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***** ИЗВЕСТИЯ *****
НИЖНЕВОЛЖСКОГО АГРОУНИВЕРСИТЕТСКОГО КОМПЛЕКСА
ЗООТЕХНИЯ И ВЕТЕРИНАРИЯ
УДК 636:553.611
EKOBENTOKORM SORPTION ABILITY I.F. Gorlov1'3, G.A. Zelenkova2, A.A. Verovsky2, A.P. Pakhomov2
1Povolzhskiy scientific-research institute of meat and milk production and processing of Russian agricultural academy 2Don State Agrarian University 3 Volgograd state agrarian university
Ekobentokorm refers to montmorinite compounds having different adsorption properties. To establish and determine the parameters of ekobentokorm adsorption activity series of experiments on inorganic (mitelen blue), organic (mycotoxins) chemical and biological objects (conditionally pathogenic) were carried out. The data obtained characterized ekobentokorm as natural enterosorbent.
Key words: ekobentokorm, sorbent, sorption capacity, mycotoxins.
The work is performed on the President's grant of the Russian Federation for State Support of Leading Scientific Schools of the Russian Federation (project NSH-2602.2014.4 "New approaches to quality assurance and of environmental food safety on the basis of the living systems across the
biotech chain management ")
In many developed world's countries, a great attention pays to the environmental protection from harmful factors which include heavy metals, a variety of pathogenic microorganisms, toxins and others.
In this regard, in the last few years, the Russian scientists began to use various kinds of sorbents based on vegetable raw materials and mineral-based, promoting water purification from heavy metals to purify water and aqueous solutions [1, p. 2, 2, p. 2, 4, p. 2, 5, p. 2, 6, p. 2, 7, p. 2, 10, p. 2].
Ekobentokorm is a natural sorbent got from bentonite clay, which is not only a source of macro-and micronutrients, but also has a high adsorption activity against mycotoxins, endotoxins, heavy metals and pathogenic organisms, improves digestion and metabolism.
Having a large specific surface area, high absorption capacity and the availability of complicated composition absorbing complex, ekobentokorm has the property of adsorbing the environmental significant amount of gases, water, inorganic and organic compounds [3, p. 2-3].
The study of ekobentokorm's sorption characteristics isperformed in several series of experiments on various compounds.
In the first series of ekobentokorm's sorption activity was determined in relation to pho-tocolorimetric methylene blue method (table 1).
Table 1 - Definition o ? ekobentokorm's sorption activity in relation yo methylene blue
Feed additive Ekobentokorm, mg The quantity of 1% solution of methylene blue, ml
Ekobentokorm 10,0 10,0
15,0
30,0
б0,0
75,0
90,0
110,0
120,0
250,0
500,0
The method is based on the ekobentokorm ability to adsorb different substances in aqueous solutions. As a test indicator from the colorants 1% solution of methylene blue was selected and used in purified running water. A sorption capacity was judged by a change in the optical density of the solution after contact with ekobentokorm. For this purpose, we used different ekobentokorm samples in doses ranging from 10 to 500 mg, which were added to the test tubes with methylene blue.
Added to the test tubes with the solution of methylene blue ekobentokorm in different concentrations was thoroughly shaken for 30 minutes. Then, the solutions were filtered and the supernatant liquor optical density was measured in the photoelectrocolorimeter with a wavelength of 370 nm.
Indicators: 500 mg 0.045 - Transparent
250 mg 0.050 - Transparent 120 mg 0.085 - slightly opalescent 110 mg of 0,09 - opalescent 90 mg 0.75 - slightly transparent 75 mg 1.45 - slightly transparent 60 mg 1,6 - nontransparent
Ekobentokorm's adsorption properties weren't practically appeared and indicators were not determined below 60 mg.
The next step was to determine the ekobentokorm's absorbency concerning with methylene blue. For this purpose, a 0.5% solution of the colorant and 200 mg of ekobentokorm (average value at which the supernatant liquor remains clear after adsorption) were used. Placed in a solution of methylene blue the ekobentokorm sample was mixed, stirred on a shaker IFKO -1 (intensity 3) for 3 minutes, centrifuged at about 2000-1 rev./ min for 5 minutes. The sample was filtered, and the optical density was measured against distilled water at a wavelength of 605 nm, comparing the results with blank sample of methylene blue .
200 mg ekobentokorma absorbed:
0,025 - 0,000897 = 0,02419 mg of methylene blue.
