1、ICS 67.100.20Bestimmung der Buttersorte durch neurale Netzanalyse kompositioneller Parameter ChemometrischesVerfahrenIn keeping with current practice in standards published by the International Organization for Standardization(ISO), a comma has been used throughout as the decimal marker.Ref. No. DIN
2、 10474 : 2003-02English price group 11 Sales No. 011112.03DEUTSCHE NORM February 200310474Continued on pages 2 to 11. No part of this translation may be reproduced without the prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusi
3、ve right of sale for German Standards (DIN-Normen).Determination of butter type by neural networkanalysis of compositional parameters usingthe chemometric methodTranslation by DIN-Sprachendienst.In case of doubt, the German-language original should be consulted as the authoritative text.ContentsPage
4、Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Normative references . . . . . . . . . . . . . . . . . . .
5、. . . . . . . . . . . . . . . . . . . . . . . 23 Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Symbol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Principle . . . .
6、 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7、. . . . . . . . . . . . . . . . 38 Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410 Evaluation . . . . . . . . . .
8、. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611 Precision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 812 Test report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9、. . . . . . . . 8Annex A Data sets for the pH value, citric acid, adenosine and uridinecontents of 30 butter samples for training the neural network (Tr),of 15 butter samples for checking the trained network (T) and of15 butter samples for validating the network (V) . . . . . . . . . . . . . . 9Bibl
10、iography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11ForewordThis standard has been prepared by Technical Committee Chemische und physikalische Milchuntersuchungof the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Foods
11、tuffs and Agricultural Prod-ucts Standards Committee).1 ScopeThe chemometric method specified in this standard serves to determine the butter type. It can be used toclassify butter as required by the Verordnung ber Butter und zur nderung milch- und margarinerechtlicherVorschriften 1 and covers soure
12、d cream butter, sweet cream butter and mildly soured butter.Page 2DIN 10474 : 2003-022 Normative referencesThis standard incorporates, by dated or undated reference, provisions from other publications. These norma-tive references are cited at the appropriate places in the text, and the titles of the
13、 publications are listed below.For dated references, subsequent amendments to or revisions of any of these publications apply to thisstandard only when incorporated in it by amendment or revision. For undated references, the latest edition ofthe publication referred to applies.DIN 10325 Enzymatic me
14、thod of determining the citric acid content of cheese spreadDIN 10349 Determination of the pH value of butter serumDIN EN ISO 707 Milk and milk products Guidance on sampling (ISO 707 : 1997)DIN EN ISO 3727-1 Butter Determination of moisture, non-fat solids and fat contents Part 1: Determina-tion of
15、moisture content (reference method) (ISO 3727-1 : 2001)1 Verordnung ber Butter und zur nderung milch- und margarinerechtlicher Vorschriften (Artikel 1 Verord-nung ber Butter und andere Milchstreichfette) (German Regulation on butter and regulation amending legis-lation on milk and margarine) (Articl
16、e 1 Regulation on butter and other spreadable milk fats, as of 3 February1997, BGBl. (German Federal Law Gazette) I, No. 7, 1997, pp. 1441533 Concepts3.1 Butter plasmaFat-free aqueous phase that is separated by centrifuging and mixing butter oil from butter melted at 65 C.3.2 Butter serumButter plas
17、ma deproteinized by centrifuging.4 SymbolHPLC High-Performance Liquid Chromatography5 PrincipleButter can be classified for commercial purposes by linking compositional parameters, including citric acid, theribonucleosides adenosine and uridine, and the pH value. A neural network is drawn up with th
18、e aid of com-puter-assisted calculations to determine the characteristics and is used to evaluate the analytical data. A three-layer, forward-linked neural network is used to classify butter chemometrically. An error-free identification ofthe particular butter type can be achieved using a minimum of
19、 two input neurons (pH value, citric acid), anintermediate layer and three simultaneous output neurons.6 ReagentsThe reagents used shall be of analytical grade and the water used shall be double distilled or at least ofequivalent purity.The following reagents shall be used.6.1 25 % (m/m) aqueous amm
