CC BY
216
VETERINARY SCIENCES / «CQLLOMUM-JlSUrMaL» #41127)), 2022
Kushnir V.I., Kushnir I.M.,
State Scientific-Research Control Institute of Veterinary Medicinal Products and Feed Additives,
Lviv, Ukraine Kutsan O. T.,
Institute of Veterinary Medicine of NAAS, Kyiv, Ukraine
Gutyj B.V., Leskiv Kh. Ya.
Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies,
Lviv, Ukraine
DOI: 10.24412/2520-6990-2022-4127-6-9
DETERMINATION OF BIOLOGICALLY ACTIVE SUBSTANCES OF THE PREPARATION PREPARED BASED ON PEPTIDOGLYCANS
Abstract.
The article presents the research results of determining the amino acid composition, water-soluble B vitamins, and qualitative and quantitative content of organic acids in biologically active agents based on peptidogly-can of lactic acid bacteria by capillary electrophoresis using the peptidoglycan the device Kapel-105/105M. When determining the amino acid composition of the research found that they include a complex of essential and substitute amino acids, which is extremely important in ensuring the biological activity of the veterinary medicinal product. Among the essential amino acids, the highest percentage of leucine + isoleucine, lysine, was found in the experimental samples and conditionally essential - arginine. Substitute amino acids are mainly alanine, proline, and serine. Substitute amino acids are mainly alanine, proline, and serine. When determining the content of water-soluble B vitamins in a biologically active agent based on peptidoglycan of lactic acid bacteria, it was found that it contains many water-soluble B vitamins, including Bj, B2, B6, B5, B3, and Bc. Organic acids in the studied biologically active agent based on peptide glycans are represented by oxalic, citric, acetic, lactic, benzoic, and sorbic acids. When determining the quantitative content of these acids, the highest concentration of acetic and lactic acids was established, which in percentage terms were 6.0 and 2.5%, respectively. Therefore, the drug's biological activity based on peptidoglycan of lactic acid bacteria is due to the uniqueness of amino acid composition and the ratio of individual amino acids, B vitamins, and organic acids.
Keywords: amino acids, B vitamins, organic acids, peptidoglycans, lactic acid bacteria, capillary electro-phoresis method.
Introduction
In the body of farm animals and birds, the microflora of the digestive tract plays a significant role. Its most important functions are counteracting pathogenic and opportunistic bacteria participation in the body's metabolic and food digestion processes. Many microorganisms represent the microflora of the digestive tract, but Lacto- and bifidobacteria are worth special attention [1, 2, 20, 21].
A characteristic feature of lactic acid bacteria is their participation in the formation of immune status and the functioning of the immune system [3-4]. The use of lactobacilli has a positive effect on the microorganism, causing the strengthening of natural immunity. In addition, they are involved in activating macrophages and increasing the level of cytokines and immunoglobulins. Therefore, lactic acid bacteria do not cause allergic reactions and inflammatory processes. In addition, they can counteract cancer [5-12].
That is why many biologically active drugs have been developed based on Lacto- and bifidobacteria, but preparations made from peptidoglycans of lactic acid microorganisms are worth special attention. [13] The use of such drugs activates the body's functional activity, helps restore impaired metabolism, and increases the body's resistance to adverse factors [1415 16]. The study aimed to determine the qualitative and quantitative composition of amino acids, B vitamins, and organic acids of Biovir-P.
Material and methods of research
Determination of the qualitative and quantitative composition of amino acids. To 0.1 g of the drug was added 10 cm3 of hydrochloric acid, sealed, and mixed. The hydrolysis was performed at a temperature of 110 °C for 14-16 hours. After hydrolysis, the mixture was cooled to room temperature and filtered. Next, 0.05 cm3 of hydrolyzate was taken from the glass flasks and evaporated, after which 0.15 cm3 of sodium carbonate solution and 0.3 cm3 of FITC solution were added, mixed thoroughly, left for 35 minutes at room temperature, and dried. Finally, the dry residue was dissolved in 0.5 cm3 of double-distilled water and used for research. Detection of amino acids was performed at a wavelength of 254 nm [17].
Determination of the qualitative and quantitative composition of B vitamins. To 0.2 g of the drug was added 5 cm3 of the working solution prepared according to the guidelines, placed in a boiling water bath for 5 minutes, cooled, filtered, and detected vitamins at wavelengths of 200 and 267 nm [18].
