1、November 2007DEUTSCHE NORM English price group 10No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 67.140.10!$JA1“139301
2、4www.din.deDDIN ISO 14502-1Determination of substances characteristic of green and black tea Part 1: Content of total polyphenols in tea Colorimetric method using Folin-Ciocalteu reagent (ISO 14502-1:2005 +Corrigendum 1:2006)English version of DIN ISO 14502-1:2007-11Bestimmung von charakteristischen
3、 Substanzen von grnem und schwarzem Tee Teil 1: Gesamt-Polyphenolgehalt in Tee Colorimetrisches Verfahren mit Folin-Ciocalteu-Reagenz (ISO 14502-1:2005 +Corrigendum 1:2006)Englische Fassung DIN ISO 14502-1:2007-11www.beuth.deDocument comprises 13 pagesDIN ISO 14502-1:2007-11 2 Contents Page National
4、 foreword 3 1 Scope .4 2 Normative references 4 3 Principle4 4 Reagents.4 5 Apparatus .6 6 Sampling.6 7 Preparation of test samples6 8 Procedure .7 8.1 General7 8.2 Determination of dry matter content7 8.3 Test portion 7 8.4 Extraction of polyphenols.7 8.5 Dilution8 8.6 Determination.8 9 Calculation
5、8 10 Precision.9 10.1 Interlaboratory test 9 10.2 Repeatability.9 10.3 Reproducibility.9 11 Test report 10 Annex A (informative) Gallic acid calibration graph 11 Annex B (informative) Results of interlaboratory test .12 Bibliography 13 DIN ISO 14502-1:2007-11 3 National foreword This standard has be
6、en prepared by Technical Committee ISO/TC 34 “Food products”, Subcommittee SC 8 “Tea” (Secretariat: BSI, United Kingdom). The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und land-wirtschaftliche Produkte (Food and Agricultural Products Standards Committee
7、), Technical Committee Tee. Technical Corrigendum 1:2006 to ISO 14502-1:2005 concerning an error in the equation for the graph given in Figure A.1 has been taken into account in this standard. Attention is drawn to the possibility that some of the elements of this document may be the subject of pate
8、nt rights. DIN shall not be held responsible for identifying any or all such patent rights. The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 3696 DIN ISO 3696 ISO 5725-2 DIN ISO 5725-2 National Annex NA (informative) Bibliography DIN ISO
9、 3696, Water for analytical laboratory use Specification and test methods DIN ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method 1 Scope This part of ISO 145
10、02 specifies a method for the determination of the total polyphenol content of leaf tea and instant tea by a colorimetric assay using Folin-Ciocalteu phenol reagent 4. It is applicable to both green and black tea products. 2 Normative references The following referenced documents are indispensable f
11、or 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 1572, Tea Preparation of ground sample of known dry matter content ISO 3696:1987, Water for analyti
12、cal laboratory use Specification and test methods ISO 7513, Instant tea in solid form Determination of moisture content (loss in mass at 103 C) 3 Principle Polyphenols are extracted with 70 % methanol from a test portion of finely ground leaf tea at 70 C. Instant teas are dissolved in hot water with
13、 10 % acetonitrile (volume fraction) added to stabilize the extract. The polyphenols in the extract are determined colorimetrically using Folin-Ciocalteu phenol reagent. The reagent contains phospho-tungstic acids as oxidants, which on reduction by readily oxidized phenolic hydroxy groups yield a bl
14、ue colour with a broad maximum absorption at 765 nm. This is due to the formation of so-called tungsten and molybdenum blues. The Folin-Ciocalteu phenol reagent reacts with a wide range of polyphenol compounds and, although the response can vary with the individual components, selection of gallic ac
15、id as a calibration standard enables useful total polyphenol data to be obtained. 4 Reagents Use only reagents of recognized analytical grade, unless otherwise specified. 4.1 Water, conforming to grade 1 of ISO 3696:1987. 4.2 Acetonitrile. 4.3 Methanol. DIN ISO 14502-1:2007-114Determination of subst
16、ances characteristic of green and black tea Part 1: Content of total polyphenols in tea Colorimetric method using Folin-Ciocalteu reagent 4.4 Methanol/water extraction mixture, 70 % methanol (volume fraction). Add 700 ml of the methanol (4.3) to a 1 litre one-mark volumetric flask. Dilute to the mar
17、k with water (4.1) and mix. 4.5 Folin-Ciocalteu phenol reagent, commercially available ready prepared. It is advisable to check the calibration linearity with respect to gallic acid in order to ascertain the suitability of the supplied reagent. 4.6 Dilute Folin-Ciocalteu phenol reagent, 10 % (volume
18、 fraction). Using a pipette, transfer 20 ml of Folin-Ciocalteu phenol reagent (4.5) to a 200 ml one-mark volumetric flask. Dilute to the mark with water and mix. Prepare fresh reagent solution daily. To avoid contamination of the concentrated Folin-Ciocalteu reagent, discard any unused dispensed rea
