FprEN 18198

SLOVENSKI STANDARD
01-julij-2025
Alge in izdelki iz alg - Merjenje vsebnosti dušika in izračun vsebnosti beljakovin v
mikro- in mikroalgah
Algae and algae products - Nitrogen content measurement and protein content
calculation for micro- and microalgae
Algen und Algenprodukte - Messung des Stickstoffgehaltes und Berechnung des
Proteingehaltes von Mikro- und Makroalgen
Algues et produits à base d'algues - Mesure de la teneur en azote et calcul de la teneur
en protéines des micro et macroalgues
Ta slovenski standard je istoveten z: prEN 18198
ICS:
13.020.55 Biološki izdelki Biobased products
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

DRAFT
EUROPEAN STANDARD
prEN 18198
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2025
ICS
English Version
Algae and algae products - Nitrogen content measurement
and protein content calculation for micro- and microalgae
Algues et produits à base d'algues - Mesure de la Algen und Algenprodukte - Messung des
teneur en azote et calcul de la teneur en protéines des Stickstoffgehaltes und Berechnung des Proteingehaltes
micro et macroalgues von Mikro- und Makroalgen
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 454.
If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations
which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.

This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other
language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
Management Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2025 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 18198:2025 E
worldwide for CEN national Members.

prEN 18198:2025 (E)
Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 General . 7
5 Kjeldahl nitrogen content . 7
5.1 Principle . 7
5.2 Reagents and materials . 7
5.3 Apparatus . 8
5.4 Sampling . 8
5.5 Preparation of test sample . 8
5.6 Procedure. 9
5.6.1 General . 9
5.6.2 5Test portion . 9
5.7 Determination . 9
5.7.1 Digestion of organic matter . 9
5.7.2 Distillation of ammonia . 9
5.7.3 Titration . 10
5.8 Blank test . 10
5.9 Check test . 10
5.10 Calculation of nitrogen content and expression of results . 11
5.10.1 Distillate collected in sulfuric acid. 11
5.10.2 Distillate collected in boric acid . 11
5.11 Precision . 11
5.11.1 Interlaboratory test . 11
5.11.2 Repeatability . 11
5.11.3 Reproducibility . 12
5.12 Test report . 12
6 Dumas total nitrogen content . 12
6.1 Principle . 12
6.2 Reagents . 13
6.3 Apparatus . 14
6.4 Sampling . 14
6.5 Preparation of test sample . 14
6.6 Procedure. 14
6.6.1 General . 14
6.6.2 Test portion . 14
6.6.3 Control of oxygen supply . 15
6.6.4 Calibration . 15
6.6.5 Determination . 15
6.6.6 Detection and data processing . 15
6.7 Calculation of total nitrogen content . 16
7 Precision . 16
7.1 Interlaboratory tests . 16
7.2 Repeatability . 16
7.3 Reproducibility . 16
8 Test report . 16
prEN 18198:2025 (E)
9 Difference between Kjeldahl and Dumas methods . 17
10 Calculation of the crude protein content . 17
11 Calculation of specific nitrogen-to-protein conversion factors . 18
Annex A (informative) Details on Interlaboratory study on two algae samples testing of
nitrogen content by Kjeldahl method and by Dumas method . 19
Annex B (informative) Examples of protein calculations . 22
Bibliography . 24

prEN 18198:2025 (E)
European foreword
This document (prEN 18198:2025) has been prepared by Technical Committee CEN/TC 454 “Algae and
algae products”, the secretariat of which is held by NEN.
This document is currently submitted to the CEN Enquiry.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
prEN 18198:2025 (E)
Introduction
This document has been prepared by the experts of CEN/TC 454 'Algae and algae products'.
The European Committee for Standardisation (CEN) was requested by the European Commission (EC) to
draft European standards or European standardisation deliverables to support the implementation of
Article 3 of Directive 2009/28/EC for algae and algae-based products or intermediates.
This request, presented as Mandate M/5471, also contributes to the Communication on "Innovating for
Sustainable Growth: A Bio economy for Europe".
The former working group CEN Technical Board Working Group 218 “Algae”, was created in 2016 to
develop a work programme as part of this Mandate. The technical committee CEN/TC 454 'Algae and
algae products' was established to carry out the work programme that will prepare a series of standards.
The interest in algae and algae-based products or intermediates has increased significantly in Europe as
a valuable source including but not limited to, carbohydrates, proteins, lipids, and several pigments.