Determination of the qualitative and quantitative composition of organic acids. To 0.5 g of the drug was added 50 cm3 of double-distilled water heated to 70 °C, stirred on a laboratory shaker for 10 minutes. Then 1 cm3 of the filtrate was centrifuged, and the quantitative and qualitative content of organic acids was indicated. Detection was performed at a wavelength of 190 nm [19].
«CQyyOMUM-JMTMaiL» #41127)), 2022 / VETERINARY SCIENCES
7
Results and discussion
The qualitative and quantitative composition of amino acids, B vitamins, and organic acids in Biovir-P was performed by capillary electrophoresis using the device "Kapel-105/105M". In indicating the amino acid composition, it was found that among the essential amino acids, leucine + isoleucine (0.75%), lysine (0.46%), valine (0.39%), and the least - histidine (0.05%) and methion (0.03%). Of the conditionally irreplaceable, arginine was found the most (0.36%). Substitute amino acids are represented mainly through alanine (0.53%), to a lesser extent by proline, serine, and glycine. The importance of amino acids for a living organism is determined by their unique role in the construction and intermediate synthesis of the main structural components of cells. In particular, leucine is necessary not only for protein synthesis but also to strengthen the body's immune system. Furthermore, an essential amino acid, lysine, is one of the essential components of carnitine formation.
In addition, lysine ensures proper absorption of calcium participates in the formation of collagen and the production of antibodies, hormones, and enzymes. Methionine plays a vital role in the metabolism of fats and proteins; the body also uses it to synthesize cysteine. It is involved in the biosynthesis of choline, adrenaline, cysteine, helps lower cholesterol, and actively removes heavy metals from the body. Histidine is part of
the active centers of RNase, chymotrypsin, transketo-lase and promotes tissue growth and repair. Serine is closely associated with the formation of glycine. Glycine is actively involved in providing oxygen to newly formed cells of the body is an essential participant in the synthesis of hormones responsible for strengthening the immune system. Alanine is involved in the biosynthesis of proteins, peptides, hormones, strengthens the immune system by producing antibodies; actively involved in the metabolism of sugars and organic acids. L-Arginine strengthens the immune system; it is necessary for protein synthesis and optimal growth. Proline ensures the proper functioning of ligaments and joints, which is also involved in maintaining performance and strengthening the heart muscle. Serine is concerned with the biosynthesis of glycine, sulfur-containing amino acids (methionine, cystine), tryptophan side chain, sphingolipids, and ethanolamine.
Nicotinic acid (0.674 g/kg), calcium pantothenate (0.636 g/kg), and riboflavin were the most abundant in the determination of water-soluble B vitamins (Bi, B2, B6, B3, B5, Bc) in the biologically active agent based on lactic acid peptidoglycan. (0.407 g/kg), slightly less -pyridoxine hydrochloride (0.138 g/kg) and folic acid (0.106 g/kg) and the least thiamine chloride hydrochloride (0.087 g/kg) (Table 1).
Vitamins Biologically active agent
Vitamin Bi, (thiamine chloride hydrochloride), g/kg 0.087±0.020
Vitamin B2, (riboflavin), g/kg 0.407±0.055
Vitamin B6 , ((pyridoxine hydrochloride), g/kg 0.138±0.024
Vitamin B3, (calcium pantothenate), g/kg 0.636±0.064
Vitamin B5, (nicotinic acid), g/kg 0.674±0.438
Vitamin Bc, (folic acid), g/kg 0.106±0.038
The role of vitamins, which are part of biologically active substances, is multifactorial. Vitamin Bi promotes the biosynthesis of nucleic acids, proteins, glucose, glycogen, and fats in animal tissues. Vitamin B2 is part of more than 60 flavin enzymes involved in cellular respiration and metabolism of proteins, nucleic acids, carbohydrates, and lipids. Vitamin B3 has a significant role in protein, carbohydrate, and lipid (fat) metabolism and participates in synthesizing acetylcholine and steroid hormones necessary for normal tissue function, growth, and hair pigmentation. Vitamin B5 regulates carbohydrate and protein metabolism in the body and the pancreas' function, stimulating digestion. The
The content of organic acids
biological role of vitamin B6 is determined by its participation in amino acid metabolism and the construction of the enzyme phosphorylase, which breaks down glycogen. It is necessary to convert linoleic acid into arachidonic acid in animals. Vitamin B6 forms arachi-donic acid from linoleic acid, niacin from tryptophan, cholesterol metabolism, and hemoglobin formation regulates fat metabolism in the liver. Vitamin Be affects hematopoiesis, stimulates the formation of erythrocytes and leukocytes, lowers blood cholesterol.