Percentage of absorption: 0,025 - 100 %
0,02419 - x, x = 96,77 %.
When reducing the ekobentokorm amount twice (100 mg), the percentage of absorption is:
0,025 - 100 %
0,02324 - x, x = 92,56 %.
Reducing the ekobentokorm concentration four times (50 mg) resulted in the following:
0,025 - 100 % 0,00764 - x, x = 30,56 %.
Thus, the ekobentokorm sorption activity against methylene blue is very high at various concentrations even at its small amount the absorptive capacity is 30.56 %.
Earlier the studies of sorbents based on natural plant materials revealed that the research of the methylene blue sorbents activity to their sorption capacity ranged from 3.8 to 4.0 mg / g, and the degree of sorption was from 84 to 87 % [8, p. 2-3, 9, p. 2-3].
Ekobentokorm sorption activity concerning to opportunistic pathogenic microflora was studied in the second series of experiments on the model of Escherichia coli (E. coli) and Staphylococcus aureus (St. aureus). To do this in advance, we used standard turbidity number 1, prepared one billion suspensions of daily agar cultures. We added 10 ml of standardized bacterial breeding suspension to 1 g of sterile ekobentokorm. The got mixture was shaken for
5 minutes and then the slurry was allowed to stand for 30 minutes.
After clarification (to let ekobentokorm larger particles settle) 0.5 ml of supernatant liquor was picked out and placed in 4.5 ml of sterile physiological solution. The sizing of germ culture till dilution 10-9 (up to ninth test tube) was made likewise from the resulting mixture. Control test tubes contained the original one billion suspension of bacteria, from which serial dilutions were also produced.
From each dilution of control and test samples of 0.1 ml were picked out and plated on agar plates Endo (for E. coli) and MPA (for St. Aureus). In 24 hours the raised colonies were counted. Results are shown in the table 2.
Table 2 - Changes in the number of CFU of bacteria after ekobentokorm
effect on the bacterial mass
Tests Lg of cultivation, CFU
9 8 7 6 5 4 3 2 1
E.coli
Experimental 20 34 58 72 70 134 Confluent growth Confluent growth Confluent growth
Control 332 288 420 522 1148 1280 Confluent growth Confluent growth Confluent growth
St. aureus
Experimental 10 18 21 29 44 63 75 Confluent growth Confluent growth
Control 328 534 440 645 824 1764 Confluent growth Confluent growth Confluent growth
The data presented in the table show that the ekobentokorm sorption capacity in concern with bacteria was enough expressed.
Number of colonies after the bacterial mass contact with the sorbent was 15-30 times less than at inoculation culture without contacting ekobentokorm. At this time the sorption capacity of the preparation ia more expressed regard to staphylococci than to E. coli.
Thus, if the reduction in the colony forming units number in each dilution of E. coli after in interaction with ekobentokorm decreased in 7-15 times, then St. Aureus did in 20-30 times.
Based on the data, one can conclude that ekobentokorm shows high sorption capacity, so it can be used to reduce microbial contamination of various biological objects or environments, such as the digestive tract of farm animals and birds.
In the third series of experiments the researches on determination of ekobentokorm adsorption properties in regard with the most common combinations of mycotoxins occurring in farms of Rostov region were carried out.
The mycotoxin concentration was determined by enzyme-linked immunosorbent ana-lisys (ELISA), according to the instruction on the use of test systems (T-2 toxine - № 13-502/0514; ochratoxin - № 13-5-02/0516; zearalenone - № 13 - 5-02/0515; fumonisin B1 - № 13-5-02/0519) (table 3).
At this time to the test tubes with mixed feed samples (with clearly known mycotoxins combination) a certain amount of ekobentokorm was added (0.5 to 3%) and adjusted pH to 2 (acidic) and 8 (alkaline) in order to determine the ekobentokorm sorption properties at significant fluctuations of pH.
Table 3 - Ekobentokorm adsorption activity
Toxine type Base material 0,5 % (pH 8) 1,0 % (pH 8) 2,0 % (PH 8) 3,0 % (pH 8) 1,0 % (pH 2) 2,0 % (pH 2)
T-2 62,9 mkg/kg 58,8 6,5 % 52,8 16 % 51,5 18 % 28,9 54 % 61,0 3 % 57,8 8 %
OA 36,2 mkg/kg 32,2 11 % 31,1 14 % 29,3 19 % 26,0 28 % 33,6 7 % 33,4 8 %
ZEN 69,0 mkg/kg 62,1 10 % 52,4 24 % 47,6 31 % 26,9 61 % 64,17 7 % 64,17 7 %
FUM 1,199 mkg/kg 1,043 13 % 0,935 24 % 0,839 31 % 0,407 66 % 1,031 14 % 1,0 16 %
Appendix: The top figure in the column is the mycotoxin concentration after adsorption, the lower figure is the percentage of mycotoxin absorption of.
