prEN 18204
(Main)Algae and algae products - Determination of the phycocyanin content in Arthrospira (Spirulina)
Algae and algae products - Determination of the phycocyanin content in Arthrospira (Spirulina)
This document specifies a laboratory method for the quantification of phycocyanin content in the genus Arthrospira (Spirulina) by a
spectrophotometric method.
Algen und Algenprodukte - Bestimmung des Phycocyaningehalts in Arthrospira (Spirulina)
Dieses Dokument legt die Extraktions- und Bestimmungsverfahren für die Phycobiliproteine C- Phycocyanin(C-PC) und das komplementäre Pigment Allophycocyanin(APC) von trockenen Proben von Arten fest, die zur Gattung Arthrospira (Cyanobakterien, Cyanoprokaryoten oder Blaugrünalgen) (mitt lerweile als Limnospira bezeichnet, [1]) gehören und im Handel als Spirulina bekannt sind. Dieses Dokument ermöglicht es Laboren, Arthrospira-Proben zu analysieren, um genaue C-PC- und APC-Konzentrationen und -Ausbeuten zu erhalten.
Algues et produits à base d’algues — Détermination de la teneur en phycocyanine d’Arthrospira (Spiruline)
Le présent document spécifie les procédures d’extraction et de détermination des phycobiliprotéines C-phycocyanine (C-PC) et du pigment complémentaire allophycocyanine (APC) à partir d’échantillons secs d’espèces appartenant au genre Arthrospira (cyanobactéries, Cyanoprokaryota ou algues bleues) (aujourd’hui appelées Limnospira, [1]), et commercialement connues sous le nom de spiruline. Ce document permet aux laboratoires qui analysent des échantillons d’Arthrospira de rapporter des concentrations et des rendements précis de C-PC et d’APC.
Alge in izdelki iz alg - Določanje vsebnosti fikocianina v Arthrospira (Spirulina)
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-september-2025
Alge in izdelki iz alg - Določanje vsebnosti fikocianina v Arthrospira (Spirulina)
Algae and algae products - Determination of the phycocyanin content in Arthrospira
(Spirulina)
Algen und Algenprodukte - Bestimmung des Phycocyaningehalts in Arthrospira
(Spirulina)
Algues et produits à base d’algues - Détermination de la teneur en phycocyanine
d’Arthrospira (Spiruline)
Ta slovenski standard je istoveten z: prEN 18204
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
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2025
ICS
English Version
Algae and algae products - Determination of the
phycocyanin content in Arthrospira (Spirulina)
Algues et produits à base d'algues - Détermination de Algen und Algenprodukte - Bestimmung des
la teneur en phycocyanine d'Arthrospira (Spiruline) Phycocyaningehalts in Arthrospira (Spirulina)
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 18204:2025 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 6
5 Apparatus . 6
6 Reagents and materials . 6
7 Procedures . 6
7.1 Handling of samples . 6
7.2 Preparation and storage of the phosphate buffer . 6
7.3 Extraction protocol . 7
7.4 Procedure for obtaining the supernatant . 7
7.5 C-PC and APC spectrophotometric determination and quantification . 7
7.6 Purity criteria . 8
8 Calculations . 8
8.1 General. 8
8.2 Interlaboratory reproducibility . 9
9 Report . 9
Annex A (informative) Results of Interlaboratory Trial and Interlaboratory Study for
phycobiliproteins . 10
A.1 Introduction . 10
A.2 Results of the Interlaboratory Trial . 10
A.3 Results of the Interlaboratory Study . 11
Bibliography . 13
European foreword
This document (prEN 18204:2025) 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 standardization request addressed to CEN by the European
Commission. The Standing Committee of the EFTA States subsequently approves these requests for its
Member States.
Introduction
This document has been prepared by the experts of CEN/TC 454 'Algae and algae products'.
The European Committee for Standardization (CEN) was requested by the European Commission (EC) to
draft European standards or European standardization deliverables to support the implementation of
Article 3 of Directive 2009/28/EC for algae and algae products.
This request, presented as Mandate M/547, 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 products 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 and fertilizer/biostimulants. Standardization 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 and algae products and will promote and support
commercialisation of the European algae industry.
1 Scope
This document specifies the extraction and determination procedures for the phycobiliproteins C-
phycocyanin (C-PC) and the complementary pigment allophycocyanin (APC) from dry samples of species
belonging to the genus Arthrospira (Cyanobacteria, Cyanoprokaryota or blue-green algae)(nowadays
referred to Limnospira, [1]), and commercially known as Spirulina. This document enables laboratories
analysing Arthrospira samples to report accurate C-PC and APC concentrations and yields.
