EN ISO 6498 – Animal Feeding Stuffs – Guidelines For .
TOP 7EN ISO 6498 – Animal Feeding Stuffs –Guidelines for Sample Preparation6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro1
EN ISO 6498 – Animal Feeding Stuffs –Guidelines for Sample PreparationSAMPLINGà take a representative lab sample from a lotSAMPLE PREPARATIONà prepare a representative test samplefrom a lab (sub) sampleANALYSISà follow the (microscopical, microbiological, chemical)method protocols6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro2
EN ISO 6498 – Animal Feeding Stuffs –Guidelines for Sample PreparationContentsForeword1Scope2Normative References3Principle4Definitions and examples of animal feeding stuffs characteristics5Considerations to sample preparation errors6Safety precautions7Equipment8Procedure9Performance tests (quality control)10Annexes: Categories of feeds – special remarks and flow chartsAnnex A (informative) Calculations, examples and tables to minimum massBibliography6./7.Dec 2011Workshop ANKARA TOP 1 2 IntroPageiv11128111113242746503
Foreword The main task of technical committees is to prepare InternationalStandards. Draft International Standards adopted by the technicalcommittees are circulated to the member bodies for voting. Publication asan International Standard requires approval by at least 75 % of the memberbodies casting a vote. ISO 6498 was prepared by Technical Committee ISO/TC 34, Foodproducts, Subcommittee SC 10, and by Technical Committee CEN/TC 327,Animal feeding stuffs in collaboration.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro4
1 ScopeThis International Standard specifies guidelines for the preparation of testsamples from laboratory samples of animal feeding stuffs including petfoods mostly quoted from Association of American Feed Control Officialsguidelines.The guidelines are overruled by special instructions and regulations forsample preparation demanded by specific analysis methods for feedingstuffs (e.g. ISO, CEN, IEC).NOTE This International Standard does not include special guidelines forsample preparation for microbiological analysis of microorganisms likeyeasts, bacteria and molds, but for microorganisms which are used as feedadditives (probiotics) some important aspects to sample preparation aregiven.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro5
2 Normative ReferencesISO 6497:2002, Animal feeding stuffs – SamplingISO 664:2008, Oilseeds – Reduction of laboratory sample to test sampleAssociation of American Feed Control Officials Incorporated (AAFCO),Guidelines for Preparing Laboratory Samples, prepared by : AAFCOLaboratory Methods and Service Committee – Sample Preparation WorkingGroup (Nancy Thiex, Lawrence Novotny, Charles Ramsey, George Latimer,Laszlo Torma, Robert Beine), Second Edition March 2003Commission Regulation (EC) No 152/2009 of 27 January 2009 layingdown the methods of sampling and analysis for the official control of feed6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro6
3 Principle (1)All the sample preparation steps depend- on the different properties of the feedstuffs and- on the parameters to be analyzed.In every case the special instructions of the analysismethods concerning sample preparation have to beconsidered !6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro7
3 Principle (2)The sample preparation guidelines describe theprocedure for preparing a sample coming to a laboratory(in general with minimum weight of 0,5 kg) to get ahomogeneous test sample (with minimum weight of100g) with the same constitution, with the samecomposition and without any contamination.NOTE In some cases the laboratory sample size couldbe less than 500 g (i.e. standards of feed additives) butstatutory regulations have to be followed and in everycase the sample size needs to be homogeneous.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro8
3 Principle (3)In general the whole laboratory sample is reduced inmass and in particle size to obtain one or more testsamples for the analysis of stable and unstableparameters, for microscopy analysis and for reserve.If the analysis protocol and the intended proceeding ofthe reserve sample permit it, the laboratory sampleshould be preferably 'pre-grinded' completely at first toan adequate coarse particle size and then mass reducedto ensure homogeneity of the test samples.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro9
3 Principle (4)From a test portion (of 0,5g up to 25 g and more) forweighing to feedstuff analysis representative results should be achieved of the laboratory sample andfinally of the whole lot from which the sample wasdrawn.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro10
3 Principle (5)Therefore all the steps for sample preparation should bedone rather quickly, under convenient and very cleanconditions so there could be no degradation of sensitiveanalytes, no contaminations and no oxidation processdue to influences of too high temperatures, daylight, airor from residues of apparatus used or from samplesprepared before or simultaneously.Especially contamination from sample to sample shouldbe prevented.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro11
