EN ISO 11702:2009
(Main)Animal and vegetable fats and oils - Enzymatic determination of total sterols content (ISO 11702:2009)
Animal and vegetable fats and oils - Enzymatic determination of total sterols content (ISO 11702:2009)
ISO 11702:2009 specifies a method for the quantitative determination of the total sterols content by means of an enzymatic staining test. The method is applicable to free and esterified sterols in animal and vegetable fats and oils, fatty foods and related products. The determination is applicable to sample quantities of 1 g to 2 g of fat.
The method is not applicable to dark coloured fats and oils, e.g. crude palm oil. The enzyme is not specific for cholesterol, but also oxidizes other 3-hydroxysterols. The method has not been tested for products fortified with sterols at higher levels.
Tierische und pflanzliche Fette und Öle - Enzymatische Bestimmung des Gesamtsterin-Gehaltes (ISO 11702:2009)
Diese Internationale Norm legt ein Verfahren zur quantitativen Bestimmung des Gesamtsterin-Gehaltes durch einen enzymatischen Farbtest fest. Das Verfahren ist anwendbar auf freie und veresterte Sterine in tierischen und pflanzlichen Fetten und Ölen, fetthaltigen Lebensmitteln und verwandten Erzeugnissen. Die Bestimmung gilt für Probenmengen von 1 g bis 2 g Fett.
Das Verfahren ist nicht auf dunkel gefärbte Fette und Ölen, wie z. B. rohes Palmöl, anwendbar. Das Enzym ist nicht spezifisch für Cholesterin, sondern es werden auch andere 3 Hydroxysterine oxidiert. Das Verfahren wurde nicht an Produkten geprüft, die mit höheren Konzentrationen von Sterinen angereichert waren.
ANMERKUNG Das Verfahren ist der IUPAC Methode 2.404 [8] und der DGF Einheitsmethode F III 2 (91) [7] technisch gleichwertig.
Corps gras d'origines animale et végétale - Détermination enzymatique de la teneur en stérols totaux (ISO 11702:2009)
L'ISO 11702:2009 spécifie une méthode pour la détermination quantitative de la teneur en stérols totaux à l'aide d'un essai de coloration enzymatique. La méthode s'applique aux stérols libres et estérifiés, contenus dans les corps gras d'origines animale et végétale, dans les aliments gras et les produits connexes. La détermination s'applique aux quantités d'échantillon de 1 g à 2 g de matière grasse.
Cette méthode ne s'applique pas aux corps gras de couleur sombre comme l'huile de palme brute. L'enzyme n'est pas spécifique du cholestérol et oxyde également d'autres 3‑hydroxystérols. La méthode n'a pas été testée pour des produits enrichis avec des stérols à des niveaux plus élevés.
Rastlinske in živalske maščobe in olja - Encimsko določevanje celotnih sterolov (ISO 11702:2009)
Ta mednarodni standard določa metodo za količinsko določevanje celotnih sterolov z encimskim preskusom obarvanja. Metoda velja za proste in esterificirane sterole v živalskih in rastlinskih maščobah in oljih, maščobnih živilih in sorodnih proizvodih. Določevanje se lahko uporablja pri vzorcih z 1 do 2 g maščobe. Metoda ne velja za temno obarvane maščobe in olja, npr. surovo palmovo olje. Encim ni specifičen za holesterol, ampak oksidira tudi druge 3-hidroksisterole. Metoda ni bila preskušena za proizvode, ojačane z večjimi koncentracijami sterolov.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-marec-2010
5DVWOLQVNHLQåLYDOVNHPDãþREHLQROMD(QFLPVNRGRORþHYDQMHFHORWQLKVWHURORY
,62
Animal and vegetable fats and oils - Enzymatic determination of total sterols content
(ISO 11702:2009)
Tierische und pflanzliche Fette und Öle - Enzymatische Bestimmung des Gesamtsterin-
Gehaltes (ISO 11702:2009)
Corps gras d'origines animale et végétale - Détermination enzymatique de la teneur en
stérols totaux (ISO 11702:2009)
Ta slovenski standard je istoveten z: EN ISO 11702:2009
ICS:
67.200.10 5DVWOLQVNHLQåLYDOVNH Animal and vegetable fats
PDãþREHLQROMD and oils
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EUROPEAN STANDARD
EN ISO 11702
NORME EUROPÉENNE
EUROPÄISCHE NORM
December 2009
ICS 67.200.10
English Version
Animal and vegetable fats and oils - Enzymatic determination of
total sterols content (ISO 11702:2009)
Corps gras d'origines animale et végétale - Détermination Tierische und pflanzliche Fette und Öle - Enzymatische
enzymatique de la teneur en stérols totaux (ISO Bestimmung des Gesamtsterin-Gehaltes (ISO 11702:2009)
11702:2009)
This European Standard was approved by CEN on 18 November 2009.
