Methods and measures


The AJINOMOTO EUROLYSINE S.A.S. customer laboratory applies standardized and / or official methods and in-house validated methods:

The analysis methods under the scope of accreditation are mentioned in the Cofrac technical annex.

 

Receipt and preparation of samples

  1. Receipt and registration of samples
    The laboratory takes delivery and registers the samples
  2. Grinding of the samples
    Samples are ground before analyses
  3. Weighing room
    Analyses start with first a weighing of the sample

Carrying out analyses

Protein determination (Standard NF EN ISO 16634-1)

Nitrogen analyser

The total nitrogen content is determined by DUMAS method. This method consists of a « flash » combustion of the sample at high temperature. The various components of the sample (C, H, N, S, O) are found in the form of gas (CO2, H2O, NOx, SO2, halogens, O2 ...). Several traps as well as oxidation and reduction catalysts make it possible to retain only N2. This gas is measured thanks to a katharometric detector. The detection principle is linked to the conductivity of the gas to be measured.

Total amino acids other than tryptophan determination in raw materials and feeds (Commission regulation N° 152/2009 - standard NF EN ISO 13903)

Determination of total amino acids is carried out in duplicate for each sample, after hydrolysis of the proteins under acidic conditions:

  • Reflux hydrolysis for 23 hours at 110°C. This hydrolysis makes it possible to obtain all the amino acids except methionine and cystine
  • Reflux hydrolysis for 23 hours at 110°C after performic acid oxidation of the methionine (transformed into methionine sulfone) and the cystine (transformed into cysteic acid). This hydrolysis makes it possible to obtain all the amino acids except tyrosine and phenylalanine, which must be determined using hydrolysis without oxidation

The hydrolysates are adjusted to a pH of 2.20 and the amino acids are separated by ion exchange chromatography and then determined using photometric detection at 570 nm (440 nm for proline) after reaction with ninhydrin. The method is not valid for dosage of methionine hydroxy analogue.

Figure 1. Protocol for total amino acid analysis

Sand bathes for
hydrolyses
pH adjustment of
hydrolysates
Amino acid analyzer

Total tryptophan determination in feeds and raw materials (In-house method from the abrogated standard AFNOR XP V18-114)

Determination of total tryptophan is carried out in duplicate for each sample, after basic hydrolysis of the proteins.

The sample is hydrolysed under alkaline conditions with barium hydroxide and heated in an autoclave at 120°C for 16 hours. The hydrolysates are acidified with chlorhydric acid to pH 3.0.

The tryptophan from the hydrolysates is separated by reverse phase high performance liquid chromatography (HPLC) and determined by fluorometric detection.

Figure 2. Protocol for total tryptophan analysis


Autoclaves for hydrolyses

HPLC for tryptophan analysis

Free amino acids and free tryptophan determination

Free amino acids determination in feeds
Free amino acids except tryptophan (Commission regulation N° 152/2009 - standard NF EN ISO 13903)

The amino acids are extracted using 0.1 mol/l chlorhydric acid. The co–extracted nitrogenous macromolecules are precipitated with sulfosalicylic acid and removed by filtration. The solution is filtered and adjusted to pH 2.2. The amino acids are separated on ion-exchange resins and dosed by photometric detection at 570 nm after reaction with ninhydrin on a specific amino acid analyser.

Free tryptophan (in-house method)

Free tryptophan is extracted using 0.1 N chlorhydric acid. The tryptophan from the extracts is separated by reverse phase HPLC and determined by fluorometric detection.

Free amino acids determination in pure products and in premixes
Free amino acids except tryptophan (Method NF EN ISO 17180 – Method AOAC 999.13)

Determination of the amino acid content in commercial products, premixes and concentrates is carried out using an amino acid analyzer. The amino acids are extracted using 0.1 mol/l chlorhydric acid, separated on ion-exchange resins and dosed by photometric detection at 570 nm after reaction with ninhydrin on a specific amino acid analyser.

Analyses are carried out under Cofrac accreditation only for lysine, threonine and methionine and when the concentration is superior to 10%.

Free tryptophan (In-house method)

Free tryptophan is extracted using 0.1 N chlorhydric acid. The tryptophan from the extracts is separated by reverse phase HPLC and determined by fluorometric detection.

 

Top