RU2403551C1 - Method to determine food value of worm-infested fishes - Google Patents

Abstract

FIELD: veterinary science.

SUBSTANCE: sample of fish muscular tissue is poured with hydrochloric acid into vessels for hydrolysis. Vessels are closed with plugs and placed into thermostat, then cooled, hydrolysate is taken by pipette into microvessel and dried. Then sodium carbonate is added, mixed, afterwards solution of phenyl isothiocyanate in isopropyl alcohol is added and left for reaction for 40 minutes. After the content dries under natural conditions, not more than 0.5 cm³ of distilled water is added, mixed, transferred to Eppendorf tube and centrifuged. Qualitative and quantitative analysis of amino acids is carried out in the sample by capillary zone electrophoresis using reaction buffer. Produced data is compared to reference result of combined amino acids concentration in clinically healthy fishes. If concentration of combined amino acids in infested fishes compared to the reference value reduces, then fish is considered to be of bad quality.

EFFECT: invention makes it possible to reduce time and to increase accuracy of combined amino acids detection in determination of fish products quality.

6 tbl, 1 dwg

Description

The invention relates to the field of veterinary medicine, in particular to veterinary and sanitary examination.

The definition of serum protein is known by electrophoresis [see Laboratory research in veterinary medicine, ed. V.Ya. Antonova and P.N. Blinova. M .: "Kolos", 1971. S. 423-427], where by electrophoresis the protein is divided into four groups: albumin, alpha, beta and gamma globulins. At pH 8.6, proteins in an electric field move toward the anode, because they are negatively charged under these conditions. Albumin, then alpha globulins, beta globulins and finally gamma globulins move most rapidly. Use a device for electrophoresis on paper; photoelectrocolorimeter for determining the amount of dye eluted from electrophoregrams, densitometer, chromatographic filter paper. Electrophoresis is carried out on strips of paper, then a colorimetric determination of the ratio of individual protein fractions is carried out by extracting ink from paper. After staining, four spots corresponding to albumin, alpha, beta and gamma globulins are detected on the electrophoregram.

With electrophoresis on paper, only the ratio between the individual fractions of whey proteins is determined. Therefore, it is necessary to determine the total amount of protein and calculate the absolute number of individual protein fractions.

The closest in technical essence is the method of analysis of amines in fish using the system of capillary electrophoresis "Drops" (see. Practical guidance on the use of systems of capillary electrophoresis "Drops", N.V. Komarova, Ya. S. Kamentsev. S-P, 2008 ., p.102), including derivatization of a biological sample, exposure to it by capillary zone electrophoresis using a working buffer, detection of amino acids and their qualitative and quantitative analysis.

The disadvantages of the known methods are the limited capabilities, the complexity of the study, the lengthy determination process, the lack of the possibility and quantitative composition of bound amino acids in the stretching of the muscles of various fish species, depending on the degree of invasion by Anisakis simplex larvae.

The technical solution to the problem is to expand the functional and technological capabilities using capillary electrophoresis of the Kapel-103R device, shorten the lead time and increase the accuracy of determination of bound amino acids when establishing the quality and safety of fish products invaded by Anisakis simplex larvae.

The problem is achieved in that in a method for determining the nutritional value of fish infected with helminths, including derivatization of a biological sample, exposure to it by capillary zone electrophoresis using a working buffer, detection of amino acids and their qualitative and quantitative analysis, according to the invention, an aqueous extract of muscle tissue is used as a biological sample fish infected with anisacidosis, pre-hydrolysis of the bioassay is carried out, for this they take a sample of 0.1-0.2 g of muscle tissue of fish and pour up to 10 cm ^{3 of a} 20% salt acid in the vessels for hydrolysis, then they are closed with stoppers and placed in a thermostat for 14 hours at a temperature of not more than 105 ° C, then cooled, not more than 0.05 cm ^{3 of the} hydrolyzate are pipetted into a micro vessel and dried to dryness, up to 0.1 is added cm ³ 0.1 M sodium carbonate solution, stirred, then add no more than 0.3 cm ³ solution of phenylisothiocyanate in isopropyl alcohol and leave to react for 40 minutes, after drying the contents under natural conditions, add no more than 0.5 cm ³ of distilled water, stirred, Perrin they are poured into an Eppendorf tube and centrifuged to remove gases and suspensions, then the data obtained as a result of qualitative and quantitative analyzes are compared with the control result of the concentration of bound amino acids of clinically healthy fish and the quality of the fish is determined, if the concentration of bound amino acids in infected fish decreases compared to the control, then fish are classified as poor quality.

