SIST EN ISO 16472:2006
(Main)Animal feeding stuffs - Determination of amylase-treated neutral detergent fibre content (aNDF) (ISO 16472:2006)
Animal feeding stuffs - Determination of amylase-treated neutral detergent fibre content (aNDF) (ISO 16472:2006)
This International Standard specifies methods for the determination of amylase-treated neutral detergent insoluble fibrous residue content in all types of animal feed. It includes a gravimetric routine method and a reference method.
Futtermittel - Bestimmung des amylase-behandelten neutral gereinigten Fasergehalts (ISO 16472:2006)
Diese Internationale Norm legt Verfahren zur Bestimmung des amylase-behandelten Faserrückstands, der in neutralem Waschmittel unlöslich ist, in allen Arten von Futtermitteln fest.
Sie beinhaltet ein gravimetrisches Routineverfahren und ein Referenzverfahren.
Aliments des animaux - Détermination du contenu en fibre détergente neutre traitée a l'amylase (ISO 16472:2006)
L'ISO 16472:2006 décrit une méthode de détermination du contenu en fibres résiduelles, insolubles, traitées à l'amylase et au détergent neutre, dans tous les types d'aliments pour animaux.
Elle inclut une méthode par gravimétrie courante et une méthode de référence.
Krma – Določevanje vlaknin z nevtralnim reagentom po predhodni obdelavi z amilazo (aNDF) (ISO 16472:2006)
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 16472:2006
01-september-2006
.UPD±'RORþHYDQMHYODNQLQ]QHYWUDOQLPUHDJHQWRPSRSUHGKRGQLREGHODYL]
DPLOD]R D1') ,62
Animal feeding stuffs - Determination of amylase-treated neutral detergent fibre content
(aNDF) (ISO 16472:2006)
Futtermittel - Bestimmung des amylase-behandelten neutral gereinigten Fasergehalts
(ISO 16472:2006)
Aliments des animaux - Détermination du contenu en fibre détergente neutre traitée a
l'amylase (ISO 16472:2006)
Ta slovenski standard je istoveten z: EN ISO 16472:2006
ICS:
65.120 Krmila Animal feeding stuffs
SIST EN ISO 16472:2006 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 16472:2006
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SIST EN ISO 16472:2006
EUROPEAN STANDARD
EN ISO 16472
NORME EUROPÉENNE
EUROPÄISCHE NORM
April 2006
ICS 65.120
English Version
Animal feeding stuffs - Determination of amylase-treated neutral
detergent fibre content (aNDF) (ISO 16472:2006)
Aliments des animaux - Détermination du contenu en fibre Futtermittel - Bestimmung des amylase-behandelten
détergente neutre traitée à l'amylase (ISO 16472:2006) neutral gereinigten Fasergehalts (ISO 16472:2006)
This European Standard was approved by CEN on 23 March 2006.
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 Central Secretariat 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 Central Secretariat has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, 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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COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36 B-1050 Brussels
© 2006 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16472:2006: E
worldwide for CEN national Members.
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SIST EN ISO 16472:2006
EN ISO 16472:2006 (E)
Foreword
This document (EN ISO 16472:2006) has been prepared by Technical Committee ISO/TC 34
"Agricultural food products" in collaboration with Technical Committee CEN/TC 327 "Animal
feeding stuffs - Methods of sampling and analysis", the secretariat of which is held by NEN.
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 October 2006, and conflicting national
standards shall be withdrawn at the latest by October 2006.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium,
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.
Endorsement notice
The text of ISO 16472:2006 has been approved by CEN as EN ISO 16472:2006 without any
modifications.
2
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SIST EN ISO 16472:2006
INTERNATIONAL ISO
STANDARD 16472
First edition
2006-04-15
Animal feeding stuffs — Determination of
amylase-treated neutral detergent fibre
content (aNDF)
Aliments des animaux — Détermination du contenu en fibre détergente
neutre traitée à l'amylase
Reference number
ISO 16472:2006(E)
©
ISO 2006
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SIST EN ISO 16472:2006
ISO 16472:2006(E)
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ii © ISO 2006 – All rights reserved
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SIST EN ISO 16472:2006
ISO 16472:2006(E)
Contents Page
Foreword. iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions. 1
4 Principle. 1
5 Reagents. 2
6 Apparatus . 2
7 Sampling. 3
8 Preparation of test sample. 3
9 Procedure . 4
9.1 Procedure for traditional method as described in Reference [1] . 4
9.2 Determination using Fibertec-type apparatus . 5
9.3 Modifications for specific types of samples . 7
9.4 Quality assurance. 8
10 Calculation and expression of results. 8
10.1 Calculation. 8
10.2 Expression of results . 9
11 Precision. 9
11.1 Interlaboratory test . 9
11.2 Repeatability. 9
11.3 Reproducibility. 10
12 Test report . 10
Annex A (informative) Results of interlaboratory test. 11
Annex B (informative) Standardization of heat-stable alpha-amylase working solution. 14
Bibliography . 16
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SIST EN ISO 16472:2006
ISO 16472:2006(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 16472 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 10, Animal
feeding stuffs.
