1、BRITISH STANDARD BS4401-11: 1995 ISO3496:1994 Methods of test for Meat and meat products Part11: Determination of hydroxyproline contentBS4401-11:1995 This British Standard, having been prepared under the directionof the Consumer Products and Services Sector Board, was published under theauthority o
2、f the Standards Board and comes into effect on 15 February1995 BSI03-1999 First published February1979 Second edition February1995 The following BSI references relate to the work on this standard: Committee reference AW/6 Draft for comment92/57397DC ISBN0 580 23750 8 Committees responsible for this
3、British Standard The preparation of this British Standard was entrusted by the Consumer Products and Services Sector Board to Technical Committee AW/6, upon which the following bodies were represented: Association of Public Analysts British Food Manufacturing Industries Research Association British
4、Meat Manufacturers Association British Poultry Federation Ltd. Department of Trade and Industry (Laboratory of the Government Chemist) Meat and Livestock Commission Ministry of Agriculture, Fisheries and Food Royal Society of Chemistry Worshipful Company of Butchers Amendments issued since publicati
5、on Amd. No. Date CommentsBS4401-11:1995 BSI 03-1999 i Contents Page Committees responsible Inside front cover National foreword ii 1 Scope 1 2 Definition 1 3 Principle 1 4 Reagents 1 5 Apparatus 1 6 Sampling 2 7 Preparation of test sample 2 8 Procedure 2 9 Calculation 3 10 Precision 3 11 Test report
6、 3 Annex A (informative) Bibliography 4 List of references Inside back coverBS4401-11:1995 ii BSI 03-1999 National foreword This Part of BS4401 has been prepared under the direction of the Consumer Products and Services Sector Board. It is identical with ISO3496:1994 Meat and meat products Determina
7、tion of hydroxyproline content, published by the International Organization for Standardization (ISO), and in the preparation of which the United Kingdom played a full part. This Part of BS4401 supersedes BS4401-11:1979 which is withdrawn. A British Standard does not purport to include all the neces
8、sary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. Cross-references International standard Corresponding British Standard ISO3100-1:1991 BS5348 Methods
9、for sampling meat and meat products Part1:1991 Taking primary samples (Identical) ISO5725:1986 BS5497 Precision of test methods Part1:1987 Guide for the determination of repeatability and reproducibility for a standard test method by inter-laboratory tests (Identical) Summary of pages This document
10、comprises a front cover, an inside front cover, pages i and ii, pages1 to4, an inside back cover and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table on theinside front cover.ISO3496:1994(E) BSI
11、03-1999 1 1 Scope This International Standard specifies a method for the determination of the hydroxyproline content of all kinds of meat and meat products, including poultry. It is applicable to meat and meat products containing less than0,5%(m/m) hydroxyproline. 2 Definition For the purposes of th
12、is International Standard, the following definition applies. 2.1 hydroxyproline content of meat and meat products content of hydroxyproline determined according to the procedure specified in this International Standard the content is expressed as a percentage by mass 3 Principle Hydrolysis of a test
13、 portion in sulfuric acid at105 C. Filtration and dilution of the hydrolysate. Oxidation of the hydroxyproline by chloramine-T, followed by the formation of a red compound with p-dimethylaminobenzaldehyde. Photometric measurement at a wavelength of558nm. 4 Reagents Use only reagents of recognized an
14、alytical quality and distilled or demineralized water or water of equivalent purity. 4.1 Sulfuric acid solution, c(H 2 SO 4 ) . 3mol/l Add750ml of water to a2 litre one-mark volumetric flask. Add slowly, with agitation,320ml of concentrated sulfuric acid ( 20 =1,84g/ml). Cool to room temperature and
15、 make up to the mark with water. 4.2 Buffer solution, pH=6,8, consisting of: 26,0g of citric acid monohydrate (C 6 H 8 O 7 .H 2 O); 14,0g of sodium hydroxide; 78,0g of anhydrous sodium acetate Na(CH 3 CO 2 ). Dissolve the reagents in500ml of water and transfer quantitatively into a1litre one-mark vo
16、lumetric flask. Add250ml of propan-1-ol and make up to the mark with water. When stored at4 C in the dark, this solution is stable for several weeks. 4.3 Chloramine-T reagent Dissolve1,41g of sodium N-chloro-p-toluene-sulfonamide trihydrate (chloramine-T) in100ml of the buffer solution (4.2). Prepar
17、e this solution immediately before use. 4.4 Colour reagent Dissolve10,0g of p-dimethylaminobenzaldehyde in35ml of perchloric acid solution60%(m/m) and then slowly add65ml of propan-2-ol. Prepare this solution on the day of use. If purification of p-dimethylaminobenzaldehyde is necessary (see note3 i