19、gent. 4.7 Sodium carbonate solution, 7,5 % (mass concentration). Weigh (37,50 0,01) g of anhydrous sodium carbonate (Na2CO3) into a 500 ml one-mark volumetric flask. Add sufficient warm water to half-fill the flask. Swirl to dissolve the sodium carbonate, cool to room temperature, dilute to the mark
20、 with water and mix. NOTE This solution will remain stable at room temperature for up to 1 month. 4.8 Gallic acid stock standard solution, corresponding to approximately 1 000 g/ml of anhydrous gallic acid. Weigh (0,110 0,001) g of gallic acid monohydrate (M = 188,14) into a 100 ml one-mark volumetr
21、ic flask. Dissolve in water, dilute to the mark and mix. Prepare a fresh standard solution daily. NOTE Gallic acid monohydrate is preferred over anhydrous, due to its greater solubility, reduced hygroscopic properties and availability of certified reagent grades, e.g. A.C.S., which is used to denote
22、 chemicals that meet specifications outlined by the American Chemical Society. If not known, the moisture content (loss in mass at 103 C) on a portion of the standard material should be determined. The concentration of the stock standard solution on a gallic acid anhydrous basis can then be calculat
23、ed. 4.9 Gallic acid standard solutions A to E Using pipettes, transfer the volumes of gallic acid stock standard solution (4.8) given in Table 1 to 100-ml one-mark volumetric flasks. Dilute to the mark with water and mix. These dilute standard solutions should be prepared on the day of use. Table 1
24、Gallic acid dilute standard solutions Gallic acid standard solution Volume of gallic acid stock solution Nominal concentration of dilute standard ml g/ml A 1,0 10 B 2,0 20 C 3,0 30 D 4,0 40 E 5,0 50 DIN ISO 14502-1:2007-1155 Apparatus Usual laboratory apparatus and, in particular, the following. 5.1
25、 Analytical balance, capable of weighing to an accuracy of 0,001 g. 5.2 Water bath, capable of being maintained at 70 C 1 C. 5.3 Dispenser, for methanol/water extraction mixture (4.4), and set at 5,0 ml. 5.4 Centrifuge, capable of 2 000 Relative Centrifugal Force (R.C.F.) (typically 3 500 r/min). 5.
26、5 Spectrophotometer, set at 765 nm and able to accommodate 10 mm path length cells, preferably in an automatic flow cell assembly. 5.6 Pipettes, glass or automatic, to cover the volume range for standard and sample extract dilutions. 5.7 One-mark volumetric flasks, of capacities 100 ml, 200 ml, 500
27、ml, and 1 litre. 5.8 Vortex mixer, for efficient mixing during extraction. 5.9 Extraction tubes, glass, of 10 ml capacity, stoppered and able to withstand being centrifuged. 5.10 Graduated tubes, glass, of 10 ml capacity with 0,1 ml graduations. As the assay is sensitive to traces of organic impurit
28、ies, extraction tubes (5.9) and graduated tubes (5.10) should all be taken through an additional cleaning procedure of washing in approx. 15 % (volume fraction) nitric acid, followed by rinsing thoroughly in water and drying in an air oven at 100 C. The use of disposable plastic tubes as an alternat
29、ive to the graduated tubes in the final colorimetic assay is recommended, as additional cleaning has not been found necessary. 6 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 t
30、he method specified in this part of ISO 14502. A recommended sampling method is given in ISO 1839 for leaf tea, and ISO 7516 for instant tea. 7 Preparation of test samples To ensure homogeneity, grind the sample of leaf tea in accordance with ISO 1572 and store samples in well-sealed containers prot
31、ected from light. Grinding of instant tea is only required on samples of a coarse granular structure. DIN ISO 14502-1:2007-1168 Procedure 8.1 General If it is required to check whether the repeatability limit (10.2) is met, carry out two single determinations in accordance with 8.2 to 8.6 under repe
32、atability conditions. 8.2 Determination of dry matter content Calculate the dry matter content from the moisture content (loss in mass at 103 C) determined on a portion of the test sample (Clause 7) in accordance with ISO 1572 for leaf tea, or ISO 7513 for instant tea. 8.3 Test portion 8.3.1 Instant
33、 tea Weigh (0,500 0,001) g of the test sample (Clause 7) into a 50 ml one-mark volumetric flask. 8.3.2 Leaf tea Weigh (0,200 0,001) g of the test sample (Clause 7) into an extraction tube (5.9). 8.4 Extraction of polyphenols 8.4.1 Instant tea 8.4.1.1 Add to the instant tea in the flask (8.3.1) appro
34、ximately 25 ml of hot water (maximum temperature 60 C). Mix to dissolve the sample and allow to cool to room temperature. 8.4.1.2 Add 5,0 ml of acetonitrile (4.2). Dilute to the mark with water and mix. 8.4.2 Leaf tea 8.4.2.1 Place the methanol/water extraction mixture (4.4) contained in the dispens