iv © ISO 2006 – All rights reserved
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SIST EN ISO 16472:2006
INTERNATIONAL STANDARD ISO 16472:2006(E)
Animal feeding stuffs — Determination of amylase-treated
neutral detergent fibre content (aNDF)
WARNING — The use of this International Standard may involve the use of hazardous materials,
operations and equipment. This International Standard does not purport to address all the safety risks
associated with its use. It is the responsibility of the user of this International Standard to establish
appropriate safety and health practices and determine the applicability of local regulatory limitations
prior to use.
1 Scope
This International Standard specifies methods for the determination of amylase-treated neutral detergent
insoluble fibrous residue content in all types of animal feed.
It includes a gravimetric routine method and a reference method.
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 6498, Animal feeding stuffs — Preparation of test samples
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
amylase-treated neutral detergent fibre content
aNDF content
mass fraction of insoluble fibre residues determined by the procedure specified in this International Standard
NOTE The aNDF content is expressed as a percentage by mass.
4 Principle
Neutral detergent (ND) solution and heat-stable alpha-amylase are used to dissolve the easily digestible
proteins, lipids, sugars, starches and pectins in feeds, leaving an insoluble fibrous residue that is primarily cell
wall components of plant materials (cellulose, hemicellulose and lignin) and indigestible nitrogenous matter in
animal products.
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SIST EN ISO 16472:2006
ISO 16472:2006(E)
5 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized
water or water of equivalent purity.
5.1 Sodium sulfite, anhydrous (Na SO ).
2 3
5.2 Dried hominy corn (corn grits, raw), ground to pass through a 1 mm screen in a cutter mill.
5.3 Iodine solution, containing 2 g of potassium iodide and 1 g of iodine in 100 ml of water.
Store the solution in an amber or opaque bottle.
5.4 Heat-stable alpha-amylase, as a solution or a water extract of lyophilised enzyme powder (approx. 1 g
of powder extracted in 100 ml of water).
EXAMPLE Termamyl 120 l from Novo Enzymes or equivalent.
Standardize the heat-stable alpha-amylase solution or enzyme powder extract so that two additions of 2 ml
will remove starch from 0,5 g of raw corn starch (5.2). For a detailed procedure on standardizing heat-stable
alpha-amylase solution, see Annex B.
5.5 Neutral-detergent (ND) solution
Pour between 400 ml and 500 ml of water into a 1 l flask. Add 4,0 g of sodium hydroxide (NaOH) 14,6 g of
EDTA, 4,56 g of sodium hydrogen phosphate (Na HPO ), and 6,81 g of sodium borate decahydrate
2 4
⋅
(Na B O 10 H O) and mix until dissolved (heat if necessary). The NaOH and EDTA may be replaced with
2 4 7 2
18,6 g of disodium EDTA.
Under a safety hood, add 30 g of sodium lauryl sulfate and, after dissolution, add 10 ml of triethylene glycol
(anti-foaming aid). Add water to about 950 ml and mix. Adjust the pH to between 6,95 and 7,05 with
concentrated hydrochloric acid (HCl) or sodium hydroxide (NaOH) and dilute to 1 000 ml with water. If the pH
is off the range by more than 0,5, discard the solution.
Store the ND solution at room temperature. If precipitation occurs, warm the solution to 25 °C and mix before
use. Record the date the ND solution was prepared, the pH measurements and any adjustments in a reagent
log book.
6 Apparatus
Usual laboratory apparatus and, in particular, the following.