These materials are suitable for use in a wide range of applications from food and feed purposes to other
sectors, such as textile, cosmetics, biopolymers, biofuel, fertilizers and biostimulants. Standardisation
was identified as having an important role in order to promote the use of algae and algae products.
The work of CEN/TC 454 should improve the reliability of the supply chain, thereby improving the
confidence of industry and consumers in algae, which include macroalgae, microalgae, cyanobacteria,
Labyrinthulomycetes, algae-based products or intermediates and will promote and support
commercialisation of the European algae industry.
Determining the nitrogen content of algae and algae products is an important method in the algae
industry. It is needed to calculate protein content, biomass gross composition, nutritional value, and also
to evaluate mass balances along algae farming.
The goal of this document is to give algae producers and algae products industries recommendations for
analytical methods for nitrogen content in compliance with food and feed regulations [1]. This document,
based on EN ISO 5983-1:2005 Animal feeding stuffs — Determination of nitrogen content and calculation
of crude protein content and EN ISO 16634-2:2016, Food products - Determination of the total nitrogen
content by combustion according to the Dumas principle and calculation of the crude protein content - Part
2: Cereals, pulses and milled cereal products, demonstrates the suitability of both methods for the
determination of nitrogen in micro- and macroalgae and highlight potential differences between the two
methods. This document also provides recommendations for the calculation of protein content and
guidance to establish accurate nitrogen to protein conversion factors in conjunction with total amino acid
analyses according to prEN 18197:2025, Algae and algae products — Determination of the amino acid
profile when required.
prEN 18198:2025 (E)
1 Scope
This document describes the application of Kjeldahl and Dumas methods for the determination of
nitrogen content in algae and their relevant products.
The method was initially tested and evaluated on the algae species Nannochloropsis sp. and Palmaria
palmata. The study validated this document for both algae species. This method can also be used for other
algae species.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
EN 17399, Algae and algae products - Vocabulary
EN 17605, Algae and algae products - Methods of sampling and analysis - Sample treatment
3 Terms and definitions
For the purposes of this document, the terms and definitions in EN 17399 and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• ISO Online browsing platform: available at https://www.iso.org/obp
• IEC Electropedia: available at https://www.electropedia.org/
3.1
Kjeldahl nitrogen content
mass fraction of the Kjeldahl nitrogen content in the sample
Note 1 to entry: Determined by the procedure specified in clause 5.
Note 2 to entry: Unless otherwise specified, the nitrogen content is expressed on as is basis, not on dry weight
3.2
Dumas total nitrogen content
mass fraction of the total nitrogen content in the sample
Note 1 to entry: Determined by the procedure specified in clause 6.
Note 2 to entry: Unless otherwise specified, the nitrogen content is expressed on as is basis, not on dry weight
3.3
crude protein content
nitrogen content (either Kjeldahl or Dumas) multiplied by a conversion factor
Note 1 to entry: The nitrogen content measurement method and the conversion factor shall be specified.
prEN 18198:2025 (E)
4 General
The Kjeldahl method for the determination of nitrogen content in microalgae and macroalgae and their
relevant products is based on EN ISO 5983-1:2005.
The Kjeldahl method does not measure oxidized forms of nitrogen or heterocyclic nitrogen compounds.
The Dumas method for the determination of Nitrogen content in microalga and seaweed algae and their
relevant products is based on EN ISO 16634-2.
The Dumas method measures all forms of nitrogen present in the sample.
The selection between Kjeldahl or Dumas method is left to the user’s discretion since either method has
advantages and drawbacks. However, the nitrogen content determined may differ depending on the
amount of oxidized nitrogen in the sample. The selected method shall be specified in the test report.
The methods described in EN ISO 5983-1:2005 and EN ISO 16634-2 do not distinguish between protein
nitrogen and non-protein nitrogen.
The utilization of a conversion factor of 6,25 is required in regulatory documents such as regulation (EU)
No 1169/2011 (food, nutrition labelling) and commission regulation (EC) No 152/2009 (feed, official
control) to convert the nitrogen content of a product (food, feed) into a protein value.
A more accurate protein content can be obtained by calculating the sum of all amino acyls, and this
protein content can then be divided by the total nitrogen content to obtain appropriate nitrogen-to-
protein conversion factors (NCFs) for the matrices of interest [4]. These matrix-specific NCFs can be
useful when a more accurate protein content needs to be calculated from their nitrogen content. Guidance
for the calculation of those nitrogen to protein conversion factor is provided in this document.