The researches results show that in a very acidic environment (pH = 2) the prepartions sorption activity is quite low, ranging from 3 to 16% by ekobentokorm. However, with increasing of pH till the medium-alkaline, these indicators rise sharply. Moreover, adsorbent concentration plays an important role in the increase of sorption activity. Thus, the most pronounced jumps are observed with increasing of ekobentokorm amounts from 0.5 to 1.0% in 1.2-2.4 times and from 2.0 to 3.0% in 1.5-3.0 times.
Thus we note that ekobentokorm has adsorption properties with respect to mycotoxins (T-2, ochratoxin, zearalenone and fumonisin B1) in the following percentages: ekobentokorm from 4.0% to 64.0% depending on the toxin in a greater degree adsorbed in a descending order FUM> ZEN> T-2. Adsorbent works better with pH increasing from acidic to alkaline, and the highest concentration input of 3.0%.
Reference list
1. Gorlov, I.F. A method for producing a sorbent / I.F. Gorlov, I.M. Osadchenko, M.M. Ko-valev / / Patent of RF № 2221585, 2004. Recorded: 24.09.2002. Application for a patient № 2002125549/15. Published: Bull. № 2, 20.01.2004. - 2 p.
2. Gorlov, I.F. Patent RF № 2221640 A method for producing a plant-based sorbents / I.F. Gorlov, I.M. Osadchenko, L.M. Myakotnykh / / Application for a patient № 2003103824/15. Published: Bull. № 2, 20.01.2004. - 2 p.
3. Zelenkova G.A., Pakhomov A.P. Bentonite clay is a natural enterosorbent / / Perspective Poultry: Theory and Practice. - 2012. - № 2. - P. 1-5.
4. Osadchenko I.M. Patent RF № 2210431 A method for producing a plant-based sorbent to clean the water solutions from heavy metals / I.M. Osadchenko, I.F. Gorlov, N.I. Mosolova, I.A. Semenova / / Application for a patient № 2002128592/12. Published: Bull. № 23, 20.08.2003. - 2 p.
5. Osadchenko I.M. Patent RF № 2221638 A method for producing a plant-based sorbent / I.M. Osadchenko, I.F. Gorlov, O.N. Kuznetsova / / Application for a patient № 2002134929/15. Published: Bull. № 2, 20.01.2004. - 4 p.
6. Osadchenko I.M. Patent RF № 2251450 A method for producing a plant-based sorbent / I.M. Osadchenko, I..F Gorlov, I.N. Penkova, T.T. Rivnyak / / Application for a patient № 200409500/15. Published: Bull. № 13, 10.05.2005. - 3p.
7. Osadchenko I.M. Patent RF № 2252818 A method for producing a plant-based sorbents / I.M. Osadchenko, I.F. Gorlov, N.I. Shigaeva / / Application for a patient № 2004112716/15. Published: Bull. № 15, 27.05.2005. - 3 p.
8. Osadchenko I.M. Patent RF № 2276620 A method for producing phytosorbents / I.M. Osadchenko, I.F. Gorlov, T.G. Serebriakova, N.I. Mosolova, A.S. Konovalov / / Application for a patient № 2005101957. Published: Bull. № 14, 20.05.2006. - 4 p.
***** ИЗВЕСТИЯ ***** № 1 (33), 2014
НИЖНЕВОЛЖСКОГО АГРОУНИВЕРСИТЕТСКОГО КОМПЛЕКСА
9. Osadchenko I.M. Patent RF № 2307706 Method of sorbent-based natural polymer / I.M. Osadchenko, I.F. Gorlov, N.I. Shigaeva, D.A. Skachkov // Application for a patient № 2006105168/15. Published: Bull. № 28, 10/10/2007. - 3 p.
10. Osadchenko I.M. Patent RF № 2311955 Method of sorbent based on minerals / I.M. Osadchenko, I.F. Gorlov, M.E. Spivak, V.G. Dikusarov // Application for a patient № 2006115100/15. Published: Bull. № 34, 10.12.2007. - 3 p.
E-mail: niimmp@maul.ru