6.1 Analytical balance, capable of weighing to the nearest 0,1 mg, with a readability of 0,1 mg.
6.2 Cyclone mill with 2 mm screen, or cutter mill with 1 mm screen, capable of grinding samples to obtain
a geometric mean particle size of 220 µm to 260 µm
6.3 Refluxing apparatus, with individual heating units and cold water condensers that fit 600 ml flasks.
Any conventional apparatus suitable for crude fibre determinations is acceptable. Calibrate the heating unit
settings so that 50 ml of water boils within 4 min to 5 min when using cold water condensers. A Fibertec type
apparatus may be used and should boil 50 ml of water within 10 min.
6.4 Fritted-disc Gooch crucibles, coarse porosity (pore size 40 µm to 60 µm) crucibles, high-form, 40 ml
to 50 ml capacity, or P2 (pore size 40 µm to 100 µm), 26 ml to 28 ml capacity.
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SIST EN ISO 16472:2006
ISO 16472:2006(E)
Clean new crucibles and ash at 500 °C for 1 h. Clean crucibles after each use by ashing at 500 °C for 3 h,
removing ash, inverting in a detergent solution and sonicating for 7 min to 10 min. Rinse crucibles in hot water,
and soak in water at room temperature for at least 30 min. Fit the top of each crucible with a rubber stopper
fitted with a port that is connected to a trap and vacuum line. Back-flush each crucible with water, by
repeatedly plunging and removing the bottom of the crucible into water to create a vigorous rinsing action.
Occasionally check the filtration rate as follows. Fill each crucible with 50 ml of distilled water (25 ml for
Fibertec P2 crucibles) and record the time required to drain completely without vacuum (should be
180 s ± 60 s for Gooch or 75 s ± 30 s for P2). If the drain time is < 100 s (or < 30 s for P2), discard the crucible.
If it is < 120 s (or < 45 s for P2), check for cracks in the fritted disc. If the filtration takes > 240 s (or > 105 s for
P2), clean the crucible with acid or alkaline cleaning solution (see Reference [1]). If cleaning does not improve
the filtration rate, discard the crucible.
Instead of P2 crucibles, stainless-steel metal crucibles with a 90 µm aperture stainless-steel metal sieve may
also be used.
6.5 Vacuum filter manifold (e.g. Fibertec type), that allows adequate soaking of fibrous residues.
The manifold should provide a vacuum-tight seal with the crucible to reduce foam formation in vacuum lines.
Use thick-walled vacuum tubing to connect the manifold to a trap (4 l to 18 l) and vacuum source. A vacuum
reservoir (18 l) between the trap and vacuum source is recommended to ensure adequate vacuum capacity to
remove the foam.
6.6 Boiling water supply
Use a continuous boiling water generator as described in Reference [1] or a suitable alternative. The
apparatus shall be capable of supplying boiling water (> 95 °C) in a quantity sufficient for all samples being
washed at one time, through a nozzle producing a fine stream (flow rate of 35 ml to 40 ml per 10 s; a 2,5 ml
disposable plastic pipette tip makes an acceptable nozzle). A fine nozzle minimizes the water needed to
transfer particles to the crucible, but provides the water pressure needed to remove residues attached to the
side of the flask. It is critical that water is boiling when added to the crucibles, especially for samples
containing starches, pectic substances, mucilages or glyco-proteins. For Fibertec type apparatus, use a
syringe with a cone-spray nozzle to rinse the condensers and a 60 ml disposable syringe with 12 gauge
needle that is 10 cm in length to dislodge any residues adhering to the condensers.
7 Sampling
Sampling is not part of the method specified in this International Standard. A recommended sampling method
is given in ISO 6497.
It is important that the laboratory receive a sample which is truly representative and has not been damaged or
changed during transport or storage.
8 Preparation of test sample
Prepare the test samples in accordance with ISO 6498.
For sample storage and ease of grinding, samples should be air-dry (about 90 % dry matter)
Dry wet samples at < 60 °C to prevent creation of artefact fibre. The amount of residue after extraction is
affected by the particle size of the sample. Grind representative samples to obtain a geometric mean particle
size of 220 µm to 260 µm (see 6.2).
Grinding segregates the sample, with highest fibre content material passing out of the grinder last. Do not
discard material in the grinder, combine it with material in the grinder receptacle. Mix the ground sample by
placing it on a square sheet of paper (approximately 40 cm × 40 cm) creased along both diagonals. Lift two
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SIST EN ISO 16472:2006
ISO 16472:2006(E)
opposite corners of the sheet to slide the sample in towards the central crease. Spread the sheet out flat again,
turn it through 90° and lift the other two corners. Repeat 11 times. Transfer the sample to a suitable container.