3 Principle (6)A loss or a change of moisture during samplepreparation should be avoided.In any case it must be taken into account that for officialcontrol results have to be corrected (to origin moisturecontent, 88 % or 100 % of dry mass).For feedstuffs with a higher moisture content (dry matter 85 %) partial drying or freeze drying before massreduction could be necessary.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro12
3 Principle (7)For feedstuffs with lumps or particle sizes 6 mmcoarser grinding of the whole laboratory sample to aparticle size of 6 mm before mass reduction /subsampling is absolutely necessary.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro13
3 Principle (8)The samples have to be stored at every stage of thesample preparation under adequate conditions (e.g. atroom temperature, refrigerated, frozen, air-tight, lightprotected / dark) to maintain their integrity.NOTE For microbiological analysis all steps of samplepreparation have to be done under aseptic conditions;laboratory samples should not be frozen or heated( 40 C) and not set to vacuum or higher oxygeninfluence.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro14
4 Definitions – concerning sample (1)lotquantity of material that is assumed to be of the sameproduction process and represented by sampling accordingto the rules of regulation (EC) No. 152/2009.laboratory sample (lab sample)sample as prepared (from the lot) for sending to thelaboratory and intended for inspection or testing.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro15
4 Definitions – concerning sample (2)test sample(sub-) sample prepared from the laboratory sample and fromwhich test portions will be taken.test portionquantity of material drawn from the test sample (or from thelaboratory sample if both are the same).reserve sampleleft material from the laboratory sample where splitted /subsampled test samples are taken away from and where nofurther particle size reduction is done.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro16
4 Definitions – concerning parameter (3)parametersanalytes or constituents or microorganisms for which thefeeding stuff is to be analysed by microscopic, (micro-)biological- or chemical procedures.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro17
4 Definitions – concerning parameter (4)stable parametersanalytes or constituents or microorganisms which do notdegrade during sample preparation on common handling orstorage at room temperature of 20 C to 25 C.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro18
4 Definitions – concerning parameter (5)unstable parametersanalytes or constituents or microorganisms which degradesduring sample preparation on common handling or storageat room temperature of 20 C to 25 C because they arevolatile, degradable, temperature sensitive or sensitive tolight, enzymatic degradation or chemical oxidation.NOTE Stability of parameters in this context refers only toinfluences of sample preparation to them like e.g. intensivegrinding and not to given minimum durability fromproducers or from labelling e.g. for a feed (additive).6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro19
4 Definitions – parameter classification (6)StableparametersUnstable parametersReason(s) for degradation / changeMoistureTemperature (volatile)Starch, sugar,lactoseAmmoniaTemperature (volatile)In vitro tests(e.g. gasproduction,enzyme solubleorganicsubstance)Organic acids (e.g. lactic acid,acetic acid, butyric acid, citricacid, fumaric acid, formic acid)Temperature (volatile)Minerals (e.g.Ca, P, Mg, Na, K,Cl)Unsaturated fatty acidsAir oxidation (might be change to shortchain fatty acids)protein,Nutrients (Crude)fat, ash, fibre6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro20
4 Definitions – parameter classification (7)FeedadditivesStable parametersUnstable parametersReason(s) fordegradation / changeTrace elements (e.g.Cu, Zn, Mn, Fe, Se,Co)Vitamins (e.g. vitamin A, C, D, E)Temperature, UV-light,air oxidation (sensitive)Amino acids (e.g.lysine, methionine,tryptophan)1,2-propandiol, glycolTemperature (volatile)Enzymes (e.g.phytases, nonestarchpolysaccharideenyzmes)Microorganisms like probiotics (e.g.Saccharomyces cerevisiae, Enterococcusfaecium)Temperature (freezing),pressure (sensitive)6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro21
4 Definitions – parameter classification (8)Stable parametersUndesirablesubstancesHeavy metals (e.g.As, Pb, Cd, Hg)Dioxins and PCB-likeDioxinsBannedsubstancesProteins of animalorigin(Other)Microorganisms6./7.Dec 2011Unstable parametersReason(s) fordegradation / changeMold growth and changeMycotoxins (e.g. aflatoxin B1,ofmycotoxins possible atdeoxynivalenol, fumonisins, ochratoxin A, roomtemperature; UVT-2 and HT-2 toxin, zearalenone, , antibiotics, pesticidesTemperature (sensitive)Hydrocyanic acidTemperature (volatile)Banned drugs, banned antibioticsTemperature (sensitive)Antibiotics, yeasts, bacteria, moldsTemperature (sensitive),dryness (Anaerobiosis),influx of oxygenWorkshop ANKARA TOP 1 2 Intro22