The novelty of the proposed proposal is due to the fact that the rapid analysis established the parameters for changing the concentration of bound amino acids of the muscle tissue of fish depending on the degree of invasion by spiral-shaped larvae of the species Anisakis, genus Anisakis simplex, in addition, the order and time of release of bound amino acids in the hood of the muscle tissue of fish was established.

The advantage of this method is as follows: high separation efficiency of components, not available with other research methods; low consumption of reagents and solvents; simplicity of equipment; high speed analysis and high reproducibility of reaction conditions; good solubility of derivatives in an aqueous medium; commercial availability; the wide possibilities of capillary electrophoresis in the analysis of bound amino acids. In addition, the proposed proposal is more economical, because does not require the use of expensive chemicals.

The invention is illustrated by the drawing, which shows a graph of the concentration of bound amino acids in the extract of muscle tissue for example hamsa.

An example of a specific implementation of the method for determining the nutritional value of fish infected with helminths.

Parasitological studies of fish are preliminarily carried out, in which spiral larvae of the species Anisakis, of the genus Anisakis simplex in fish were identified: from the cod family - northern blue whiting (Micromesistius poutassou) and pollock (Theragra); from the herring family - Atlantic herring (Clupca barengus), from the anchovy family (Engraulidae), herring-like order - Black Sea hams (Engraulis encrasicholus ponticus); family smelt (Osmeridae) - capelin (Mallotus villosus). Then, depending on the degree of invasion by Anisakis larvae, fish samples are divided into groups. Blue whiting samples are divided into four groups: the first - during invasion from one to two Anisakis larvae., The second - from 9 to 13, the third - from 20 to 36, the fourth - from 44 to 63. Pollock samples - into two groups: the first - when detection of Anisakis larvae from one to two, the second - from 7 to 8. Herring samples - into three groups: the first - when detecting Anisakis larvae from 10 to 14, the second - from 15 to 20 and the third - from 31 to 42. Hamsa samples - into four groups: the first - non-invasive, the second - invasive from 1 to 4 Anisakis larvae, the third - from 15 to 20 and the fourth - from 21 to 27. Capelin samples - into two groups: the first - not invasive ovannaya larvae Anisakis, second - from 4 to 8.

The concentration of bound amino acids is determined in the stretch of muscle tissue above these fish, non-invasive and invasive by larvae of the species Anisakis, of the genus Anisakis simplex. For this purpose, the selected muscle tissue samples are preliminarily hydrolyzed. To do this, take a sample of 0.1-0.2 g of muscle tissue of fish and pour 10 cm ^{3 of} 20% hydrochloric acid in special vessels for hydrolysis. The vessels are closed with stoppers and placed in a thermostat for 14 hours at a temperature of 105 ° C. Then it is cooled, taken with a pipette into a microvessel (glass or bottle) of 0.05 cm ^{3 of} hydrolyzate and dried to dryness. Then add 0.1 cm ³ 0.1 M sodium carbonate solution, mix, then add 0.3 cm ³ solution of phenylisothiocyanate (FITC) in isopropyl alcohol (0.2 cm ³ FITC in 12 cm ^{3 of} isopropyl alcohol) and leave to pass reaction for 40 minutes. After the contents have dried under natural conditions, 0.5 cm ^{3 of} distilled water is added, mixed, transferred to an Eppendorf tube and centrifuged to remove gases and suspensions. For analysis, a Capill-103 R capillary electrophoresis device is used, which is equipped with an ultraviolet detector with a detector wavelength of 254 nm (see drawing).

Analysis conditions: quartz capillary, 0.5 m long to the detector, inner diameter 75 × 10 ^-6 m; adjustable high voltage source of positive polarity 3-25 kV; hydrostatic injection of a sample at a pressure of 30 mbar for 5 sec; β-cyclodextrin-based buffer working solution (54 mg in 12.5 cm ^{3 of a} buffer solution of dihydrogen phosphate and sodium hydrogen phosphate); voltage "plus 10 kV"; analysis time passes within 40 minutes; forced air cooling of the capillary to room temperature; output and processing of results on a computer. The method of capillary electrophoresis to determine the mass concentration of amino acids is based on the separation of anionic forms of N-phenylthiocarbamyl derivatives of amino acids under the influence of an electric field due to their different electrophoretic mobility. To identify and quantify the analyzed components, ultraviolet absorption is recorded at a wavelength of 254 nm.

