0.02% and trypsin 0.25% in a ratio of 3: 1 are added to the vials, for better detachment of cells from the substrate, the vials are placed on a horizontal thermostatic shaker at 37 ° C with a rotation speed of 40-50 rpm for 10 min; the degree of disaggregation of the cell monolayer is monitored visually, with insufficient transition to the suspension, sterile scrapers lifters or scrapers are used; upon reaching the detachment, 90-100% of the cells are well resuspended by pipetting and spread; in a new vial, a suspension of cells with a density of about 6000-7000 cells / cm ² and 10 ml of EMEM, DMEM or 199 nutrient medium, including 10-15% bovine or cattle serum and 0.05% antibiotic gentamicin or penicillin-streptomycin; the vials are placed in a CO ₂ incubator at a temperature of 37 ° C and a gas concentration of 5.0%.

In the process of reseeding after trypsinization, the state of the cell culture is evaluated by eliminating the dye and counting the total number of cells, as well as identifying dead and living cells among them: an aliquot of the cell suspension (~ 40 μl) is mixed with an equal amount of a 0.4% trypan blue dye solution, 3 μl of the suspension is taken with a pipette, placed under the coverslip of the Goryaev chamber, and cells are counted under a small magnification of the microscope.

The proposed method is illustrated by the following examples.

Example 1

Study of the effect of the natural plant dye shikonin on the state of cell lines HEK 293 - human embryo kidney, FLECH - human lung embryo fibroblasts, HEP-2 - epidermoid carcinoma of the human larynx, MN-22a (MG-22a) - mouse hepatoma of the C3NA line, NCTC - subcutaneous connective tissue, mouse, clone 929.

A portion of the pigment is introduced into a sterile tube and sterilized for 1 h with 96% ethanol at the rate of 0.1 ml of alcohol per 5 mg of shikonin powder. With poor solubility, a few drops of DMSO are added. The method of double serial dilutions is used: a number of double serial dilutions of decreasing concentrations of the drug from the initial concentration of shikonin of 16 μg / ml to 0.5 μg / ml are prepared from this tube.

To prepare the test system, cells are grown on appropriate nutrient media according to table 1 with the addition of 0.05% gentamicin antibiotic for 10-14 days depending on the line; nutrient medium is removed from the formed monolayer by a laboratory aspirator; wash the cell monolayer twice with phosphate-buffered saline.

Nutrient media according to table 1 with alcoholic solutions of shikonin of decreasing concentrations are added to vials with a formed monolayer, incubated for 1 h in a CO ₂ incubator at 37 ° C and a gas concentration of 5.0%, after which the nutrient medium is removed from the vials, washed twice with phosphate saline buffer, add 10 ml of fresh nutrient medium, the bottles are placed in a CO ₂ incubator for 24 hours. After a day, the nutrient medium is drained, the cells are disaggregated with Versene 0.02% and trypsin 0.25% in a 3: 1 ratio on a rocking chair in within 10 minutes, removing the monolayer cells, staining and counting of living and dead cells using a Goryaev’s camera, according to the results of the calculations, the TC _{50 of} shikonin is determined for each line.

The data obtained are compared with the control - the number of monolayer cells grown on a nutrient medium without the use of shikonin extracts. Background exposure to 96% ethanol in the range of applied concentrations is negligible. The results of determining the viability of cell lines when exposed to alcoholic extracts of shikonin are presented in table 2.

In relation to the selected cell lines, the dose-dependent effect of naphthoquinone extracts is manifested. The effect of shikonin is significantly more pronounced in relation to tumor cells of mouse hepatoma and human laryngeal carcinoma. Cytopathic concentration naphthoquinone against various cellular test systems made, ug / ml: HEK-293 - about 8, NCTC and FLECH - about 4, MH-22a and HEp-2 - less than 2. Thus, the index TC _50/10 reached, %: HEK-293 - about 0.8, NCTC and FLECH - about 0.4, MN-22a and HEP-2 - less than 0.2. Thus, shikonin has low cytotoxicity, because in terms of pure substance, the cytopathic activity exceeds 0.03%, and it can be assumed that the drug is safe for use in food production.

Example 2

Determining the effect of spicy herbs marjoram, dill, parsley, black pepper (peas), cilantro (coriander), caraway seeds on the state of cell lines and choosing among them the object with the most gentle effect.

Formula for the preparation of alcoholic extracts: herbs are finely chopped, filled with 96% ethyl alcohol in a ratio of 1: 1.5 by weight and placed in a dark glass dish, tightly corked to prevent alcohol weathering. To intensify the extraction process, ultrasonic treatment of the extracts with an ultrasonic bath of 5.7 L Sapphire TTZ is used for 30 minutes at room temperature for each extract; supply voltage - 220 V / 50-60 Hz, operating frequency - 35 kHz. The prepared solutions are placed in a dark warm place for 2 weeks, periodically shaking. The resulting alcoholic extract was filtered through a paper filter and centrifuged for 5 minutes at 12,000 rpm.

VERO cell lines — the African green monkey kidney cell line, HEP-2 — human laryngeal epidermoid carcinoma, and FLECH — human lung embryo fibroblasts, are selected as cell test systems.

Cells are cultured in appropriate culture media according to table 3 with the addition of 0.05% penicillin-streptomycin antibiotic for 10-14 days depending on the line; nutrient medium is removed from the formed monolayer by a laboratory aspirator; washed with a monolayer of cells twice with phosphate-buffered saline; make 10 ml of the appropriate nutrient medium, including 10% serum according to table 3.