4 Example of feed characteristics (9)For identification and grouping a laboratory sample to theterms and annexes used within these guidelines someexamples of animal feeding stuffs characteristics are given inthis document.NOTE Definitions of animal feeding stuffs are given bylegislation worldwide. As an example definitions of Europeandirectives and for straight feeds in an alphabetical list from aGerman committee are mentioned within the bibliography.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro23
4 Example of feed characteristics (10)dry feedsfeed ingredient or complete animal feed which typicallycontains not more than 15 % moisture.NOTE Dry feed pellets are an agglomerated dry feed by amechanical process.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro24
4 Example of feed characteristics (11)green fodderedible parts of plants, other than separated grain, that canprovide feed for grazing animals or that can be harvested forfeeding, including browse, herbage, and mast.NOTE Generally, the term refers to more digestible material(i.e. what is called pasturage, hay, silage, dehydrated andgreen chop) in contrast to less-digestible plant material,known as roughage.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro25
4 Example of feed characteristics (12)silageforage preserved in a succulent condition by organic acidsproduced by anaerobic fermentation of sugars in the forage.roughagefibrous, coarsely textured parts of plants.NOTE Examples for fibrous, coarsely textured parts of plantsare stovers, straws, hulls, cobs, and stalks.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro26
4 Example of feed characteristics (13)haythe aerial portion of grass or herbage especially cut andcured for animal feeding.haylageforage preserved in a succulent condition by organic acidsproduced by anaerobic fermentation of sugars in the foragewith about 45 % moisture.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro27
4 Example of feed characteristics (14)total mixed ration (TMR)a single mixture of all feed ingredients (forages, grains, andsupplements) that is supplied to an animal for a 24-hourperiod.NOTE In practice, the 24-hour allotment of the mixture maybe offered in one or more feedings.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro28
4 Example of feed characteristics (15)by-productproduct which remaining during process-procedures for theproduction of ingredients from plant material.EXAMPLEDried destillers grains with solubles (DDGS)from fermentation.oilseedany seed from which oil is expressed.EXAMPLE6./7.Dec 2011sunflower seedsWorkshop ANKARA TOP 1 2 Intro29
4 Example of feed characteristics (16)mineral mixsupplementary feed that mainly consists of mineralingredient in either granular, bead, or small pelleted formand which is as entire mix free flowing.NOTE Mineral pellets are an agglomerated mineral feedformed by a mechanical process.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro30
4 Example of feed characteristics (17)premixtureuniform mixture of one or more micro-ingredients withdiluent and/or carrier.NOTE Premixtures are used to facilitate uniform dispersionof the micro-ingredients (i.e. vitamins, probiotics, drugs,antibiotics, and/or pharmaceuticals) in a large mix.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro31
4 Example of feed characteristics (18)canned pet foodfeed product for pets which has been processed, packaged,sealed, and sterilized for preservation in cans or similarcontainers.semi-moist feedmeat based feed product for pets or aquatic animals that hasbeen partially dried to prevent microbial decomposition.NOTE The moisture content may range from 15 % to 40 %.The product generally is in the form of strips or cubes and isdesigned to be stored at room temperature.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro32
4 Example of feed characteristics (19)dog chewmeat and skin/peel strips that have been completely dried toa leather-like consistency.NOTE Dog chews are also known as rawhide bones .6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro33
4 Example of feed characteristics (20)range cube and alfalfa hay cubesagglomerated feed formed by compacting and forcing themix through die openings by a mechanical process.NOTE The pellets are about 2 cm of diameter and 5 cm oflength ( /- 16 cm3) and may contain molasses; this definitionapplies also to alfalfa cubes (chopped alfalfa hay) larger thanthe mentioned dimension.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro34
4 Example of feed characteristics (21)texturized and sticky feedmix of assorted grains and commercial feed (generallypelleted) all of which has been treated with a coating ofmolasses.NOTE Some of the grains may have been steam heated and /or rolled prior to incorporating into the texturized fee6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro35