Organic acids in the study agent are represented by oxalic, citric, acetic, lactic, benzoic, and sorbic acids (Table 2).
Table 2
№ n/n The organic acid The organic acids content
Concentration, mg/l %
1 oxalic acid 1.59±0.87 0.016±0.009
2 citric acid 22.1±3.21 0.23±0.041
3 acetic acid 575.8±39.5 6.05±0.327
4 lactic acid 236.3±33.4 2.47±0.315
5 benzoic acid 1.47±0.21 0.015±0.001
6 sorbic acid 5.03±0.73 0.052±0.005
Table 1
The content of B vitamins in the drug (n±6, M±m)
8
VETERINARY SCIENCES / «ШУУШУПУМ-ШУШаУ» #4(И27)), 2022
The data in Table 2 shows that the concentration of acetic and lactic acids was the highest and was, respectively - 575.8 and 236.3 mg/l, which were - 6.05 and 2.47% in percentage terms. The value of acetic acid in metabolism is very high because it is a source of ac-etyl-CoA used in the Krebs cycle. Lactic acid, which is used as an energy source and is involved in lipogenesis, is also essential. Its high antibacterial action, active stimulation of pancreatic enzyme secretion, and intestinal villi regeneration processes have also been established. At the same time, its presence creates favorable conditions for developing lactobacilli. The latter actively populate the small intestine's mucous membrane, preventing its colonization by pathogenic bacteria.
In calculating citric and sorbic acids, their content was - 0.23 and 0.05%. Citric acid is involved in the tricarboxylic acid cycle and promotes calcium absorption in the body, and improves its use. In addition, it is involved in hematopoiesis and promotes better absorption of iron. In combination with formic and acetic acid, sorbic acid has a bactericidal effect on E. coli, Salmonella spp., Campylobacter spp.
The oxalic and benzoic acid concentration was almost equal and was 0.016 and 0.015%, respectively. Although oxalic and benzoic acids have antimicrobial action, oxalic acid also has a pronounced immunostim-ulatory effect.
Determination of the qualitative and quantitative composition of organic acids in the drug Biovir reveals the mechanism of its immunostimulatory action.
Conclusion
The drug's biological activity based on peptidogly-can of lactic acid bacteria is due to the uniqueness of the amino acid composition and the ratio of individual amino acids, B vitamins, and organic acids.
References
1. Havkin, A.I. (2003). Intestinal microbiocenosis and immunity. Ros. honey. Magazine, 11(3), 33-40.
2. Kushnir, V.I., Kushnir, I.M., & Semen, I.S. (2014). Influence of biologically active agent based on peptidoglycan on the intestinal microflora of laboratory animals. Nauk.-tehn. byul. IBT and DNDKI veterinary drugs and feed additives, 15(2-3), 162-166.
3. Markova, T.P. & Chuvirov, D.G. (1998). Mechanisms of action of bacterial immunomodulators. Ukr. honey. Magazine, 6(8), 27-32.
4. Khaitov, R.M., & Pinegin, B.V. (2000). Modern immunomodulators: basic immunomodulators: basic principles of their application. Immunology, 5, 4-7.
5. Steward-Tule, D.A. (1980). The Immunological activities of bacterial peptidoglicans. Ann. Rev. Micro-biol, 34, 311-340.
6. Shida, K., Kiyoshima-Shibata, J., Nagaoka, M., et al. (2006). Induction of interleukin-12 by Lactobacil-lus strains having a rigid cell wall resistant to intracel-lular digestion. J Dairy Sci, 89, 3306-3317.
7. Mosienko, V.S., Mosienko, M.D., & Savtsova, Z.D. (1999). Blasten new domestic immunomodulator of biological origin. Journal. Academy of Medical Sciences of Ukraine, 5(1), 79-86.
8. Dong, Y., Wang, S., Wang, C., Li, Z., Ma, Y., & Liu, G. (2017). Antagonizing NOD2 signaling with
conjugates of paclitaxel and muramyl dipeptide derivatives sensitizes paclitaxel therapy and significantly prevents tumor metastasis. Journal of Medicinal Chemistry, 60(3), 1219-1224.
9. Tarutinov, V.I., Mosienko, V.S., & Kasya-nenko, I.V. (2001). Survival of patients with breast cancer in complex treatment with a natural immunomodulator from Lactobacillus Delbrueckii. Ukr. chemothera-pist. Magazine, 2(10), 51-56.