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 given in EN 17399 and the following apply.
ISO and IEC maintain terminology 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
Phycobiliproteins
PBP
water soluble pigments present in cyanobacteria and certain algae groups such as glaucophytes,
cryptomonads, and red algae (Rhodophytes) used for capturing light
Note 1 to entry: Phycobiliproteins capture light energy, which is then passed on to chlorophylls during
photosynthesis. Phycobiliproteins are formed of a complex between proteins and covalently bound phycobilins that
act as chromophores (the light-capturing part). They are most important constituents of the phycobilisomes.
3.2
Phycocyanin
C-PC
blue-green phycobilin pigment found in cyanobacteria, glaucophytes, cryptomonads, and red algae
Note 1 to entry: Nomenclature for phycocyanin specifically obtained from cyanobacteria is identified as C-PC.
3.3
allophycocyanin
APC
type of phycobilin photosynthetic accessory pigment found in cyanobacteria, glaucophytes,
cryptomonads, and red algae
4 Principle
Phycobiliproteins (PBP) like C-PC and APC can be extracted from dried biomass by vortexing this with a
phosphate buffer solution. The C-PC and APC concentration can be spectrophotometrically determined
and calculated in the resulting supernatant.
5 Apparatus
5.1 Analytical balance, with an accuracy of 0,1 mg
5.2 Vortex
5.3 Magnetic stirrer
5.4 Centrifuge, over 5,000 × g, preferably with temperature operation mode at 4 °C
5.5 UV-VIS Spectrophotometer, for 1 cm cuvettes
6 Reagents and materials
6.1 Phosphate buffer (0,1 M) plus NaCl (0,15 M) at a pH of 6.8
6.2 Stock solutions
6.2.1 0.2 M solution of monobasic sodium phosphate (27,8 g of NaH PO in 1000 ml distilled water)
2 4
6.2.2 0.2 M solution of dibasic sodium phosphate (53,65 g of Na HPO • 7H O or 71,7 g of Na HPO
2 4 2 2 4
• 12H O in 1000 ml distilled water)
7 Procedures
7.1 Handling of samples
Sample treatment for dry matter (DM) determination shall be performed according to EN 17605.
Additionally, samples should be stored in the dark prior to analysis.
In general, most of the cyanobacteria Arthrospira/Spirulina clean samples are nowadays obtained by
cultivation at different scales. Samples can be obtained as fresh/wet biomass but dry biomass can also be
obtained through different methodologies including oven(heating)-dried, freeze-dried or spray-dried.
In case flake samples are obtained special attention is needed considering the homogeneity of the
samples. If there are doubts on the homogeneity of the samples, it is required to perform the several
sample preparation steps described in EN 17605.
7.2 Preparation and storage of the phosphate buffer
Mix 51,0 ml of stock buffer 6.2.1 with 49,0 ml of stock buffer 6.2.2 to obtain a final pH of 6,8 (Table 1, ref).
Dilute to a total of 200 ml with distilled water. The final concentration will be 0,1 M.
To prepare the final saline phosphate buffer solution, add sodium chloride to a final concentration of 0,15
M (1,75 g NaCl). The stocks and buffer solutions will be stored at 4 °C during a maximum of 3 months.
Table 1 — Overview of volumes of stock buffers to use according to the pH [2]
6.2.1 (ml) 6.2.2 (ml) pH
68,5 31,5 6,5
62,5 37,5 6,6
56,5 43,5 6,7
51,0 49,0 6,8
45,0 55,0 6,9
39,0 61,0 7,0
7.3 Extraction protocol
For statistical purposes the extraction protocol should be performed in triplicate for each sample.
Procedures should be performed under dim light to avoid the photochemical degradation of C-PC that
also can be important for other molecules extracted in the process. However, C-PC is much more sensitive
to increased temperature than to photodegradation. This sample treatment should be performed with
temperatures below 40 °C.
Both glass and/or plastic materials can be used to perform the analysis.
7.4 Procedure for obtaining the supernatant
— Weigh 20 mg sample and resuspend in 10 mL saline phosphate buffer (0,1 M) plus sodium chloride
(0,15 M) at pH 6,8 as the extraction solvent (proportion 2:1000 w:v, biomass:solvent). Vortex the
mixture for 1 min maximum and repeat this twice. It is important to avoid overheating while
conducting the procedure.
Cells disruption is obtained by 1 freeze–thaw cycle: freezing at a temperature of −20 °C in darkness
(incubated until solid), followed by the thawing at 4 °C under low light cond
...
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