1、BRITISH STANDARD BS 6248-6: 1982 Caseins and caseinates Part 6: Method for determination of lactose content (photometric method) NOTEThis Part should be read in conjunction with Part 1 “General introduction, including preparation of laboratory samples”, published separately. UDC 637.147.2.044:543.42
2、:547.458.227.3BS6248-6:1982 This British Standard, having been prepared under the directionof the Dairying Standards Committee, was published under the authority ofthe Board of BSI and comesintoeffect on 30 June 1982 BSI 12-1999 The following BSI references relate to the work on this standard: Commi
3、ttee reference DAC/3 Draft for comment 79/53463 DC ISBN 0 580 12750 8 Foreword This Part of BS 6248, which has been prepared under the direction of the Dairying Standards Committee, is related to ISO 5548:1980 “Caseins and caseinates Determination of lactose content Photometric method”, prepared by
4、ISO/TC 34, Agricultural food products, of the International Organization for Standardization (ISO) in cooperation with the International Dairy Federation (IDF) and the Association of Official Analytical Chemists (AOAC). This British Standard differs from ISO 5548 principally in its inclusion of safe
5、ty warnings in 5.6 and 8.5.1. However, for ease of reproduction the ISO text has been used, with changes made as appropriate. Some terminology and certain conventions are not identical with those used in British Standards; attention is especially drawn to the following. The comma has been used throu
6、ghout as a decimal marker. In British Standards it is current practice to use a full point on the baseline as the decimal marker. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compl
7、iance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, pagesi andii, pages1 to4, an inside back cover and a back cover. This standard has been updated (see copyright date) and may have ha
8、d amendments incorporated. This will be indicated in the amendment table on the inside front cover. Amendments issued since publication Amd. No. Date of issue CommentsBS6248-6:1982 BSI 12-1999 i Contents Page Foreword Inside front cover 1 Scope and field of application 1 2 References 1 3 Definition
9、1 4 Principle 1 5 Reagents 1 6 Apparatus 1 7 Sampling 1 8 Procedure 1 9 Expression of results 3 10 Test report 3 Publications referred to Inside back coverii blankBS6248-6:1982 BSI 12-1999 1 1 Scope and field of application This British Standard describes a photometric method for the determination o
10、f the lactose and other soluble carbohydrates content of caseins and caseinates containing less than 2.0 % of total soluble carbohydrates. 2 References The publications referred to in this standard are listed on the inside back cover. 3 Definition lactose content of caseins and caseinates the conten
11、t of total soluble carbohydrates, expressed as anhydrous lactose as a percentage by mass, determined by the procedure described in this British Standard 4 Principle Dissolution of a test portion a) in hot water in the case of caseinates; b) in hot water with the addition of sodium hydrogen carbonate
12、 in the case of acid casein; c) in hot water with the addition of pentasodium triphosphate in the case of rennet casein. Precipitation of the casein with acetic acid and sodium acetate solution at pH 4,6, followed by filtration to obtain a protein-free solution of the carbohydrates. Addition of phen
13、ol solution and concentrated sulphuric acid to an aliquot portion of the filtrate, thus producing a colour which is proportional to the amount of carbohydrate present, and photometric measurement at a wavelength of490 nm. 5 Reagents All reagents shall be of recognized analytical quality. The water u
14、sed shall be distilled water or water of at least equivalent purity. NOTEWater complying with BS 3978 is suitable. 5.1 Sodium hydrogen carbonate (NaHCO 3 ) (for analysis of acid casein). 5.2 Pentasodium triphosphate (Na 5 P 3 O 10 ) (for analysis of rennet casein). 5.3 Hydrochloric acid or sulphuric
15、 acid, c(HCl) or c(1/2 H 2 SO 4 ) = 0,1 mol/l. 5.4 Acetic acid, 100 g/l solution. 5.5 Sodium acetate solution, c(CH 3 COONa) =1 mol/l. 5.6 Phenol, 80 % (m/m) solution. Heat a mixture of 8 g of phenol and 2 g of water until the mixture is homogeneous. WARNING NOTE. Appropriate precautions to protect