20、onium solution, NH3.6.2 98 % to 100 % (m/m) formic acid, HCOOH.6.3 HPLC eluents.6.3.1 0,1 mol/l diammonium hydrogenphosphate solution (eluent A), (NH4)2HPO4, prepared by dissolving13,2 g of diammonium hydrogenphosphate in about 80 ml of water in a 100 ml beaker while stirring using amagnetic stirrer
21、, transferring the solution to a 1 000 ml graduated cylinder, making up to about 980 ml withwater, adjusting the pH value to 9,8 with ammonia solution and making up to the mark with water while keepinga check on the pH value.6.3.2 0,03 mol/l ammonium formate solution (eluent B), HCOONH4, prepared by
22、 dissolving 1,9 g of ammo-nium formate in about 50 ml of water in a 100 ml beaker while stirring using a magnetic stirrer, transferring thesolution to a 1 000 ml graduated cylinder, making up to about 980 ml with water, adjusting the pH value to 3,4with formic acid and making up to mark with water w
23、hile keeping a check on the pH value.6.3.3 Methanol, suitable for HPLC (eluent C) and water (eluent D).6.4 Other reagentsSee DIN 10325 and DIN EN ISO 3727-1.Page 3DIN 10474 : 2003-027 ApparatusIn addition to standard laboratory equipment the following shall be used.7.1 Ribonucleoside HPLC analysis s
24、ystem7.1.1 ApparatusThe analysis system (see figure 1) consists of a two-column high-performance liquid chromatograph in whicha precolumn (C1) is coupled to a separating column (C2) by an automatic column switching valve (AS), twopumps (P1 and P2), a control module (SM), a sample injection system (P
25、A), a UV detector (UV) and an evaluationmodule (AM).Figure 1: Ribonucleoside HPLC analysis system7.1.2 Automatic column switching valveIn the LOAD position, the precolumn is connected to the sample injection system and the separating column(C2) can be operated in parallel using pump P2 (see figure 2
26、).In the INJECT position, the switching valve couples the precolumn to the separating column to transfer theribonucleosides from the former to the latter (see figure 2). On switching back to the LOAD position, theribonucleosides adenosine and uridine are analytically separated by the separating colu
27、mn and the precolumnis re-equilibrated at the same time.Figure 2: Column switching valve positionsPage 4DIN 10474 : 2003-027.1.3 Chromatographic conditionsPrecolumn (4 mm internal diameter, 30 mm long), packed with a nitrated phenylboronic acid gel1).Separating column (4,6 mm internal diameter, 250
28、mm long), packed with RP-18 reverse-phase material ofparticle size 5 m.7.2 Neural networkA three-layer, forward-linked neural network having two (calculation path I) or three (calculation paths II andIII) input neurons (input layer), an intermediate (concealed) layer and three simultaneous output ne
29、urons (outputlayer).7.3 Centrifuges7.3.1 Centrifuge, capable of being adjusted to 12 000 g to 15 000 g.7.3.2 Centrifuge, capable of being adjusted to (350 t 50) g.7.4 Centrifuge vessels7.4.1 Conical plastic centrifuge vessels, measuring about 10 mm 40 mm, designed to suit the centrifuge asin subclau
30、se 7.3.1.7.4.2 Cylindrical glass centrifuge vessels, measuring about 22 mm 110 mm, sealable at both ends withstoppers, designed to suit the centrifuge as in subclause 7.3.2.7.5 pH meter (as in DIN 10349).7.6 Magnetic stirrer.7.7 Graduated cylinder, of nominal capacity 1 000 ml.7.8 Beakers, of nomina
31、l capacity 100 ml.7.9 Analytical balance.7.10 Volumetric flasks, of nominal capacity 10 ml.7.11 Double-beam spectrometer, capable of determining absorption at a wavelength of 260 nm or 262 nm.7.12 Membrane filters, of pore sizes 0,2 m and 1,2 m.8 SamplingSee DIN EN ISO 707.9 Procedure9.1 Preparation
32、 of test solution9.1.1 Butter plasmaMelt 80 g to 90 g of butter (stored at 4 C) at 65 C. Separate the butter plasma from the butter oil by centrifugingfor ten minutes at (350 t 50) g and, after removal from the centrifuge, cool the butter sample until the fat phasesolidifies. Transfer the butter pla
33、sma to a test tube or beaker and mix it. The butter plasma may be stored at20 C prior to analysis.9.1.2 Butter serumTo determine the adenosine and uridine content (see subclause 9.2.3), adjust the pH value of the butter plasmato 3,5 using formic acid, centrifuge it for 20 minutes at 12 000 g to 15 0
34、00 g and then filter the supernatantthrough a membrane filter having a pore size of 0,2 m. The soured butter serum may be stored at 20 C priorto analysis.9.1.3 Adenosine and uridine stock solutionsWeigh about (1 t 0,2) mg of adenosine and about (1 t 0,2) mg of uridine into separate, sealable reactio