Alcoholic extracts of herbs in an amount of 0.5 ml are added to the medium with the resulting monolayer, incubated for 1 h in a CO ₂ incubator at 37 ° C and a gas concentration of 5.0%. Then, the nutrient medium is replaced and after 24 hours the number of living and dead cells and the viability index are counted. As a control, the number of living and dead monolayer cells grown without the use of plant extracts and alcohol.

The results of determining the viability of cell lines when exposed to 5% alcohol extracts of herbs are presented in table 4. As the background effect, the results obtained using 0.4 ml of 96% ethanol are taken. The data obtained are compared with the control, which is used as the number of living and dead cells of the monolayer grown without the use of plant extracts and alcohol.

The most gentle effect on cell test cultures was exerted by alcoholic extracts of marjoram, dill and black pepper. Plant extracts negatively affect the human laryngeal carcinoma HEP-2 cell line, especially marjoram, which is important in the suppression of cancer cells and in the fight against cancer.

Since in the case of processing cell test systems with 5% spice extracts, in no case did 50% of the population die, it can be concluded that tinctures of plants of these concentrations have low cytotoxicity, and we can assume that these drugs are safe for use in food production .

List of sources used

1. GOST 31674-2012 Feed, animal feed, animal feed. Methods for determining total toxicity. - M .: Standartinform, 2013 .-- 33 p.

2. Guidelines for toxicological evaluation of meat, meat products and milk using Tetrachimena pyrimiformis infusoria (express method): approved. GUV of the Ministry of Agriculture of the Republic of Belarus on 10.20.1997. - Vitebsk. - 1997. - 13 p.

3. MU 2.3.2.2789-10 Guidelines for the sanitary-epidemiological assessment of the safety and functional potential of probiotic microorganisms used for food production. - M .: Federal Center for Hygiene and Epidemiology of Rospotrebnadzor, 2010. - 93 p.

4. MUK 2.3.2.721-98 Determination of the safety and effectiveness of biologically active food additives. - M.: Federal Center for Sanitary Inspection of the Ministry of Health of Russia, 1999. - 47 p.

5. RF patent No. 2266015, IPC A23K, G01N. A method for a comprehensive assessment of the toxicity of feed and food / Grozdov A.O. (RF). - declared. 08/16/01 .; publ. 08/20/03.

6. RF patent No. 2415417, IPC G01N 33/03. A method for determining the safety of vegetable oils / Frantseva T.P., Prudnikova T.N., Shirokoryadova O.V. (RF), - declared. 08/03/09 .; publ. 03/27/11.

Claims (1)

A method for determining the safety of food ingredients, in which mammalian and human cell cultures are used as test systems, comprising three stages:
preparation of the studied object: preparation of a number of double serial dilutions of decreasing concentrations from the initial concentration of the drug; selection of a sample by weight or volume method for the preparation of dilutions from each sample of an ingredient or product; the weight (volume) of the sample is individual for each specific ingredient; preparation of dilutions of water-soluble objects in saline, soluble in organic solvents - in ethanol, oils, if necessary - in solvents (DMSO, etc.) that do not have cytotoxic activity;
preparation of the test system: growing a monolayer of cells in a culture bottle with an area of 25 cm ² on an appropriate nutrient medium EMEM, DMEM or 199, including 10% serum of fetal bovine or cattle, 0.05% of the antibiotic gentamicin or penicillin-streptomycin; removal of the nutrient medium from the formed monolayer; washing the cell monolayer twice with phosphate-buffered saline, or Versen's solution, or saline;
determining the safety of an object for a test system: introducing into a vial with a formed monolayer of cells 0.5 ml of a solution of the test object and 10 ml of EMEM, DMEM or 199 nutrient medium, including 10% bovine or cattle serum; cultivation for 1 h in a CO ₂ incubator at a temperature of 37 ° C and a gas concentration of 5.0%; removing the culture medium and washing the cell monolayer twice with phosphate-buffered saline, or Versen's solution, or saline; 10 ml of fresh EMEM, DMEM or 199 nutrient medium including 10% bovine or cattle serum; cultivation for 24 hours in a CO ₂ incubator at a temperature of 37 ° C and a gas concentration of 5.0%; nutrient removal; removal of a monolayer of 1 ml of a mixture of Versen solutions 0.02% and trypsin 0.25% in a ratio of 3: 1; staining cells with trypan blue dye solution; counting living and dead cells using a Goryaev’s camera; determination of viability and TS ₅₀ , where
viability (G) is expressed as a percentage according to formula (3) as the ratio of the number of living cells grown on the medium with the addition of the test ingredient to the total number of cells:

where KZ is the arithmetic mean of the number of living cells at the end of the experiment, pcs .;
To - the arithmetic mean of the total number of cells at the beginning of the experiment, pcs .;
100 - conversion factor in percent,
TC ₅₀ is a cytopathic activity that characterizes the concentration of the test sample in a nutrient medium, which leads to the destruction of the cell population by 50%, moreover, the object has low cytotoxicity and is considered safe for use in food products if, in terms of the content of pure substance, the concentration that leads to in a 50% destruction of the cellular system is greater than 0.03% (TC _50/10); otherwise the object has a high cytotoxicity (as pure substance based on its concentration, which results in a 50% destruction of the cell system does not exceed 0.03% (TC _50/10)) is safe as a food ingredient and not recommended for use in food production.