doi: 10.15389/agrobiology.2016.2.223eng

UDC 636.087.7:577.15:57.088



A.A. Komarov, L.Ya. Telishevskaya, A.A. Sarkhanova, L.V. Vasil’eva,
E.G. Titova, A.N. Panin

All-Russian State Center for Quality and Standartization of Veterinary Drugs and Feed, Federal Agency of Scientific Organizations,
5, Zvenigorodskoe sh., Moscow, 123022 Russia,

Received September 3, 2015


Worldwide a large number of enzymes, including those with endo-β-glucanase activity are produced by different companies as fodder additives. In Russia, there is no uniform method for endo-β-glucanase activity analysis that causes difficulties in assessing quality of the enzyme preparations. The aim of the study was to develop such uniform method. In this, we optimized a viscometric determination based on change of the relative viscosity of a substrate mixed with enzyme preparation using Ostwald capillary viscometer. The following regime has been accepted: the diameter of the capillary is 0.73 mm; the temperature of hydrolysis is 30 °C; the solvent is the acetate buffer (pH 4.7); time of incubation is 5, 10, 15 min in accordance with the kinetics of the enzymatic reaction; and β-glucane (0.4 %) was used as substrate. The carboxymethyl cellulose is shown not to be relevant so far as it gives results incomparable with those obtained with β-glucane due to estimation of the cellulase but not the β-glucanase activity, that does not correspond to the objectives of forage production. The unit used in the developed method is the amount of enzyme which increases the relative substrate flow rate in 1 min-1 under standard conditions of hydrolysis. The measurement of accuracy of the method was carried out according to GOST R ISO 5725-2002 (the state standards). In this, 5 certified samples with different activity were prepared from a commercial sample of β-glucanase (Sigma-Aldrich, USA) with known activity (GLA/g). The activity of the standard was expressed in GLA/g as glucose equivalents corresponding to the amount of enzyme which acts on b-glucane, releasing 1 µmol of sugars in terms of glucose. Certified samples were analyzed under intermediate precision conditions with three factors changed (i.e., operator, matrix, reagents). The matrices were mineral fillers, the calcium carbonate and zeolite. To calculate the accuracy of the method the viscosity of certified samples was expressed in GLA units using the calibration curves. Expanded uncertainty of measurements ranged within 10-22 % for the coverage factor k = 2.

Keywords: endoglucanase activity, non-starch polysaccharides, hemicellulose, viscosity, viscosimetry, β-glucane, carboxymethyl cellulose, matrix, intermediate precision, expanded uncertainty.


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  1. Grishutin S.G., Dzedzyulya E.I., Kondrat'eva E.G., Zorov I.N., Sinitsyna O.A., Sinitsyn A.P. Sravnitel'noe testirovanie kommercheskikh fermentnykh preparatov dlyakormoproizvodstva [Comparative estimation of commercial enzymes for feed production (in Russ.). Available http://biovet-ferment. ru/index.php?id=50. Accessed 25 March 2016].
  2. Komarov A.A., Telishevskaya L.Ya., Shevchenko A.A., Vasil'eva L.V., Panin A.N. Vetkorm, 2013, 3: 8-10 (in Russ.).
  3. Choct M. Enzymes for the feed industry: past, present and future. Worlds Poult. Sci. J., 2006, 62: 5-15 CrossRef
  4. Zhang Z., Marquardt R.R., Guenter W. Evaluating the efficacy of enzyme preparations and predicting the performance of Leghorn shicks fed rye-based diets with a dietary viscosite assay. Poultry Sci., 2000, 79: 1158-1167 CrossRef
  5. Geraert P.-A., Palais F., Grenier S., Jakob S. Dietary carbohydrates: a review of their physicochemical properties and digestibility in poultry and swine. Proc. Eastern Nutrition Conference (May 10-11, 2005). Canada, Ontario, 2005: 1-18.
  6. Olukosi O.A., Cowieson A.J., Adeola O. Energy utilization and growth of broilers receiving diets supplemented with enzymes containing carbohydrase or phytase activity individually or in combination. Br. J. Nutr., 2008, 99(3): 682-690.
  7. Carvalho J.C.C., Rios R.L., Lima E.M.C., Bertechini A.G., Piracés F., Sorbara J.O.B. Differences in corn energy with the use of α-amylase and β-glucanase for broilers in various production stages. Proc. Poultry Science Association Annual Meeting. Canada, Ontario, 2008: 67.   
  8. RovabioTMEksel' AP (
  9. Natugrein TS. Natugrein TS.mht [Natugrain TS. Available No date (in Russ.)].
  10. [Hostasim (Biovet AD, Huvefarma AD, Bulgaria. Available No date (in Russ.)]
  11. Kemzyme. Kemin products. Available Accessed March 25, 2016.
  12. Ziebal R. Pitfalls and challenges for the official control of enzymes in feed. In: Antimicrobial growthpromoters. Wageningen Academic Publishers, the Netherlands, 2006: 249-254.
  13. Telishevskaya L., Sarkhanova A., Vasil'eva L., Khvatova D., Titova E. Kombikorma, 2015, 9: 103-104 (in Russ.).
  14. Engelen A.J., van der Heeft F.C., Randsdorp P.H.G. Viscometric determination of beta-glucanase and endoxylanase activity in feed. Journal of AOAC International, 1996, 79: 5.
  15. McCleary B.V. Analysis of feed enzymes. In: Enzymes in farm nutrition. M. Bedford, G. Partrige (eds.). CAB International, 2001: 85-107.
  16. Polygalina G.V., Cherednichenko V.S., Rimareva L.V. V sbornike: Opredelenie aktivnosti fermentov [In: Enzyme assay (in Russ.)]. Moscow, 2003: 180-181.
  17. Vlasenko E.Yu., Ryan A.I., Shoemaker Ch.F., Shoemaker Sh.P. The use of capillary viscometry, reducing end-group analysis, and size exclusion chromatography combined with multi-angle laser light scattering to characterize endo-1,4-β-D-glucanases on carboxymethylcellulose: a comparative evaluation of the three methods. Enzyme and Microbial Technology, 1998, 23: 350-359.
  18. Ankudimova N.V. Biokhimicheskie i fiziko-khimicheskie svoistva klyuchevoi topoliticheskoi endoglyukanazy tsellyulaznogo kompleksa Chaetomium cellulolyticum. Kand. dis. [Biochemical, physical and chemical characterization of the key topolytic endoglucanase of the Chaetomium cellulolyticum cellulase complex. PhD Thesis (in Russ.)]. Moscow, 2000.
  19. GOST R ISO 5725-2002 [Accuracy (correctness and precision) of mesuarements and obtained data. State Standard GOST R ISO 5725-2002 (in Russ.)]. Moscow, 2002.
  20. RMG 61-2010 [RMG 61-2010. Recommendations on Interstate Standardization. State system for ensuring the uniformity of measurements. Indicators of correctness, accuracy, precision of the quantitative chemical analysis techniques. Assessment methods (enacted by the Order of the Rosstandart of December 13, 2011, No 1064st (in Russ.)]. Moscow, 2013.