35、n vesselsof nominal capacity 1,5 ml and add 1 000 l of ammonium formate. Store these solutions at 21 C.1) Information on sources of supply is obtainable from Normenausschuss Lebensmittel und landwirtschaftlicheProdukte of DIN, 10772 Berlin, Germany.Page 5DIN 10474 : 2003-029.1.4 HPLC standard soluti
36、onFor the standard HPLC analyses, pipette 50 l of each of the adenosine and uridine stock solutions into a 10 mlvolumetric flask and make up to the mark with ammonium formate. Store these solutions at 21 C prior toanalysis.9.2 Analyses9.2.1 Water contentDetermine the water content of the butter samp
37、le by the method specified in DIN EN ISO 3727-1 in duplicate.9.2.2 Citric acid contentMix 100 l of butter plasma (obtained as in subclause 9.1) with 900 l of water in a centrifuge vessel (as insubclause 7.4.1) and centrifuge at 12 000 g to 15 000 g. Filter the supernatant through a membrane filter o
38、f poresize 1,2 m. Perform a duplicate determination enzymatically in 0,2 ml of dilute butter plasma solution usingthe method described in DIN 10325. When using commercially available ready-to-use reagents, observe themanufacturers instructions. Report the content of citric acid in mg per 100 g of bu
39、tter.9.2.3 Adenosine and uridine contentsDetermine the adenosine and uridine contents of butter serum by liquid chromatography in duplicate (seesubclause 9.3). Report the nucleoside contents in g per 100 g of butter.9.2.4 pH valueDetermine the pH value of butter plasma as specified in DIN 10349.9.3
40、Use of eluentsEquilibrate the precolumn with eluent A and, after injecting the 100 l butter serum sample, wash for fourminutes with eluent A at a flow rate of 0,4 ml/min. The ribonucleosides will be retarded in the precolumn, whilethe remaining constituents of the butter serum will be washed out (se
41、e figure 2).Then couple the precolumn to the separating column and transfer the ribonucleosides in a narrow zone fromthe former to the latter using eluent B. After decoupling the precolumn, separate the ribonucleosides witheluent B while increasing the concentration of eluent C as organic modifier.
42、During the separation, regeneratethe precolumn with eluent A using pump P1.9.3.1 Elution programmeThe ribonucleoside HPLC elution programme for separating and determining adenosine and uridine is specifiedin table 1, the percentages being proportions by volume.Table 1: Elution programmeTime, in minE
43、luent B, Eluent C,Flow rate, in ml/minas a percentage as a percentage0 100 0 1,24 100 0 1,24,11) 100 0 0,57,2) 100 0 0,57,1 100 0 1,414 99 1 1,417 98 2 1,420 95 5 1,420,1 95 5 1,624 94 6 1,634 84 16 1,637 71 29 1,640 65 35 1,61) Valve switched from LOAD to INJECT (see figure 2).2) Valve switched bac
44、k from INJECT to LOAD.Page 6DIN 10474 : 2003-0210 Evaluation10.1 Calculation of water content(See DIN EN ISO 3727-1)Calculate the water content of butter, wm, in duplicate, as a percentage by mass, using equation (1):wm= ()()()023142mmmmmm. 100(1)wherem0is the mass of the dish prepared, in g;m1is th
45、e mass of the dish used for the blank test before drying, in g;m2is the mass of the dish including the sample before drying, in g;m3is the mass of the dish used for the blank test after drying, in g;m4is the mass of the dish including the sample after drying, in g.Report the arithmetic mean of the i
46、ndividual results obtained, wm, to one decimal place as the result if therepeatability limit specified in DIN EN ISO 3727-1 is not exceeded.10.2 Calculation of citric acid contentDetermine the citric acid content of butter, wC, in a given volume of butter serum (V2) in duplicate by measuringthe chan
47、ge in absorbance, DEC, by reduction of the coenzyme NAD+during enzymatic reaction of the citric acidas in DIN 10325. Calculate the change in absorbance as follows:DEC= (E1 E2)test solution (E1 E2)blank(2)Calculate the citric acid content, wC, in mg/100 g of butter using equation (3).wC= 000121.VdFwV
48、MEmCCD(3)whereDECis the change in absorbance due to the cleavage of citric acid or oxidation of the cleavage product,oxalacetic acid;MCis the molar mass of anhydrous citric acid (here, MC= 192,1 g/mol);V1is the volume of test mixture, in ml;wmis the water content2) of the butter sample as calculated
49、 in subclause 10.1, expressed as a percentage;F is a dilution factor (here, F = 10);e is the molar absorptivity of NADH, in l/mmol.cm (6,3 at 340 nm, 3,4 at Hg 365 nm, and 6,18 at Hg334 nm);d is the path length of the measuring cell, in cm (here, d = 1 cm);V2is the volume of the test solution in the test mixture, in ml.Report the arithmetic mean of the individual results, wC, to one decimal place as the result.10.3 Calculation of nucleoside content10.3.1 GeneralDetermine the adenosine content, cAdo, and uridine