Alpha amino acids, when reacted with a phenylisothiocyanate in an alkaline medium, produce N-phenylthiocarbamyl (FTK) derivatives, which are acids that exist under basic conditions in the form of anions. Due to the presence in the structure of the obtained compounds of the benzene ring, phenylcarbamyl derivatives have an absorption band at 254 nm. The content of bound amino acids is determined through its derivatives with phenylisothiocyanate by capillary electrophoresis.

In the process of analysis, the order and time of release of bound amino acids in the extract of fish samples were established (Table 1).

Table 1 The order and time of release of bound amino acids in the extraction of fish samples No. Component Exit time, min No. Component Exit time, min one 2 3 four 5 6 one Arginine 15,53 8 Valine 28.38 2 Lysine 21.37 9 Proline 29.13 3 Tyrosine 22.00 10 Threonine 30.19 four β-phenylalanine 23.28 eleven Tryptophan 30.71 5 Histidine 23.49 12 Series 32.03 6 Leucine 27.00 13 α-alanine 32.26 7 Methionine 28.00 fourteen Glycine 35.31

As a result of the experiment, the following distinctive criteria were established for the concentration of bound amino acids in the muscle tissue of fish, depending on the degree of invasion by their larvae of the species Anisakis, genus Anisakis simplex. The total concentration of bound amino acids in the blue whiting muscle extract, affected from one to two larvae, was 143332.32 mg / kg of minced fish, with an invasion of 9 to 13 Anisakis larvae - 117059.46 mg / kg of minced fish, with an invasion of 20 to 36 - 98655.25 mg / kg of minced fish, with an invasion of 44 to 63 - 83,363.16 mg / kg of minced fish (Table 2).

table 2 The concentration of bound amino acids in the extract of the blue whiting muscle (M ± m; n = 10) Associated amino acids, mg / kg minced fish The number of Anisakis larvae on viscero blue whiting 1-2 9-13 20-36 44-63 Arginine 6929.11 ± 122.19 6272.34 ± 86.45 *** 5334.22 ± 96.11 *** 4471.07 ± 114.29 *** Lysine 326.01 ± 3.13 264.89 ± 2.88 *** 240.66 ± 2.81 *** 217.88 ± 1.01 *** Tyrosine 56465.39 ± 1053.05 44808.64 ± 1254.56 *** 35817.88 ± 1192.96 *** 29602.44 ± 713.63 *** β-phenylalanine 171.18 ± 1.61 154.01 ± 1.60 *** 130.85 ± 1.10 111.37 ± 2.07 *** Histidine 5384.37 ± 86.68 5685.16 ± 110.64 * 4432.04 ± 136.88 *** 3296.78 ± 98.66 *** Leucine 11009.17 ± 193.01 9189.60 ± 118.81 *** 7699.76 ± 126.38 *** 6855.51 ± 180.65 *** Methionine 11576.88 ± 266.24 9878.88 ± 172.74 *** 8355.79 ± 231.71 *** 6479.28 ± 92.79 *** Valine 4702.09 ± 92.60 3670.68 ± 106.77 *** 3331.93 ± 115.76 *** 3760.93 ± 75.16 *** Proline 4982.38 ± 88.07 4067.99 ± 69.49 *** 3464.57 ± 95.41 *** 2951.27 ± 57.30 *** Threonine 14178.04 ± 141.13 12086.00 ± 236.66 *** 8780.16 ± 115.58 *** 7490.64 ± 92.28 *** Tryptophan 221.17 ± 1.34 189.21 ± 4.44 *** 180.22 ± 1.34 *** 110.91 ± 1.66 *** Series 3435.89 ± 82.36 2358.72 ± 61.71 *** 1710.41 ± 22.53 *** 1400.67 ± 23.01 *** α-alanine 18217.92 ± 166.08 15155.83 + 237.69 *** 16040.46 ± 115.17 *** 13302.55 ± 280.80 *** Glycine 5732.06 ± 83.73 3277.51 ± 85.40 *** 3136.30 ± 68.77 *** 3311.86 ± 92.31 *** * P <0.05; *** P> 0.001

Among all the associated amino acids in the blue whiting muscle extract, the maximum percentage concentration was attributable to tyrosine (39-43%), α-alanine (10-12%), threonine (9-11%), histidine (4-5%), and vice versa the minimum - for lysine (0.20-0.30%), tryptophan (015, -0.20%).