10. Wang, S., Han, X., Zhang, Y., et al. (2018). Whole peptidoglycan extracts from the Lactobacillus paracasei subsp. Paracasei M5 strain exert anticancer activity in vitro. Biomed Res Int, 2018, 2871710.
11. Matsuzaki, T. Shimizu, J., & Yokokura, T. (1990). Augmentation of antimetastatic effect on Lewis Lung carcinoma (3LL) in C57Bl/6 mice by priming with Lactobacillus casei. Med. Microbiol. Immunol, 179(3), 161-168.
12. De Pablo, M.A., Gafortio, J.J., & Gallego, A. (1999). Evaluation of immunomodulatory effects of nisin-containing diets on mice. FEMS Immunol. Med Microbiol., 24, 35-42.
13. Kotsyumbas, I.I., Kushnir, V.I., Kushnir, I.M., & Tchaikovsky, O.I. (2014). Pharmacological aspects of peptidoglycan use. Institute of Animal Biology, DNDKI veterinary drugs and feed additives, scientific and technical bulletin, 15(1), 288-294.
14. Bondarenko, V.M., Rubakova, E.I., & Lavrova, V.A. (1998). Immunostimulatory action of lactobacilli used as a basis for probiotics. Journal. mi-crobiol., epidemiol. and immunol., 5, 107-112.
15. Chumakov, H. (1990). Lactic acid bacteria and their effect on the immune system. International. agronom. Journal, 3, 116-119.
16. Ermolenko, E.I. (2014). Immunomodulatory action of probiotic bacteria in diseases of the gastrointestinal tract. Bulletin of St. Petersburg University, Series 11: Medicine, 4, 5-18.
17. Kotsyumbas, I.J., Levitsky, T.R., Ryvak, G.P., etc. (2013). Feed and feed raw materials. Determination of amino acid content by capillary electrophoresis using the system of capillary electrophoresis "Drops-105/105M" (Guidelines). Considered and approved by TC 132 of Derzhspozhyvstandart of Ukraine "Animal protection products, feed and feed additives" (Minutes № 10 of 22.10.2013) and NMR of the State Veterinary and Phytosanitary Service of Ukraine (Minutes № 1 of 19.12.2013). Lviv, 32.
18. Kotsyumbas, I.J., Levitsky, T.R., Ryvak, G.P., etc. (2013). Premixes. Determination of the content of vitamins of group B (B1, B2, B3, B5, B6 and Bc) by the method of capillary electrophoresis using the system of capillary electrophoresis "Krapel-105/105M" (Methodical recommendations). Considered and approved by TC 132 of Derzhspozhyvstandart of Ukraine "Animal protection products, feed, and feed additives" (Minutes № 10 of 22.10.2013) and NMR of the State Veterinary and Phytosanitary Service of Ukraine (Minutes № 1 of 22.12.2013). Lviv, 26.
19. Kotsyumbas, I.J., Levitsky, T.R., Rivak, G.P., etc. (2013). Feeds, compound feeds, feed additives. Determination of the content of organic acids by capillary
«©syyomum-jmtmail» 2022 / veterinary sciences
electrophoresis using the system of capillary electro-phoresis "Krapel--105/105M" (Guidelines). Considered and approved TC 132 of Derzhspozhyvstandart of Ukraine "Animal protection products, feed, and feed additives" (Minutes № 10 of 22.10.2013) and NMR of the State Veterinary and Phytosanitary Service of Ukraine (Minutes № 1 of 22.12.2013). Lviv, 29.
20. Ostapyuk, A.Y., Holubieva, T.A., Gutyj, B.V., Slobodian, S.O. (2021). The effect of sylimevit, metifen, and milk thistle on the intensity of the processes of peroxidation of lipids in the body of laying hens in
9_
experimental chronic cadmium toxicosis. Ukrainian Journal of Ecology, 11 (4), 57-63. doi: 10.15421/2021_199
21. Bashchenko, M.I., Boiko, O.V., Honchar, O.F., Gutyj, B.V., Lesyk, Y.V., Ostapyuk, A.Y., Ko-valchuk, 1.1., Leskiv, Kh.Ya. (2020). The effect of milk thistle, metiphen, and silimevit on the protein-synthesizing function of the liver of laying hens in experimental chronic cadmium toxicosis. Ukrainian Journal of Ecology, 10(6), 164-168. doi: 10.15421/2020_276
