1、Ref. No. DIN 10754 : 2002-08English price group 06 Sales No. 010602.03DEUTSCHE NORM August 200210754Continued on pages 2 to 4. 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
2、 exclusive right of sale for German Standards (DIN-Normen).Determination of proline content of honeyTranslation by DIN-Sprachendienst.In case of doubt, the German-language original should be consulted as the authoritative text.ICS 67.180.10Untersuchung von Honig Bestimmung des ProlingehaltesIn keepi
3、ng with current practice in standards published by the International Organization for Standardization(ISO), a comma has been used throughout as the decimal marker.ForewordThis standard has been prepared by Technical Committee Honiguntersuchung of the NormenausschussLebensmittel und landwirtschaftlic
4、he Produkte (Foodstuffs and Agricultural Products Standards Committee).AmendmentsThis standard differs from the March 1993 edition in that table 1 has been amended and it has been editoriallyrevised and brought in line with current standards practice.Previous editionDIN 10754: 1993-03.1 ScopeThis st
5、andard specifies a method of determining the proline content of honey, which may be used, in additionto other methods, for detecting adulterations.2 ConceptProline contentThe content of constituents of the honey capable of coupling with ninhydrin.3 PrincipleProline forms a coloured complex compound
6、with ninhydrin. After 2-propanol has been added, the prolinecontent is calculated from the ratio of the absorbance maxima of the sample solution and a reference solution.4 Reagents4.1 GeneralUnless otherwise specified, analytical grade reagents shall be used. The water used shall be either distilled
7、or of equivalent purity.The term solution is used in this standard to mean an aqueous solution.Supersedes March 1993edition.Page 2DIN 10754 : 2002-084.2 98 % to 100 % (m/m) formic acid, HCOOH.4.3 1,5 g/50 ml solution of ninhydrin in ethylene glycol monomethyl ether.NOTE: A fresh solution shall be ma
8、de up prior to every analysis.4.4 Proline reference solution4.4.1 40 mg/50 ml proline stock solution.4.4.2 0,8 mg/25 ml proline standard solution, prepared by making up 1 ml of the solution as in subclause4.4.1 to 25 ml.4.5 50 % (V/V) 2-propanol, prepared by mixing one part of 2-propanol with one pa
9、rt of water.5 Apparatus5.1 GeneralIn addition to standard laboratory equipment, the equipment specified in subclauses 5.2 to 5.7 shall be used.5.2 Stainless-steel screen, of aperture size 0,5 mm.5.3 Spectrophotometer, if possible capable of recording for analyses at wavelengths from 500 nm to 520 nm
10、.5.4 Glass cells, with a path length of 1 cm.5.5 Test tubes, with screw cap or ground stopper, of nominal capacity 20 ml.5.6 Volumetric flasks, of nominal capacity 100 ml.5.7 Beaker.6 SamplingPending the publication of a standard on sampling, the sampling procedure shall be agreed upon.A representat
11、ive laboratory sample of not less than 200 g shall be taken for the analysis.7 Procedure7.1 Sample preparation7.1.1 Pure liquid or set honeyAdequately homogenize the laboratory sample by stirring it vigorously for not less than three minutes, taking careto minimize the amount of air included, especi
12、ally if the sample is to be used to determine hydroxymethylfurfural.7.1.2 Impure liquid or set honeyAfter removing coarse impurities, stir the honey at ambient temperature until smooth and pass it through a screen(as in subclause 5.2), using a spatula in the case of set honey.7.1.3 Comb honeyDe-cap
13、the combs if still capped, then separate the honey completely from the combs without heating, usinga screen (as in subclause 5.2).7.2 Preparation of sample solutionWeigh into a small beaker about 5 g of honey to an accuracy of 1 mg, dissolve it in 50 ml of water and transferthe solution quantitative
14、ly to a 100 ml volumetric flask. Then make the solution up to the mark, seal the flask andshake it vigorously.7.3 AnalysisSince the absorbance fluctuates, its mean value for the proline standard solution shall be determined at leastin triplicate for every series of analyses.Pipette 0,5 ml of the sam
15、ple solution into a test tube, 0,5 ml of water into a second test tube (as blank) and 0,5 mlof proline standard solution into each of three test tubes. Add 1 ml of ninhydrin solution and 1 ml of formic acidto each test tube, seal the test tubes and, after mixing vigorously for 15 minutes, place them
16、 in a boiling waterbath, ensuring that the level of water in the bath is higher than that of the liquid in the test tubes.Following that, place the test tubes for ten minutes in a water bath at a temperature of 70 C. During this time,add 5 ml of 2-propanol as in subclause 4.5 to each of the test tub
17、es and immediately reseal them. To developthe colour fully, cool to ambient temperature and determine the maximum absorbance of the sample andstandard solutions 45 minutes after removing the test tubes from the water bath by recording the absorbanceof the solutions in cells as in subclause 5.1 at wa
18、velengths from 500 nm to 520 nm, using the blank as reference.Page 3DIN 10754 : 2002-08The absorbance maximum will be at around 510 nm.Observe the specified times.8 Evaluation8.1 CalculationCalculate the content by mass of proline, wP, in mg/kg of honey, using the following equation:wp= spEE m1 280m
19、whereEpis the absorbance of the sample solution;ESis the mean absorbance of the proline standard solution;m1is the initial sample mass of the proline stock solution, in mg (here, m1= 40 mg);80 is the dilution factor per 1 g of honey;m2is the initial sample mass of honey, in g (here, m2= 5,0 g).8.2 P
20、recision8.2.1 GeneralThe precision data obtained in an interlaboratory test are shown in table 1.NOTE: The interlaboratory test was evaluated as specified in ISO 5725-1 and ISO 5725-2.8.2.2 Repeatability limit(same operator, same equipment)The absolute difference between two successive results obtai
21、ned under repeatability conditions will not exceedthe repeatability limit, r, in more than 5 % of cases. Values of r and the repeatability standard deviation, sr, aregiven in table 1.8.2.3 Reproducibility limit(different operators, different equipment)The absolute difference between two individual r
22、esults obtained under reproducibility conditions will not exceedthe reproducibility limit, R, in more than 5 % of cases. Values of R and of the reproducibility standard deviation,sR, are given in table 1.Table 1: Results of interlaboratory testing (values given in mg/kg)Type of honey Acacia honey Yu
23、catan honey Oak honeyMean, x 175 293 762Repeatability limit, r 10,3 12,5 24,4Repeatability standard deviation, sr3,6 4,4 8,6Reproducibility limit, R 38,2 35,7 58,4Reproducibility standard deviation, sR13,5 12,6 20,79 Test reportThe test report shall refer to this standard and include the following i
24、nformation:a) type, origin and description of sample;b) type of sampling and date;c) dates of receipt and analysis;d) proline content, in mg/kg of sample, to the nearest integer;e) reasons for any deviation from this standard.Page 4DIN 10754 : 2002-08Normative referencesISO 5725-1 : 1994 Accuracy (t
25、rueness and precision) of measurement methods and results Part 1: Generalprinciples and definitionsISO 5725-2 : 1994 Accuracy (trueness and precision) of measurement methods and results Part 2: Basicmethod for the determination of repeatability and reproducibility of a standard measure-ment method.BibliographyMartin, P. G., Manuals of Food Quality Control 3. CommoditiesRomse., Food and Agriculture Organization of the United Nations, 1979, p. 162 (FAO Food and Nutrition Paper,14/3).Ough, C. S., Rapid determination of proline in grapes and wines, in: Journal of Food Science 1969: 34,pp. 228230.