16、the eyes and skin should be taken. 5.7 Sulphuric acid, concentrated, ( 201,84 g/ml). 5.8 Lactose, 20 g/l solution. Weigh 2,105 0,001 g of lactose monohydrate, corresponding to 2,00 g of anhydrous lactose, into a100 ml volumetric flask, dissolve in water, makeup to volume and mix well. Store the solu
17、tion at 0 C. 6 Apparatus Usual laboratory equipment, and in particular: 6.1 Analytical balance 6.2 Conical flasks, of capacity 100 ml. 6.3 One-mark pipettes, of capacity 1, 2 and 10 ml. 6.4 Micropipettes, of capacity 0,2 ml, with 0,001 ml divisions. 6.5 Graduated pipettes, of capacity 25 ml. 6.6 Tes
18、t tubes, of capacity about 40 ml, with ground necks and fitted with ground glass stoppers. 6.7 Automatic dispenser, capable of dispensing 5 ml of concentrated sulphuric acid within 1 s. 6.8 Water bath, capable of being controlled at 60 to70 C. 6.9 Photometer, suitable for making measurements at a wa
19、velength of 490 nm, provided with cells of optical path length 1 to 2 cm. 6.10 Mixer, suitable for mixing inside the test tubes(6.6), with a stirrer resistant to strong acid. 6.11 Grinding device, for grinding the laboratory sample, if necessary (see 8.1.4), without development of undue heat and wit
20、hout loss of moisture. A hammer-mill shall not be used. 6.12 Test sieve, wire cloth, diameter 200 mm, nominal size of aperture 500 4m, with receiver, complying with BS 410. 6.13 Volumetric flasks, of capacity 100 ml. 6.14 Water bath, capable of being controlled at20 C. 7 Sampling See BS 809 and BS 6
21、248-1. 8 Procedure 8.1 Preparation of the test sample 8.1.1 Thoroughly mix the laboratory sample by repeatedly shaking and inverting the container (if necessary, after having transferred all of the laboratory sample to an air-tight container of sufficient capacity to allow this operation to be carri
22、ed out).BS6248-6:1982 2 BSI 12-1999 8.1.2 Transfer about 50 g of the thoroughly mixed laboratory sample to the test sieve (6.12). 8.1.3 If the 50 g portion passes completely or almost completely through the sieve, use for the determination the sample prepared in 8.1.1. 8.1.4 Otherwise, grind the 50
23、g portion, using the grinding device (6.11), until it passes through the sieve. Immediately transfer all the sieved sample to an air-tight container of sufficient capacity and mix thoroughly by repeatedly shaking and inverting. During these operations, take precautions to avoid any change in the wat
24、er content of the product. 8.1.5 After the test sample has been prepared, carry out the determination (8.5) as soon as possible. 8.2 Preparation of a blank solution Prepare a blank solution containing 0,1 0,001 g of sodium hydrogen carbonate or 0,1 0,001 g of pentasodium triphosphate, as appropriate
25、, using the same apparatus, the same reagents in the sameamounts, and the same procedure as described in 8.4.2 to 8.5.1 inclusive, but omitting the test portion and omitting those operations in connection with the presence of a test portion. NOTEFor the most accurate results, prepare the blank solut
26、ion, the test solution and the standard solutions for the calibration graph (see 8.6) simultaneously. 8.3 Test portion Weigh, to the nearest 1 mg, about 1 g of the test sample (8.1) into a conical flask (6.2). 8.4 Test solution 8.4.1 In the case of acid casein, add 0,1 0,001 g of the sodium hydrogen
27、 carbonate (5.1). In the case of rennet casein, add 0,1 0,001 g of the pentasodium triphosphate (5.2). 8.4.2 Add 25 ml of water, place in the water bath(6.8), controlled at 60 to 70 C, and mix occasionally by shaking. 8.4.3 When the test portion is completely dissolved generally this takes 10 to 15
28、min cooland add successively: 15 ml of water; 8 ml of the hydrochloric acid or sulphuric acid solution (5.3); 1 ml of the acetic acid solution (5.4). Stopper and mix the contents by shaking after each addition. 8.4.4 Leave for 5 min and then add 1 ml of the sodium acetate solution (5.5). Mix by shak
29、ing. 8.4.5 Allow the casein precipitate to settle, then filter through a dry filter paper. Discard the first few millilitres of the filtrate. 8.5 Determination 8.5.1 Pipette 2 ml of the filtrate (8.4.5) into a test tube (6.6), add 0,2 ml of the phenol solution (5.6) by means of a micropipette (6.4),