5 Kjeldahl nitrogen content
5.1 Principle
The organic matter is digested by sulfuric acid in the presence of a catalyst. The reaction product is
rendered alkaline, then the liberated ammonia is distilled and titrated. The nitrogen content is calculated.
The result may be multiplied by a conversion factor to obtain the crude protein content.
5.2 Reagents and materials
Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or deionized
water or water of equivalent purity.
The reagents [except the standard materials (5.2.6)] shall be free from nitrogenous compounds.
5.2.1 Potassium sulfate
5.2.2 Catalyst
5.2.2.1 Copper(II) oxide (CuO).
5.2.2.2 Copper(II) sulfate pentahydrate (CuSO4·5H2O).
5.2.3 Sulfuric acid, c(H2SO4) = 18 mol/l, d20 (H2SO4) = 1,84 g/ml.
5.2.4 Paraffin wax.
5.2.5 Saccharose.
5.2.6 Standard materials
prEN 18198:2025 (E)
5.2.6.1 Acetanilide, with melting point 114 °C; nitrogen (N) content 103,6 g/kg.
5.2.6.2 Tryptophan, with melting point 282 °C; nitrogen (N) content 137,2 g/kg. Dry before use.
5.2.7 Sodium hydroxide solution, w(NaOH) = 33 % (mass fraction).
5.2.8 Collecting liquid
5.2.8.1 Sulfuric acid, standard volumetric solution, c(H2SO4) = 0,05 mol/l or c(H2SO4) = 0,125
mol/l.
5.2.8.2 Boric acid, d20 (H3BO3) = 40 g/l.
5.2.9 Solutions for titration.
5.2.9.1 Sodium hydroxide, standard volumetric solution, c(NaOH) = 0,1 mol/l or c(NaOH) = 0,25
mol/l.
5.2.9.2 Sulfuric acid, standard volumetric solution, c(H2SO4) = 0,05 mol/l or c(H2SO4) = 0,125
mol/l.
The molarity of standard volumetric solutions should be known to the fourth decimal point.
5.2.10 Mixed indicator, neutral point at pH 4,4 to 5,8.
Dissolve 2 g of methyl red and 1 g of methylene blue in 1000 ml of ethanol [c(C2H5OH) = 95 % (volume
fraction)].
5.2.11 pH indicator paper.
5.2.12 Boiling aids, such as granulated pumice stone, or glass beads of diameter 5 mm to 7 mm, or
carborundum chips, washed in hydrochloric acid and in distilled water, and ashed.
5.3 Apparatus
Usual laboratory apparatus and, in particular, the following.
5.3.1 Analytical balance.
5.3.2 Digestion, distillation and titration apparatus.
5.4 Sampling
A representative sample should have been sent to the laboratory. It should not have been damaged or
changed during transport or storage.
Sampling is not part of the method specified in this document. A recommended sampling method is given
in EN 17980 and EN 17605:2022.
Store the sample closed in tight container at refrigerated temperature in such a way that deterioration
and change in its composition are prevented.
5.5 Preparation of test sample
The test sample shall be dry i.e. with a moisture content < 15%, and preferably below 10 % homogeneous
and finely ground according to EN 17605:2022, 3,7, 3.9 and 3.13.,
prEN 18198:2025 (E)
5.6 Procedure
WARNING — The operations described in 5.6.1 and 5.6.2 should be carried out under a well-ventilated
hood or in a fume cupboard which is resistant to sulfuric acid.
5.6.1 General
For general directions on the application of the Kjeldahl method, see ISO 1871:2009 [7]
5.6.2 5Test portion
Weigh, to the nearest 1 mg, a mass of the test sample chosen according to the expected nitrogen content
so that the test portion contains between 0,005 g and 0,2 g of nitrogen and, preferably, more than 0,02 g.
The mass of the test portion of homogeneous air-dry samples should be between 0,5 g and 2,0 g.
5.7 Determination
5.7.1 Digestion of organic matter
Transfer the test portion quantitatively into a Kjeldahl digestion flask of suitable size (usually 800 ml).
Add 15 g of potassium sulfate (5.2.1).
Add an appropriate quantity of catalyst as follows: 0,3 g of copper(II) oxide (5.2.2.1) or 0,9 g to 1,2 g of
copper(II) sulfate pentahydrate (5.2.2.2).
Add 25 ml of sulfuric acid (5.2.3) for the first gram of dry matter of the test portion and 6 ml to 12 ml for
each additional gram of dry matter. Mix thoroughly, ensuring complete wetting of the test portion.
Support the flask so that its axis is inclined at an angle of 30° to 45° to the vertical. Maintain the flask in
this position throughout heating.