NOTE Wet samples can be analysed for aNDF; however this is not a routine approach because it is difficult to grind
the samples to the equivalent particle size as above.
9 Procedure
9.1 Procedure for traditional method as described in Reference [1]
9.1.1 Test portion
Dry the empty crucibles at 105 °C ± 1 °C for 4 h then weigh them. Record the empty crucible mass for
samples (m ) or blanks (m ) to the nearest 0,000 1 g.
c b
Mix the material thoroughly and weigh 1 g ± 0,001 g of air-dry feed, or the equivalent amount of wet test
sample (m ), into a crucible or refluxing flask, depending on preliminary defatting.
s
Inhomogeneous samples that need grinding shall be dried (see Clause 8). Only wet samples that can easily
be homogenized may be weighed in directly.
If results are to be reported on a dry matter basis, weigh a second sample at the same time for determination
of the dry matter.
Include an in-house reference sample and two blanks for the first 20 to 30 samples in a run, and add one
reference and one blank for each additional 20 to 30 samples.
9.1.2 Preliminary defatting
Samples containing > 5 % fat should be pre-extracted. Those with > 10 % fat shall be pre-extracted to remove
the fat.
To pre-extract with acetone, put a test portion into a crucible and weigh it. Place it on the filter manifold and
extract four times with 40 ml to 50 ml of acetone (allow material to soak at least 5 min and stir three times
during each soaking). Apply vacuum to remove traces of acetone, air-dry for 10 min to 15 min to ensure that
all traces of acetone are removed and transfer to a reflux flask. Use the same crucible to collect the fibre
residue for the test sample after ND extraction.
If a filtering aid is used, it shall be dried and weighed with the crucible, then transferred to another container
before the test sample is weighed into the crucible and extracted with acetone. Replace the filtering aid in the
crucible before filtration of fibre residues after ND extraction.
9.1.3 Digestion
Add 0,5 g ± 0,1 g of sodium sulfite (5.1) using a graduated scoop and 50 ml ± 5 ml of ND solution (5.5) to
each refluxing flask and swirl (this is critical for starchy feeds that stick to the bottom during refluxing). Do not
add the ND and sodium sulfite to samples more than 60 min before refluxing.
Heat to boiling within 4 min to 5 min, add 2 ml of standardized amylase solution (5.4), resuspend any particles
stuck to the bottom or sides, and swirl.
Reflux for 60 min at a rate that creates vigorous particle movement, but not excessive foaming that would
carry particles up the side of the flask. Samples may foam vigorously for 1 min to 2 min (do not reduce the
temperature of the heating unit). Rinse the sides of the flask with a minimum amount of ND solution, using a
bottle with a fine nozzle, 5 min to 10 min after adding the amylase, and rinse as needed to resuspend particles
on the side of the flask (twice max.).
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SIST EN ISO 16472:2006
ISO 16472:2006(E)
9.1.4 Filtration
Remove the extracted sample from the heating unit and allow particles to settle for 30 s to 60 s. Before
transfer, observe the mixture to determine if lipid globules are present on the surface or if the solution is milky,
which indicates a high-fat material that should be rerun after acetone pre-extraction (9.1.2).
Place a Teflon stirring rod in the crucible and preheat by adding 40 ml of boiling water for 30 s to 60 s.
Remove the water with vacuum and immediately decant the top 30 ml to 40 ml of the solution from the flask,
keeping the flask inverted over the crucible. Use minimum vacuum to evacuate excess liquid and close
vacuum before residue becomes dry.
NOTE Excessive vacuum and evacuating to dryness causes some samples to clog the crucible and so not wash
properly.
Rinse all unattached particles into the crucible using a fine stream of boiling water. Fill the crucible half-full
with hot water. Add 2 ml of working amylase solution (5.4) and stir.
React with amylase for a minimum of 45 s to 60 s while scraping particles from the bottom and sides of the
reflux flask using a rubber policeman. Evacuate the amylase solution and transfer any remaining residue from
the reflux flask into the crucible with 20 ml to 30 ml boiling water. Two rinses are usually sufficient. After
transferring residues from the flask, fill the crucible three-quarters full with boiling water and soak for 3 min.
Evacuate the water, add 40 ml to 50 ml of boiling water, soak for 3 min to 5 min, and repeat. If residues are
difficult to filter after the first soak, add an additional 2 ml of working amylase solution. If residues appear
translucent and become more difficult to filter with each additional soaking, eliminate the third water soak. If
plugged, the crucible may be back-flushed by removing it from the filter manifold and reinserting it.