30、 and mix by shaking. Then add from the automatic dispenser (6.7), in lessthan 1 s, 5 ml of the concentrated sulphuric acid(5.7), directing the stream of acid against the liquid surface rather than against the side of the test tube in order to obtain good mixing. Immediately mix, using the mixer (6.1
31、0), and allow to stand for 15 min. Cool for 5 min in the water bath(6.14) at 20 C. Wipe the tube and proceed immediately as described in 8.5.2. WARNING NOTE. The addition of the concentrated sulphuric acid produces a violent reaction which should be contained within the test tube. Appropriate precau
32、tions should be taken, particularly to protect the eyes and skin, in case of splashing. 8.5.2 Measure the absorbance of the solution (8.5.1) at 490 nm using the blank solution (8.2) as reference. 8.5.3 If the absorbance is above the upper limit of the calibration graph (see 8.6), repeat steps 8.5.1
33、and 8.5.2, using 2 ml of a suitable dilution of the filtrate (8.4.5) instead of 2 ml of the filtrate itself. NOTEIf such a dilution is made, the formula given in 9.1 has to be modified accordingly. 8.6 Preparation of calibration graph 8.6.1 Pipette 10 ml of the lactose solution (5.8) into a 100 ml v
34、olumetric flask (6.13) and dilute to the mark with water (solution A); 1 ml of solution A corresponds to 2 mg of anhydrous lactose. Prepare three standard solutions by pipetting 1, 2 and 3 ml of solution A into three 100 ml volumetric flasks and making up the volumes with water. The anhydrous lactos
35、e concentrations of the standard solutions obtained are respectively 20, 40 and 60 4g/ml. 8.6.2 Using four test tubes (6.6), proceed in accordance with 8.5.1, but replace the 2 ml of filtrate respectively by 2 ml of each of the three standard solutions and by 2 ml of water. 8.6.3 Measure the absorba
36、nces of the three standard matching solutions using the solution obtained by treatment of the 2 ml of water as the reference liquid. 8.6.4 Construct a calibration graph by plotting the absorbances of the standard matching solutions against their anhydrous lactose concentrations in micrograms per mil
37、lilitre. Draw the best-fitting line through the calibration points.BS6248-6:1982 BSI 12-1999 3 9 Expression of results 9.1 Method of calculation and formula The lactose content of the sample, expressed as anhydrous lactose as a percentage by mass, is equal to where c is the concentration, in microgr
38、ams per millilitre, of anhydrous lactose in the test solution (8.4.5), read from the calibration curve(8.6.4); m is the mass, in grams, of the test portion(8.3). 9.2 Repeatability 1) For lactose contents less than or equal to 0,2 % (m/m), the difference between two single results found on identical
39、test material by one analyst using the same apparatus within a short time-interval will exceed 0,03 g of lactose per 100 g of product on average not more than once in 20 cases in the normal and correct operation of the method. 9.3 Reproducibility 1) For lactose contents less than or equal to 0,2 % (
40、m/m), the difference between two single and independent results found by two operators working in different laboratories on identical test material will exceed 0,04 g of lactose per 100 g of product on average not more than once in 20 cases in the normal and correct operation of the method. 10 Test
41、report The test report shall show the method used and the result obtained. It shall also mention all operating conditions not specified in this British Standard, or regarded as optional, as well as any circumstances that may have influenced the result. The report shall include all details necessary
42、for complete identification of the sample. 1) At higher lactose contents, this difference will be proportionately greater. c 10 6 -50 m -100 4 blankBS6248-6:1982 BSI 12-1999 Publications referred to BS 410, Specification for test sieves. BS 809, Methods for sampling milk and milk products. BS 3978,
43、Water for laboratory use. BS 6248, Caseins and caseinates. BS 6248-1, General introduction, including preparation of laboratory samples. BS 6248-6: 1982 BSI 389 Chiswick High Road London W4 4AL BSIBritishStandardsInstitution BSI is the independent national body responsible for preparing BritishStand
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