Table 3 The concentration of bound amino acids in the pollock muscle extract (M ± m; n = 10) Associated amino acids, mg / kg minced fish Number of Anisakis Viscero Pollock Larvae 1-2 7-8 Arginine 7527.86 ± 92.42 6558.58 ± 112.31 *** Lysine 334.60 ± 1.47 277.27 ± 1.74 *** Tyrosine 66746.94 ± 1298.05 39857.92 ± 1004.76 *** β-phenylalanine 2573.52 ± 69.12 1820.23 ± 28.76 *** Histidine 8022.69 ± 196.54 6464.98 ± 243.66 *** Leucine 12158.98 ± 115.03 10292.96 ± 155.47 *** Methionine 13931.84 ± 309.86 11013.63 ± 156.98 *** Valine 6074.15 ± 236.78 4482.47 ± 140.58 *** Proline 7089.47 ± 267.35 5194.37 ± 90.36 *** Threonine 17547.65 ± 193.97 14281.48 ± 267.81 *** Tryptophan 445.82 ± 8.06 280.11 ± 4.87 *** Series 4606.82 ± 112.62 3331.17 ± 118.67 *** α-alanine 22,248.38 ± 456.89 18236.08 ± 185.04 *** Glycine 9039.85 ± 172.49 7157.06 ± 120.95 *** *** P> 0.001

The total concentration of bound amino acids in the pollock muscle extract, affected from one to two larvae, amounted to 178348.57 mg / kg of minced fish, with an invasion of 7 to 8 Anisakis larvae - 129248.31 mg / kg of minced fish (Table 3).

Among all the associated amino acids in the pollock muscle extract, affected from one to two larvae, the maximum percentage concentration was tyrosine (30-37%), α-alanine (12-14%), threonine (9-11%), methionine (7 -8%), leucine (6-8%), histidine (4-5%), and, conversely, the minimum - for tryptophan (0.22-0.25%), lysine (0.19-0.21%) )

The total concentration of bound amino acids in an extract of herring muscles affected by 10 to 14 larvae was 152,103.64 mg / kg of minced fish, with an invasion of 15 to 20 Anisakis larvae - 93,538.3 mg / kg of minced fish, with an invasion of 31 to 42 - 99027.89 mg / kg of minced fish (table 4).

Table 4 The concentration of bound amino acids in the muscle extract of villages (M ± m; n = 10) Associated amino acids, mg / kg minced fish Number of Anisakis Viscero herring larvae 10-14 15-20 31-42 Arginine 12442.33 ± 285.79 5949.16 + 83.09 *** 5596.551200.15 *** Lysine 886.18 ± 23.77 402.77111.48 *** 253.8916.31 *** Tyrosine 1487.61 ± 36.59 903.47116.28 *** 1057.36124.98 *** β-phenylalanine 3832.01 ± 84.88 1257.82147.39 *** 1374.94149.05 *** Histidine 33547.64 ± 873.54 21331.3711355.39 *** 21,241.981988.47 *** Leucine 12200.29 ± 450.32 7870.011361.15 *** 8377.381398.58 *** Methionine 11615.87 ± 354.68 9813.681474.35 ** 11307.551407.14 Valine 5224.57 ± 138.37 3419.431136.78 *** 3582,461103,76 *** Proline 6177.73 ± 53.23 4711.791245.90 *** 4943.271202.47 *** Threonine 28369.53 ± 529.26 13593.471589.83 *** 15254.361559.75 *** Tryptophan 1063.28 ± 31.73 860,68110,27 *** 868.97135.08 *** Series 4742.13 ± 122.50 3086.771107.02 *** 3503.14189.99 *** α-alanine 21878.12 ± 1052.04 14518.181619.35 *** 16059.961703.78 *** Glycine 8636.35 ± 451.99 5819,701404.80 *** 5606.081416.01 *** ** P> 0.01; *** P> 0.001

Among all the associated amino acids in the herring muscle extract, the maximum percentage concentration was accounted for by histidine (21-23%), threonine (14-19%), α-alanine (14-16%) and, conversely, the minimum by lysine (0.30 -0.43%), tryptophan (0.69-0.92%) and tyrosine (0.97-1.07%).