4 Definitions – sample prep procedure (22)homogeneitydegree to which a property or a constituent is uniformlydistributed throughout a quantity of material.NOTE Homogeneity may be considered to have been achieved in apractical sense when the sampling error of the processed portion isnegligible compared to the total error of the measurement system; sincehomogeneity depends on the size of the units under consideration, amixture of two materials may be inhomogeneous at the molecular oratomic level, but homogenous at the particulate level; however, uniformvisual appearance does not ensure compositional homogeneity.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro36
4 Definitions – sample prep procedure (23)partial dryingpart of the sample preparation procedure for feedstuffsamples with a high moisture content (dry mass 85 %) inwhich the sample is carefully dried to allow subsequentsample preparation procedures to be applied like particlesize reduction by grinding with a mill.NOTE 1 Partial drying procedure depends on the heat stability of the parameters(e.g. 70 C 10 C for drugs and antibiotics trace elements and heavy metals).NOTE 2 Samples for microbiological analysis should not be dried (attemperatures 40 C).NOTE 3 Partial drying can also be done by freeze drying procedure which is acareful drying process in the vacuum in order to sublimate the moisture.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro37
4 Definitions – sample prep procedure (24)coarse grindinga firstly grinding step of the whole sample in cases when thelaboratory sample contains large lumps or its particle size ishigh of about 6 mm before mass reduction.NOTE Coarse grinding finally is a kind of particle sizereduction to ensure homogeneity of the laboratory samplefor subsampling purposes.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro38
4 Definitions – sample prep procedure (25)mass reductionpart of the sample preparation procedure to reduce themass of a laboratory sample by splitting or subsampling it bythe use of (stationary or rotary) dividers or by fractional(alternate) shoveling without changing the consistence ofthe sample.NOTE After mass reduction all subsamples should have thesame properties like the original laboratory sample.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro39
4 Definitions – sample prep procedure (26)particle size reductionpart of the sample preparation procedure done by chopping,crushing, cutting, blending (homogenizing), macerating,milling (grinding), pressing, pulverizing to obtain ahomogenous test sample for further analysis.NOTE In general the particle size reduction follows the massreduction step of the sample preparation procedure withdifferent sieve-size-options to ensure integrity of the testsample(s).6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro40
5 Considerations to sample prep errors (1)Sample preparation steps have been shownto be some of the largest error of laboratory error.This error, which is generally overlooked, may be muchlarger than the error in subsequent analytical procedures.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro41
5 Considerations to sample prep errors (2)5.1 Subsampling and other errorsErrors deriving from sample heterogeneity may add to thetotal subsampling error on two levels:5.1.1 Constitution heterogeneity5.1.2 Distributional heterogeneity6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro42
5 Considerations to sample prep errors (3)5.1.1 Constitution heterogeneityOn a first level this is a result when not all of the particles ofthe laboratory sample have the same composition (shape,size, density, etc.).If a great overall composition-wise difference between theindividual fragments exists, the constitution heterogeneity islarge, but if the fragments are more homogeneousconstitution heterogeneity is lower. The total contribution toheterogeneity is never zero, however, as that would be thecase of all fragments being strictly identical.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro43
5 Considerations to sample prep errors (4)5.1.1 Constitution heterogeneityMixing and blending does not change constitutionheterogeneity.The only way to alter the constitution heterogeneity of anygiven material would be by cominution (crushing/cutting) orby other methods changing the physical properties of asample. The reduction of the average grain-size is thedominating factor in reducing constitution heterogeneity bysuch means.6./7.Dec 2011Workshop ANKARA TOP 1 2 Intro44
5 Considerations to sample prep errors (5)5.1.1 Constitution heterogeneityTherefore a firstly coarse grinding ('pre-grinding') of thewhole laboratory sample before subsampling/splittingreduces constitution heterogeneity.This fundamental subsampling error can be controlled by theappropriate choice of the test sample mass.Therefore collect enough mass to ensure that all of theparticles of different composition are contained in thesubsample/split.The larger the particle size of a material the larger the massof the subsample must be to minimize error.6./7.Dec 2011Workshop ANK
Guidelines for Sample Preparation 6./7.Dec 2011 Workshop ANKARA TOP 1 2 Intro 1. EN ISO 6498 – Animal Feeding Stuffs – Guidelines for Sample Preparation SAMPLING àtake a representative lab sample from a lot SAMPLE PREPARATION
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