The nitrogen content of different proteins is relatively constant and
varies only slightly from an average value of 16%. The level of protein
in a feedstuff is usually measured by determining the nitrogen
content according to the conventional Kjeldahl or Dumas method;
the protein level of the feed can then be estimated by multiplying
the analysed N level by a factor of 6.25. Since NPN compounds are
also included in the N-determination, the protein level estimated in
this way is correctly referred to as crude protein. Other procedures
can be employed to differentiate true and crude proteins (for example
precipitation reactions).
The amount of crude protein in a feed provides a certain amount of
information concerning its nutritional value. However, it must be used
with care since it contains the NPN portion which cannot be utilised
by monogastrics. Furthermore, protein level and protein quality are
not correlated with each other (see chapter 3.3 Protein quality).
6.1. Protein-bound and free amino acids in raw materials
and feeds
One of the most important prerequisites for the formulation of mixed
feeds according to an animal’s dietary requirements is precise knowledge
of the quantitative amino acid composition of feed ingredients.
6.1.1. Total amino acids except tryptophan
The method of determination of total amino acids contents except
tryptophan which is destroyed during acid hydrolysis, in raw materials
and feeds is standardized (EN ISO 13903) and has been also
published by the European Commission regulation N° 152/2009.
The total amino acid determination includes the free amino acids
and the protein bound amino acids.
The method is specific to amino acids based on the fact that amino
acids are cations at pH 2.2 and give a specific coloured reaction
with ninhydrin. It does not enable the determination of methionine
hydroxy-analogue.
Determination of total amino acids (from proteins and free sources)
is carried out after hydrolysis of the proteins under acidic conditions
for 23 hours at 110° C. This hydrolysis makes it possible to
obtain all the amino acids except methionine and cystine. Preliminary
performic acid oxidation of the methionine (transformed into
methionine sulfone) and of the cystine (transformed into cysteic
acid) makes it possible to obtain all the amino acids except tyrosine
which must be determined using hydrolysis without oxidation.
The hydrolysates are then adjusted to a pH of 2.2 where the amino
acids are in a cationic form for the resin exchange step. Amino
acids are separated by ion exchange chromatography using an
amino acid analyser or High Performance Liquid Chromatography
(HPLC) with post column reaction (Figure 10).
Amino acid contents are determined, after reaction with ninhydrin
at high temperature, using photometric detection at 440 nm for
proline and 570 nm for all other amino acids.
6.1.2. Free amino acids except tryptophan
EN ISO 13903 is the standardized method of determination of
free amino acids contents except tryptophan. The amino acids
are extracted using 0.1 mol/l hydrochloric acid. Co-extracted nitrogenous
macromolecules are precipitated with sulfosalicylic acid
and removed by filtration. The solution is filtered and adjusted at
pH 2.2. The amino acids are separated on ion-exchange resins
and dosed by photometric detection at 570 nm after reaction with
ninhydrin in the same way as total amino acids.
6.1.3. Methionine hydroxy analogue
The methionine hydroxy analogues are extracted from the feeds
with an aqueous extraction agent and determined by reversephase-
HPLC with UV detection (method reference Z030.I06, approved
in 2012 by EURL).
6.1.4. Total tryptophan
Tryptophan is destroyed by acidic hydrolysis and therefore requires
a specific method of analysis. The principles are described
in the standard method NF EN ISO 13904 and in the corresponding
official method published by the European Commission Regulation
N° 152/2009.
Determination of total tryptophan is carried out after basic hydrolysis
of the proteins: The sample is hydrolysed under alkaline
conditions with barium hydroxide. The hydrolysates are acidified
with hydrochloric acid at pH 3.0 to perform the separation of the
peak by chromatography.
The tryptophan from the hydrolysates is separated by reverse
phase HPLC and does not need any colorimetric reaction. Indeed,
tryptophan contains an indol functional group which gives
spectroscopic properties of absorption and fluorescence in the
UV spectrum.
6.1.5. Free tryptophan
The method is based upon – as above – NF EN ISO 13904 and
European Commission Regulation N° 152/2009. Free tryptophan
is extracted using 0.1 N hydrochloric acid. Tryptophan is separated
by reverse phase HPLC and determined by fluorometric detection.
Amino acids in pure products and premixes
6.2.1. Amino acids other than tryptophan
The method is standardised (EN ISO 17180 and AOAC 999.13)
The amino acid trade products or premixes are dissolved in diluted
hydrochloric acid and diluted with sodium citrate buffer.
The solution is filtered. The amino acids are separated on ionexchange
resins and dosed by photometric detection at 570 nm
after reaction with ninhydrin in the same way as total amino acids.
Calibration must be done only with a reference solution containing
the amino-acids to be determined.
6.2.2. Tryptophan
The analysis is similar to free tryptophan in feeds. The preparation
of the sample takes into account the high content of supplemented
tryptophan. Free tryptophan is extracted using 0.1 N
chlorhydric acid. Tryptophan is separated by reverse phase HPLC
and determined by fluorometric detection.