35、er (5.3) in the water bath (5.2) set at 70 C, and allow at least 30 min for the extraction mixture to equilibrate. 8.4.2.2 Place the extraction tube containing the sample (8.3.2) in the water bath set at 70 C. Dispense 5,0 ml of hot methanol/water extraction mixture from 8.4.2.1 into the extraction
36、tube, stopper and mix on the vortex mixer (5.8). 8.4.2.3 Continue heating the extraction tube in the water bath for 10 min, mixing on the vortex mixer after 5 min and 10 min. It is important to mix the samples thoroughly to ensure complete extraction. 8.4.2.4 Remove the extraction tube from the wate
37、r bath and allow it to cool to room temperature. Remove the stopper and place the tube in the centrifuge (5.4) at 3 500 r/min for 10 min. 8.4.2.5 Carefully decant the supernatant into a graduated tube (5.10). 8.4.2.6 Repeat extraction steps 8.4.2.2 to 8.4.2.5. Combine the extracts, make up to 10 ml
38、with cold methanol/ water extraction mixture (4.4) and mix the contents. DIN ISO 14502-1:2007-1178.4.2.7 The extract from 8.4.2.6 is stable for at least 24 h if stored at 4 C. Allow the extract to attain room temperature before proceeding with the assay. Resuspension of the small amount of particula
39、te material that may settle during storage is not necessary. 8.5 Dilution Using a pipette, transfer 1,0 ml of the sample extract (instant tea extract from 8.4.1.2 or leaf tea extract from 8.4.2.6) into a one-mark 100 ml volumetric flask. Dilute to the mark with water and mix. 8.6 Determination 8.6.1
40、 Using a pipette, transfer 1,0 ml of the gallic acid standard solutions A, B, C, D and E (4.9), in duplicate, into separate plastic or graduated tubes (5.10). NOTE These correspond to approximately 10 g, 20 g, 30 g, 40 g and 50 g of anhydrous gallic acid. 8.6.2 Using a pipette, transfer 1,0 ml of wa
41、ter, in duplicate, into separate tubes (5.10). These are reagent blanks. 8.6.3 Using a pipette, transfer 1,0 ml of diluted sample extract (8.5), in duplicate, into separate tubes. 8.6.4 Using a pipette, add 5,0 ml of dilute Folin-Ciocalteu phenol reagent (4.6) into each tube and mix. 8.6.5 Within 3
42、min to 8 min after the addition of the dilute Folin-Ciocalteu phenol reagent, pipette 4,0 ml of sodium carbonate solution (4.7) into each tube. Stopper and mix. 8.6.6 Allow to stand at room temperature for 60 min, and then measure the optical densities in 10-mm path length cells against water on the
43、 spectrophotometer (5.5) set at 765 nm. 8.6.7 The reagent blank (8.6.2) should have an optical density of 0,04 should be investigated. For example, check the gallic acid moisture content, standard preparation or pipette calibration. The total polyphenol content, wT, expressed as a percentage by mass
44、 on a sample dry matter basis, is given by the formula: ()sample intercept sampleTstd sample DM, sample10010 000DD VdwSm w=where Dsampleis the optical density obtained for the sample test solution; Dinterceptis the optical density at the point the best-fit linear calibration line intercepts the y-ax
45、is; Sstdis the slope obtained from the best-fit linear calibration; msampleis the mass, in grams, of the sample test portion; Vsampleis the sample extraction volume, in millilitres (50 ml for instant tea and 10 ml for leaf tea); d is the dilution factor used prior to the colorimetric determination (
46、typically 1,0 ml to 100 ml, thus a dilution factor of 100); wDM, sampleis the dry matter content, expressed as a mass fraction in percent, of the test sample, determined in accordance with 8.2. 10 Precision 10.1 Interlaboratory test Details of the interlaboratory test to determine the precision of t
47、he method are summarized in Annex B. The values derived from this interlaboratory test may not be applicable to concentration ranges and matrices other than those given. 10.2 Repeatability The absolute difference between two independent single test results, obtained using the same method on identica
48、l test material in the same laboratory by the same operator using the same equipment within a short interval of time, will in not more than 5 % of cases be greater than the repeatability limit values, r, given in Table B.1. 10.3 Reproducibility The absolute difference between two single test results
49、, obtained using the same method on identical test material in different laboratories with different operators using different equipment, will in not more than 5 % of cases be greater than the reproducibility limit values, R, given in Table B.1. DIN ISO 14502-1:2007-11911 Test report The test report shall specify: all information necessary for the complete identification of the sample; the sampling method used, if known; the test method used, with reference to this part of ISO 14502; all operating details not speci