Heat the flask moderately at first to prevent foam from rising into the neck of the flask or escaping from
the flask.
NOTE 1 It can be advisable to add an anti-foaming agent such as paraffin wax (5.2.4).
Heat moderately, swirling from time to time, until the mass has carbonized and the foam has disappeared.
Then heat more intensively until the liquid is boiling steadily.
NOTE 2 Heating is adequate if the boiling acid condenses towards the middle of the neck of the Kjeldahl flask.
Avoid overheating of the walls of the flask not in contact with liquid.
NOTE 3 If a naked flame is used, such overheating can be prevented by placing the flask on a sheet of heat-
resistant material with an aperture of diameter slightly less than that of the flask at the liquid level.
After the liquid has become clear with a light green-blue colour, heat for another 2 h.
Leave to cool. If the digest starts to solidify, add some water and mix by swirling.
5.7.2 Distillation of ammonia
5.7.2.1 Carefully add 250 ml to 350 ml of water to dissolve the sulfates completely. If necessary,
facilitate dissolving by heating the flask in warm water. Mix by swirling and allow to cool.
Add a few boiling aids (5.2.12).
For some specific samples, the sulfates may not completely dissolve in the added water. In that case, it is
recommended to repeat the digestion with a reduced mass of potassium sulfate (5.2.1).
prEN 18198:2025 (E)
Pipette, into the collecting flask of the distillation apparatus, 25 ml of the sulfuric acid (5.2.8.1), choosing
the concentration according to the expected nitrogen content of the test portion. Add 100 ml to 150 ml of
water. Add a few drops of the mixed indicator (5.2.10). Proceed in accordance with 5.7.2.4.
5.7.2.2 Alternatively, transfer into the collecting flask 100 ml to 250 ml of boric acid (4.8.2). Add a
few drops of mixed indicator (5.2.10).
Simultaneous titration of the ammonia (see 5.7.3.3) during distillation is recommended since it facilitates
verification of the end of distillation.
5.7.2.3 Immerse the end of the condenser in the liquid contained in the collecting flask, to a depth of
at least 1 cm.
Slowly pour 100 ml of sodium hydroxide solution (5.2.7) into the digestion flask along the wall.
Immediately connect the flask to the distillation apparatus.
Heat the flask in such a manner that approximately 150 ml of distillate is collected in 30 min. At the end
of this time, check the pH of the distillate at the tip of the condenser using litmus paper (5.2.11). If the
reaction is alkaline, continue distillation.
WARNING — Lift the condenser from the liquid just before the end of the distillation, to prevent backflow.
If, during distillation using sulfuric acid as collecting liquid, the contents of the collecting flask become
alkaline, recommence the determination, making appropriate adjustments.
5.7.3 Titration
5.7.3.1 Titration with automatic endpoint indication using a pH-meter is recommended. Otherwise,
the endpoint is indicated by the change in colour of the mixed indicator (5.2.10) added in 5.7.2.
5.7.3.2 If sulfuric acid is used as the collecting liquid, titrate, in the collecting flask, the excess sulfuric
acid with sodium hydroxide solution (5.2.9.1), c(NaOH) = 0,1 mol/l or c(NaOH) = 0,25 mol/l as
appropriate, until the endpoint is indicated by the pH-meter or until the colour changes from violet to
green.
5.7.3.3 If boric acid is used as the collecting liquid, titrate the ammonia with sulfuric acid (5.2.9.2),
c(H2SO4) = 0,05 mol/l or c(H2SO4) = 0,125 mol/l as appropriate, until the endpoint is indicated by the
pH-meter or the colour changes from green to violet.
If simultaneous titration is not possible (see 5.7.2.3), the titration should be carried out as soon as
possible after the distillation is complete, ensuring that the temperature of the distillate does not exceed
25 °C. Under these conditions, losses of ammonia are avoided.
5.8 Blank test
Perform a blank test using about 1 g of saccharose (5.2.5) in place of the test portion.
5.9 Check test
Perform a check test by determining the nitrogen content of acetanilide (5.2.6.1) or tryptophan (5.2.6.2)
after addition of 1 g of saccharose (5.2.5).
The choice of the substance for the check test should be related to the digestibility of the samples to be
analyzed. Acetanilide is easily digested, whereas the digestion of tryptophan is more difficult, e.g. the case
may be dealing with microalgae with strong cell wall.
The recovery of nitrogen from acetanilide or tryptophan should be at least 99,5 % for acetanil
...