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. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the CEN Management Centre or to any CEN member.
This European Standard exists 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 Management Centre has the same status as the
official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
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EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2009 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11702:2009: E
worldwide for CEN national Members.
Contents Page
Foreword .3
Foreword
This document (EN ISO 11702:2009) has been prepared by Technical Committee ISO/TC 34 "Food products"
in collaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats and oils and their
by-products - Methods of sampling and analysis”, the secretariat of which is held by AFNOR.
This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by June 2010, and conflicting national standards shall be withdrawn at
the latest by June 2010.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech
Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and the United Kingdom.
Endorsement notice
The text of ISO 11702:2009 has been approved by CEN as a EN ISO 11702:2009 without any modification.
INTERNATIONAL ISO
STANDARD 11702
First edition
2009-12-15
Animal and vegetable fats and oils —
Enzymatic determination of total sterols
content
Corps gras d'origines animale et végétale — Détermination
enzymatique de la teneur en stérols totaux
Reference number
ISO 11702:2009(E)
©
ISO 2009
ISO 11702:2009(E)
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ii © ISO 2009 – All rights reserved
ISO 11702:2009(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 11702 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 11, Animal
and vegatable fats and oils.
INTERNATIONAL STANDARD ISO 11702:2009(E)
Animal and vegetable fats and oils — Enzymatic determination
of total sterols content
1 Scope
This International Standard specifies a method for the quantitative determination of the total sterols content by
means of an enzymatic staining test. The method is applicable to free and esterified sterols in animal and
vegetable fats and oils, fatty foods and related products. The determination is applicable to sample quantities
of 1 g to 2 g of fat.
The method is not applicable to dark coloured fats and oils, e.g. crude palm oil. The enzyme is not specific for
cholesterol, but also oxidizes other 3-hydroxysterols. The method has not been tested for products fortified
with sterols at higher levels.
[8] [7]
NOTE The method is technically equivalent to IUPAC method 2.404 and DGF standard method F-III 2 (91) .
2 Normative references
The following referenced documents are indispensable for the application 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.
ISO 661, Animal and vegetable fats and oils — Preparation of test sample
ISO 3696, Water for analytical laboratory use — Specification and test methods
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
total sterols content
w
sterols
mass fraction of sterols determined by the method specified in this International Standard
NOTE 1 For vegetable fats and oils, the sterols content is expressed as β-sitosterol; for animal fats, as cholesterol.
NOTE 2 The total sterols content is expressed in milligrams per 100 g of fat.
4 Principle
The test sample is saponified and the sterols in the unsaponifiable matter are determined enzymatically. They
are oxidized by cholesterol oxidase to cholestenone. The equimolar amount of hydrogen peroxide produced in
the process oxidizes in the presence of catalase methanol to formaldehyde. In the presence of ammonium
ions, with acetylacetone, it forms a yellow lutidine dye (3,5-diacetyl-1,4-dihydrolutidine). The latter is
determined spectrophotometrically in the visible range at 405 nm. The concentration of the dye is equivalent
to the amount of sterols.
NOTE Cholesterol oxidase oxidizes cholesterol and other sterols having a hydroxy group in the 3β-position.
Therefore, phytosterols like stigmasterol and sitosterol are also determined.
