- Publication date
- 18 May 2011 (Last updated on: 14 December 2018)
Colorimetric method based on the quantification of the inorganic phosphate released by the enzyme from the sodium phytate
In the current application authorisation is sought under articles 4(1) for Optiphos under the category "zootechnical additives", functional groups 4(a) "digestibility enhancers" according to Annex I of Regulation (EC) No 1831/2003. Authorisation is sought for chickens and turkeys for fattening, laying hens, chickens reared for laying, turkeys reared for breeding, other birds for fattening and laying, weaned piglets, pigs for fattening and sows. The active agent is 6-phytase (E.C. 22.214.171.124), produced by the strain Pichia pastoris. According to the Applicant, enzymatic activity of the active agents is expressed in "FTU" units, where: one FTU is defined as the amount of enzyme that catalyzes the release of 1.0 micromole of inorganic phosphate per minute from 5.1 mM sodium phytate in pH 5.5 buffer at 37°C. The feed additive is intended to be marketed in two granulate formulations (Optiphos G 4000 and Optiphos CT 4000) and one liquid formulation (Optiphos L 8000). Optiphos G 4000 is composed by pregelatinised starch, wheat meal and 6-phytase, with a minimum activity of 4000 FTU/g. Optiphos CT 4000 is composed by pregelatinised starch, wheat meal, distilled monoglyceride, palm oil, corn grid and 6-phytase, with a minimum activity of 4000 FTU/g. Optiphos L 8000 is composed by sucrose, sodium benzoate, purified water and 6-phytase, with a minimum activity of 8000 FTU/mL. The Applicant proposed two minimum activities for 6-phytase in complete feedingstuffs: - 125 FTU/kg for chickens for fattening, other birds for fattening, chickens reared for laying, laying hens, other laying birds, pigs for fattening and sows; - 250 FTU/kg for turkeys for fattening, turkeys reared for breeding and piglets (weaned).
For the determination of the activity of 6-phytase in the feed additive, premixtures and feedingstuffs, the Applicant proposed a single-laboratory validated and further verified colorimetric method based on the quantification of the inorganic phosphate released by the enzyme from the sodium phytate (phytic acid dodecasodium salt, C6H6O24P6Na12). Released inorganic phosphate is determined measuring the absorbance of the blue phosphomolybdate complex by a spectrophotometry at 820 nm. The released inorganic phosphate is quantified against a phosphate standard. The following method performance characteristics were recalculated by the EURL, based on the experimental data provided by the Applicant:
For the feed additive:
- a relative standard deviation for repeatability (RSDr) ranging from 1.07 to 1.85 %;
- a relative standard deviation for intermediate precision (RSDip) ranging from 2.54 to 4.42 %;
- a recovery rate (RRec) ranging from 97 to 105 %.
For premixtures and feedingstuffs: RSDr ranging from 4.98 to 6.96 %; RSDip ranging from 4.98 to 7.27 %; RRec ranging from 95 to 105 %.
Furthermore, the Applicant calculated a limit of detection (LOD) and quantification (LOQ) for feedingstuffs of 8.9 and 29.8 FTU/kg, respectively; both are well below the minimum activity proposed by the Applicant.
Aware of the existence of the ISO 30024 standard method the EURL requested the Applicant to apply this method to Optiphos. The Applicant reported precisions (RSDr and RSDip) of the order of 6%, and measured activity values about three times higher than those expected. Such a bias could be due to differences in the experimental protocols of the two methods investigated. The EURL emphasizes that due to this bias, the phytase activity measurements obtained with Applicant's method on this specific product cannot be compared with phytase activity measurements of other phytase products, for which the ISO standard is applicable.
Taking into account the international effort of harmonisation of the analytical method for phytase determination the EURL cannot presently recommend for official control any of the two methods mentioned above. However complying with article 10 (Commission Regulation (EC) No 378/2005) the EURL considers necessary the organisation of an inter-laboratory comparison to determine an accurate conversion factor (f)1 allowing the applicability of the ISO method in the frame of official control when determining phytase (Optiphos) in the feed additive, premixtures and feedingstuffs.
Furthermore the EURL considers that the Applicant should provide a different abbreviation for optiphos phytase activity unit in the registry entry. This will avoid confusion with the phytase activity unit (FTU) defined by the ISO method.
1f = phytase activity determined by ISO 30024 / phytase activity determined by Applicant method