18、n8.4), proceed as follows. Prepare a saturated solution of p-dimethylaminobenzaldehyde in hot70%(V/V) ethanol. Cool, first at room temperature and finally in a refrigerator. After about12h, filter on a Buchner funnel. Wash with a little70%(V/V) ethanol. Again dissolve in hot 70%(V/V) ethanol. Add co
19、ld water and agitate thoroughly. Repeat this procedure until a sufficient quantity of milk-white crystals has been formed. Place in the refrigerator overnight. Filter on the Buchner funnel. Wash with50%(V/V) ethanol and vacuum dry over phosphorus(V) oxide. 4.5 Hydroxyproline, standard solutions Prep
20、are a stock solution by dissolving50mg of4-hydroxypyrrolidine- -carbonic acid (hydroxyproline) in water in a100ml one-mark volumetric flask. Add1 drop of the sulfuric acid solution (4.1) and make up to the mark with water. This solution is stable for at least1 month at4 C. On the day of use, transfe
21、r5ml of the stock solution to a500ml one-mark volumetric flask and make up to the mark with water. Then prepare four standard solutions by diluting10ml, 20ml, 30ml and40ml of this solution to100ml with water to obtain hydroxyproline concentrations of 0,54g/ml, 14g/ml, 1,54g/ml and24g/ml respectively
22、. 5 Apparatus Usual laboratory apparatus and, in particular, the following. 5.1 Electric meat mincer, with horizontal blades, capable of rotating at high speed. 5.2 Round- or flat-bottomed hydrolysis flasks, of capacity about200ml, wide-necked. 5.3 Drying oven, capable of being operated at105 C 1 C
23、5.4 Filter paper discs, of diameter12,5cm. 5.5 pH-meter. 5.6 Aluminium or opaque plastic foil. 5.7 Water bath, capable of being maintained at60 C 0,5 C. 5.8 Spectrometer, suitable for use at a wavelength of558nm 2nm, or a photoelectric colorimeter with an interference filter with an absorption maxim
24、um at558nm 2nm. 5.9 Glass cells, of10mm optical path length.ISO3496:1994(E) 2 BSI 03-1999 5.10 Analytical balance, capable of weighing to an accuracy of 0,001 g. 5.11 Volumetric flasks, of capacity250ml. 5.12 Watch glasses, of diameter5cm to6cm. 6 Sampling It is important that the laboratory receive
25、 a sample which is truly representative and has not been damaged or changed during transport or storage. Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO3100-1. Proceed from a representative sample of at least200g. Store the s
26、ample in such a way that deterioration and change in composition are prevented. 7 Preparation of test sample 7.1 Raw meat and meat products Reduce intact meat into small cubes (approx. 0,5cm 3 , i.e.sides of length approx.8mm) by cutting it while it is cold (just below0 C), using a sharp knife. Eith
27、er place the sample in a container and seal the latter hermetically, or vacuum-pack the sample in heat-resistant plastic film. Then heat the container and sample so as to maintain a temperature of atleast70 C for at least30min. Cool and proceed as in7.2. During the remaining stages of preparation of
28、 the test sample and the weighing out of the test portions, ensure that the sample is kept well mixed and, in particular, that any fat or fluid is kept evenly distributed. NOTE 1The heat treatment softens the raw connective tissue and makes it less resistant to homogenization by mincing. However, it
29、 may also lead to separation of a fluid containing gelatine. The presence of fat may also demand special attention for the production of a homogeneous test sample. 7.2 Cooked meat and cooked meat products Homogenize the sample in the meat mincer (5.1). Keep the homogenized sample in a completely fil
30、led, air-tight, closed container and store it in such a way that deterioration and change in composition are prevented. Analyse the test sample as soon as possible, but always within24h. 8 Procedure 8.1 Test portion Weigh, to the nearest0,001g, about4g of the test sample into a hydrolysis flask (5.2
31、). Ensure that no sample material adheres to the side walls of the flask. 8.2 Hydrolysis 8.2.1 Add30ml 1ml of sulfuric acid solution(4.1) to the flask. Cover the flask with a watch glass (5.12) and place in the oven (5.3) for16h (conveniently, overnight) at105 C. 8.2.2 Filter the hot hydrolysate thr
32、ough filter paper(5.4) into a250ml one-mark volumetric flask(5.11). Wash the flask and filter paper three times with10ml portions of hot sulfuric acid solution(4.1) and add the washings to the hydrolysate. Make up to the mark with water and mix. 8.3 Colour development and measurement of absorbance 8
33、.3.1 Using a pipette, transfer to a one-mark250ml volumetric flask (5.11) a volume (V) of the hydrolysate (8.2.2) so that, after dilution to 250ml, the hydroxyproline concentration will be within the range0,54g/ml to24g/ml. Make up to the mark with water. NOTE 2In most cases, V will be in the order
34、of5ml to25ml depending on the amount of connective tissue present in the sample. 8.3.2 Transfer4,00ml of this solution(8.3.1) to a test tube and add 2,00ml of chloramine-T reagent(4.3). Mix and leave at room temperature for20min 1min. 8.3.3 Add2,00ml of the colour reagent(4.4), mix thoroughly and ca
