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SOME PROBLEMS IN THE DETERMINATION OF TANNINS AND POSSIBLE SOLUTIONS
TA from five commercial sources (Merck, Sigma, Aldrich, Fluka and Apin) were compared using various tannin assays.
The regression coefficients of absorbance vs TA concentration were different for different TA using the Folin-Ciocalteu and the ferric chloride assays, and the variation in the values obtained for one feed could be up to 20% using different TA as standards.
Similar results were obtained using the protein precipitation assays (PPAs). The slopes of ‘BSA precipitated per unit TA’ linear regression were different by up to 30% for the TA studied.
In general, the slopes were higher for Merck, followed by Sigma, Aldrich, Fluka and Apin.
These results show that the use of TA from different commercial sources as a standard influence the tannin levels to a large extent.
It is suggested that the commercial source of TA used and the relationship for convertion of absorbance to TA level should be mentioned, and the values obtained by different laboratories should be compared with caution.
Vanillin-HCl method is widely used for the determination of condensed tannins (CT). Tannins are generally extracted using aqueous acetone.
However, acetone reacted with acidified vanillin to produce chromogen with max at 548nm which produced a substantial error in the determination of CT. In presence of acetone, the time course of the reaction for catechin and tannins was different depending on the acetone concentration and the reaction temperature.
Reaction conditions for catechin and tannins which enable their determination in the presence of acetone are presented.
The PPAs take into account only the slope of the ‘BSA precipitated per unit TA or per unit feed sample’ linear regression and not the y-intercept.
We have observed that the y-intercepts of the linear regression for TA and for several feeds especially leaves of trees and shrubs are significantly different from zero.
This calls for a change in the present approach (addition of different quantities of tannins to a fixed amount of the protein). The addition of a different quantity of protein to a fixed amount of tannin/tannin-containing extract and determination of the maximal protein bound by the tannin could be a solution to this problem.
However, more research is required on the stoichiometry of protein bound to tannins under these conditions.