Evacuate the water, refill the crucible with 40 ml to 50 ml acetone, stir to disperse particles, soak for 3 min to
5 min, and repeat. Rinse the stirring rod to remove any attached fibre particles. Do not evacuate water
completely from the fibre residues with vacuum before adding the acetone. Excessive drying clumps the
residues and makes particle dispersion in acetone difficult, which hampers acetone extraction.
Apply a vacuum to dry the sample. Remove the crucible from the manifold and air dry for 10 min to 60 min to
remove acetone.
9.1.5 Drying
Dry crucibles at 105 °C ± 1 °C for a minimum of 8 h. Leave to cool in the dessicator and weigh to the nearest
0,000 1 g (m and m ).
ce be
9.1.6 Ashing
Ignite the crucible with the residue in a furnace at 500 °C ± 20 °C for 5 h or until carbon-free. Leave to cool in
the dessicator and weigh to the nearest 0,000 1 g (m and m ).
ca ba
9.2 Determination using Fibertec-type apparatus
9.2.1 Test portion
Add the filtering aid to the P2 crucible, dry at 105 °C ± 1 °C for 2 h to 4 h and weigh to the nearest 0,000 1 g
(m or m ). Mix material thoroughly and weigh 0,5 g ± 0,050 0 g of air-dry feed, or an equivalent amount of wet
c b
test sample (m ), into the crucible.
s
If results are to be reported on a dry matter basis, weigh a second sample at the same time for determination
of the dry matter.
Include an in-house reference sample and two blanks for the first 20 to 30 samples in a run, and add one
reference and one blank for each additional 20 to 30 samples.
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SIST EN ISO 16472:2006
ISO 16472:2006(E)
9.2.2 Preliminary defatting
Generally samples with unknown fat contents should be pre-extracted. Those with > 10 % fat shall be pre-
extracted to remove the fat.
Place the crucible on the cold-extraction unit and extract four times with 20 ml to 30 ml of acetone (allow the
material to soak for at least 5 min and stir three times during each soaking). Apply vacuum to remove traces of
acetone, air-dry for 10 to 15 min to ensure that all traces of acetone are removed.
9.2.3 Digestion
Start up the Fibertec-type apparatus, following the instructions of the manufacturer.
Add 0,5 g ± 0,1 g of sodium sulfite and 50 ml ± 5 ml of ND solution (5.5) to each crucible and mix using back-
pressure (this is critical for starchy feeds that stick to the bottom during refluxing). Do not add the ND and
sodium sulfite to samples more than 60 min before refluxing. Add 2 ml of standardized amylase solution (5.4)
and heat to boiling within 10 min. Use back pressure to mix the amylase with the ND solution and the sample.
Boil for 60 min. Samples may foam vigorously for 1 min to 2 min (do not reduce the temperature of the heating
unit). Rinse the sides of the flask with a minimum amount of ND, using a bottle with fine nozzle, 5 min to
10 min after adding the amylase, and rinse as needed to resuspend particles on the side of the flask (twice
max.).
9.2.4 Filtration
Before the initial filtration, observe the mixture to determine if lipid globules are present on the surface or if the
solution is milky, which indicates a high-fat material that should be rerun after acetone pre-extraction (9.2.2).
Evacuate the solution without allowing residues to become dry. Use minimum vacuum to evacuate excess
liquid, but close vacuum before residue becomes dry.
NOTE 1 Excessive vacuum and evacuating to dryness causes some samples to clog the crucible and so not wash
properly.
Add 30 ml of hot water (80 °C) and 2 ml of standardized amylase solution (5.4). Use back-pressure to mix the
amylase in the initial water soak. Remove amylase-water soak after a minimum of 60 s of reaction.
NOTE 2 Crucibles can be removed from the hot to the cold filtration unit for the remaining hot water soaks for samples
that are easy to filter. This allows the next set of samples to begin ND extraction on the hot filtration unit. Samples that are
difficult to filter can be washed on the Fibertec heating unit with heat reduced to minimize particle agitation.
Add 30 ml of hot water, soak for 3 min to 5 min, and remove the water. If residues are difficult to filter after the
first soak, add an additional 2 ml of amylase solution. If residues appear translucent and become more difficult
to filter with each additional soaking, eliminate the third water soak. If plugged, the crucibles may be back-
flushed using minimum back-pressure.
Do no
...
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