35、p the tube with aluminium or plastic foil(5.6). 8.3.4 Transfer the tube quickly into the water bath(5.7), set at60 C, and heat for exactly20min. 8.3.5 Cool the tube under running tap water for atleast3min and leave at room temperature for30min. 8.3.6 Measure the absorbance at558nm 2nm in a glass cel
36、l(5.9) against water, using the spectrometer or the photoelectric colorimeter equipped with an interference filter(5.8). 8.3.7 Subtract the absorbance measured for the blank solution(8.4) and read the hydroxyproline concentration of the diluted hydrolysate from the calibration graph obtained as desc
37、ribed in8.5. 8.4 Blank test Carry out in duplicate the procedure described in8.3.2 to8.3.7 inclusive, substituting water for the diluted hydrolysate. NOTE 3If the absorbance of the blank exceeds0,040, a fresh colour reagent(4.4) should be prepared and, if necessary, the p-dimethylaminobenzaldehyde s
38、hould be purified (see note1 in4.4).ISO3496:1994(E) BSI 03-1999 3 8.5 Calibration graph 8.5.1 Carry out the procedure described in8.3.2 to8.3.7 inclusive, substituting in turn4,00ml of each of the four diluted standard hydroxyproline solutions(4.5) for the diluted hydrolysate. 8.5.2 Plot the measure
39、d absorbance values, corrected for the blank value, against the concentrations of the standard hydroxyproline solutions. Construct the best-fitting straight line through the plotted points and the origin. Prepare a new calibration graph for each series of analyses. 9 Calculation For each test portio
40、n, calculate the hydroxyproline content, as a percentage by mass, from the formula where Report the result to the nearest0,01%. 10 Precision The precision of this method has been established by an international interlaboratory test carried out in accordance with ISO5725. For the values obtained for
41、repeatability and reproducibility, a probability level of95% holds. 10.1 Repeatability The absolute difference between two independent test results, obtained using the same method on identical test material in the same laboratory by the same operator using the same equipment within a short interval
42、of time, should not be greater than the value of r given by the formula: where is the mean of the two test results for the hydroxyproline content, expressed as a percentage by mass. Reject both results if the difference exceeds the above value and carry out two new single determinations. 10.2 Reprod
43、ucibility The absolute difference between two single test results, obtained using the same method on identical test material in different laboratories with different operators using different equipment, should not be greater than the value of R given by the formula: where is the mean of the two test
44、 results for the hydroxyproline content, expressed as a percentage by mass. 11 Test report The test report shall show the method in accordance with which sampling was carried out, if known, the method used, the test result obtained, and if the repeatability has been checked, the final quoted result
45、obtained. It shall also mention any operating conditions not specified in this International Standard, or regarded as optional, as well as any circumstances that may have influenced the test result. The report shall include all details necessary for the complete identification of the sample. w h is
46、the hydroxyproline content, expressed as a percentage by mass, obtained from the formula; c is the hydroxyproline concentration, in micrograms per millilitre, of the diluted hydrolysate as read from the calibration graph; m is the mass, in grams, of the test portion(8.1); V is the volume, in millili
47、tres, of the aliquot portion of the hydrolysate taken for dilution to250ml (see8.3.1). wh6,25c mV - = r 0,032 1 0,032 2 w h + = wh R 0,019 5 0,052 9 wh + = whISO3496:1994(E) 4 BSI 03-1999 Annex A (informative) Bibliography 1 ISO3100-1:1991, Meat and meat products Sampling and preparation of test sam
48、ples Part1:Sampling. 2 ISO5725:1986, Precision of test methods Determination of repeatability and reproducibility for a standard test method by inter-laboratory tests.BS4401-11:1995 BSI 03-1999 List of references See national foreword.BS4401-11: 1995 ISO3496:1994 BSI 389 Chiswick High Road London W4
49、 4AL BSIBritishStandardsInstitution BSI is the independent national body responsible for preparing BritishStandards. It presents the UK view on standards in Europe and at the international level. It is incorporated by Royal Charter. Revisions BritishStandards are updated by amendment or revision. Users of BritishStandards should make sure that they possess the latest amendments or editions. It is the constant aim of BSI to improve the quality of our products and services. We would be grateful if anyone findi
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