The total concentration of bound amino acids in the anchovy muscle extract that was not affected by Anisakis larvae was 214630.99 mg / kg of minced fish, with an invasion of 1 to 4 Anisakis larvae - 193158.58 mg / kg of minced fish, with an invasion of 15 to 20 - 173418 , 87 mg / kg of minced fish, with an invasion of 21 to 27 - 151973.78 mg / kg of minced fish (Table 5).

Table 5 Concentration of Bound Amino Acids in Hamsa Muscle Extract (M ± m; n = 10) Associated amino acids, mg / kg minced fish The number of larvae of Anisakis a viscero hamsa 0 15-20 21-27 Arginine 10456.73 ± 137.59 8993.56 ± 86.01 *** 7457.80 ± 128.67 *** 6174.55 ± 83.84 Lysine 306.38 ± 2.81 287.52 ± 3.12 268.35 ± 8.39 *** 243.87 ± 7.61 *** Tyrosine 641.28 ± 6.05 604.65 ± 5.18 *** 624.36 ± 3.53 * 511.62 ± 8.70 *** β-phenylalanine 758.23 ± 11.91 610.49 ± 10.10 682.85 ± 7.48 *** 481.89 ± 9.76 *** Histidine 50,302.33 ± 380.35 46303.65 ± 632.82 *** 44805.24 ± 1167.30 *** 34289.18 ± 1085.20 *** Leucine 16349.40 ± 491.70 15159.42 + 114.75 * 13194.15 ± 307.56 *** 10853.28 ± 158.50 *** Methionine 22205.60 ± 399.37 20011.14 ± 197.66 *** 18151.36 ± 261.61 *** 14858.64 ± 205.31 *** Valia 8889.71 ± 116.9 8 8156.38 ± 112.72 *** 6334.96 ± 95.07 *** 6199.31 ± 193.33 *** Proline 11965.10 ± 181.61 10915.16 + 118.22 *** 9926.86 ± 141.20 *** 8471.13 ± 120.44 Threonine 28622.76 ± 388.58 26779.71 ± 335.43 *** 22066.42 ± 531.49 *** 20180.24 ± 19.14 *** Tryptophan 799.60 ± 26.82 611.23 ± 9.02 *** 570.94 ± 15.30 *** 374.49 ± 8.57 *** Series 6481.59 ± 122.76 6146.50 ± 40.34 * 5500.81 ± 109.41 *** 3631.23 ± 113.64 α-alanine 39 140.30 ± 573.68 33484.70 ± 500.40 *** 30740.11 ± 650.65 *** 25174.18 ± 927.81 *** Glycine 17711.98 ± 555.87 15094.47 ± 109.44 *** 13094.66 ± 240.39 *** 10947.58 ± 151.24 *** * P <0.05 *** P> 0.001

Of all the associated amino acids in the anchovy muscle extract, the maximum percentage concentration was accounted for by histidine (22.5-23.4), α-alanine (16.56-18.24%), threonine (13.2-13.3%), methionine (9.8-10.4%), glycine (7.2-8.25%), leucine (7.1-7.61%), proline (5.57%), arginine (4.06- 4.9%), and, on the contrary, the minimum was in lysine (0.14-0.16%), tyrosine (0.30-0.33%), tryptophan (0.25-0.37%).

The total concentration of bound amino acids in the extract of capelin muscle not affected by Anisakis larvae was 137828.58 mg / kg of minced fish, while invasion of capelin from 4 to 8 Anisakis larvae was 102580.22 mg / kg of minced fish (Table 6).

The maximum percentage of all bound amino acids in the capelin muscle extract was accounted for by tyrosine (34-36%), a-alanine (12-13%), threonine (11-12%), methionine (6-8%), leucine (7 -8%), histidine (3-4%), and, conversely, the minimum - for tryptophan (0.17-0.18%), lysine (0.14-0.20%).