ISO 11702:2009(E)
5 Reagents
WARNING — Attention is drawn to the regulations which specify the handling of hazardous
substances. Technical, organizational and personal safety measures shall be followed.
Unless otherwise stated, use only reagents of recognized analytical grade.
5.1 Water, complying with ISO 3696, grade 3 or better.
5.2 Isopropanol.
5.3 Acetone.
5.4 Acetyl acetone.
1)
5.5 Suspension of cholesterol oxidase (EC 1.1.3.6) from Nocardia erythropolis, 15 U/ml.
1)
5.6 Suspension of catalase (hydrogen peroxide oxido-reductase) (EC 1.11.1.6) from bovine liver.
5.7 Hydrochloric acid, c(HCl) = 8 mol/l.
5.8 Methanolic potassium hydroxide solution, c(KOH) = 0,5 mol/l.
Dissolve 2,8 g potassium hydroxide in a small amount of hot methanol, cool, and dilute with methanol to
100 ml.
5.9 Ammonium phosphate buffer solution, adjusted to pH 7.
5.10 Solution 1.
Add 19,1 ml acetone (5.3) and 230 000 U catalase (5.6) to 50 ml buffer solution (5.9) in a 100 ml one-mark
volumetric flask (6.4), and make up to the mark with water (5.1).
5.11 Solution 2.
Add 0,26 ml acetylacetone (5.4) and 1,10 ml acetone (5.3) to 25 ml water (5.1) in a 50 ml one-mark volumetric
flask (6.4), and make up to the mark with water.
5.12 Solution 3.
Prior to use, mix 3 volumes of solution 1 (5.10) with 2 volumes of solution 2 (5.11).
NOTE Solution 3 can be kept in amber bottles for 3 months at 4 °C provided it is prepared under sterile conditions.
6 Apparatus
6.1 Test tubes, of diameter 18 mm.
6.2 Filter funnel.
6.3 Fluted filter suitable for the filter funnel (6.2).
1) A suitable ready-made test kid for the colorimetric determination of cholesterol in foodstuffs and other materials is
available from R-Biopharm. This information is given for the convenience of users of this International Standard and does
not constitute an endorsement by ISO of this product. Equivalent products may be used if they can be shown to lead to the
same results.
2 © ISO 2009 – All rights reserved
ISO 11702:2009(E)
[2]
6.4 One-mark volumetric flasks, of capacities 25 ml, 50 ml, and 100 ml, ISO 1042 class A.
[6] [1]
6.5 Enzyme pipettes, of capacities 0,02 ml, ISO 7550 , to 1 ml, ISO 648 class A.
[1]
6.6 Pipette, of capacity 5 ml, ISO 648 class A.
6.7 Round bottomed flask, standard ground joint, of capacity 50 ml.
6.8 Test tubes with ground stoppers.
6.9 Spectrophotometer, set to 405 nm.
6.10 Glass cuvettes, pathlength 1 cm, suitable for the spectrophotometer (6.9).
6.11 Water bath, thermostatically controlled at 37 °C to 40 °C.
6.12 Refrigerator, capable of maintaining a temperature of 4 °C.
6.13 Glass beads.
6.14 Reflux condenser, standard ground joint.
7 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 International Standard. A recommended sampling method
[3]
is given in ISO 5555 .
8 Preparation of the test sample
Prepare the test sample in accordance with ISO 661. Specific treatment of the test sample (filtration, melting,
etc.) shall be mentioned in the test report.
9 Procedure
9.1 Saponification
9.1.1 Weigh 1 g to 2 g of the sample accurately to within 0,001 g into a 50 ml round bottomed flask (6.7).
The sterol concentration in the test solution shall be between 0,02 g/l and 0,4 g/l. This requirement shall be
taken into account during the weighing and diluting steps. In the case of saturated fats, the amount weighed
shall be reduced, as otherwise free fatty acids formed after saponification and acidification are not completely
removed during filtration and affect the determination. Ensure at all times that the solution obtained is clear.
9.1.2 Add 10 ml of methanolic potassium hydroxide solution (5.8) and some glass beads (6.13). Heat the
mixture and, when boiling, reflux for 25
...
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