Table 6 Concentration of bound amino acids in capelin muscle extract (M ± m; n = 10) Associated amino acids, mg / kg minced fish Number of Anisakis larvae on viscero capelin 0 4-8 Arginine 6660.09 ± 146.83 4714.88 ± 96.87 *** Lysine 275.07 ± 1.67 149.18 ± 1.14 *** Tyrosine 47,697.34 ± 716.51 37158.45 ± 959.38 *** β-phenylalanine 1453.13 ± 30.32 1124.90 ± 39.52 *** Histidine 5262.00 ± 71.35 3799.70 ± 125.54 *** Leucine 10173.79 ± 159.68 8004.11 ± 173.42 *** Methionine 11203.33 ± 218.77 6962.93 ± 156.61 *** Valia 4547.34 ± 94.88 3674.13 ± 107.44 *** Proline 5661.71 ± 118.98 4304.36 ± 84.73 *** Threonine 16100,05 ± 221,43 11941.06 ± 225.03 *** Tryptophan 241.37 ± 1.32 181.18 ± 1.85 *** Series 3598.49 ± 145.34 1986.97 ± 38.90 *** α-alanine 17,612.16 ± 362.75 12986.79 ± 216.30 *** Glycine 7342.71 ± 204.70 5591.58 ± 117.06 *** *** P> 0.001

During invasion of fish by Anisakis larvae, a decrease in bound amino acids occurred depending on both the degree of invasion and the species of fish. During herring infestation by Anisakis larvae, a decrease in the concentration of bound amino acids was observed, which is most likely due to the products of helminth activity. However, during invasion from 15 to 20 Anisakis larvae, there was a decrease in tyrosine, β-phenylalanine, leucine, methionine, threonine, serine, α-alanine and, conversely, an increase in lysine relative to the affected herring from 31 to 42 larvae. As a result of the studies, we found that in the herring muscle extract invaded from 10 to 14 Anisakis larvae, the total concentration of bound amino acids was 1.6 times and 1.5 times higher than with invasions from 15 to 20 and from 31 to 42 larvae respectively.

In the blue whiting muscle extract infested with one to two Anisakis larvae, the total concentration of bound amino acids was 1.4 times, 1.7 times and 2 times higher than with invasions from 9 to 13, from 20 to 36 and from 44 up to 63 larvae, respectively. In the pollock muscle extract invaded from one to two Anisakis larvae, the total concentration of bound amino acids was 1.4 times higher than with 7 to 8 larvae invasion. In an extract of capelin muscle that was not invaded by Anisakis larvae, the total concentration of bound amino acids was 1.3 times higher than with an invasion of 4 to 8 larvae.

As a result of the studies, the authors found that in fish affected by Anisakis larvae, the concentration of bound amino acids in the hamsa muscle extract was higher than that of herring and capelin by 1.5 times, blue whiting - 1.8 times, pollock - 1.2 times. A decrease in the concentration of bound amino acids occurred due to the breakdown of polypeptide bonds between amino acids and the formation of free amino acids, which indicated the process of protein breakdown. This process is due to the influence of waste products of larvae of nematodes of the genus Anisakis. The low concentration of bound amino acids in muscle pollock muscle is caused not only by the presence of Anisakis larvae, but also by the structure of the tissue, in contrast to other studied fish species. Based on this, we found that fish with intensive invasion (blue whiting, herring and hamsa - more than 20; pollock and capelin - more than 7) cannot be used for food purposes.

Claims (1)

A method for determining the nutritional value of fish infected with helminths, including derivatization of a biological sample, exposure to it using capillary zone electrophoresis using a working buffer, the detection of amino acids and their qualitative and quantitative analysis, characterized in that an aqueous extract of muscle tissue of fish infected with anisidosis is used as a biological sample. hydrolysis of the bioassay is preliminarily carried out, for this a sample of 0.1-0.2 g of fish muscle tissue is taken and up to 10 cm ^{3 is} filled with 20% hydrochloric acid into the vessels for hydrolysis, then close with stoppers and place in a thermostat for 14 hours at a temperature of no more than 105 ° C, then cool, take no more than 0.05 cm ^{3 of} hydrolyzate with a pipette into a micro vessel and dry it to dryness, add up to 0.1 cm ^{3 of a} 0.1 M sodium carbonate solution , mix, then add no more than 0.3 cm ^{3 of a} solution of phenylisothiocyanate in isopropyl alcohol and leave to react for 40 minutes, after drying the contents under natural conditions, add no more than 0.5 cm ^{3 of} distilled water, mix, transfer to a test tube Eppendorf and centrifuge d To remove gases and suspensions, then the data obtained as a result of qualitative and quantitative analyzes are compared with the control result of the concentration of bound amino acids of clinically healthy fish and the quality of the fish is determined, if the concentration of bound amino acids in infected fish decreases compared to the control, then the fish is classified as poor quality.

Images