1、 g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58rennetsICS 67.100.10Milk Determination of total milk-clotting activity of bovine BRITISH STANDARDBS
2、 ISO 11815:2007BS ISO 11815:2007This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 July 2007 BSI 2007ISBN 978 0 580 56336 2Amendments issued since publicationAmd. No. Date CommentsCompliance with a British Standard cannot confer immunity from
3、 legal obligations.National forewordThis British Standard is the UK implementation of ISO 11815:2007. The UK participation in its preparation was entrusted to Technical Committee AW/5, Chemical analysis of milk and milk products.A list of organizations represented on this committee can be obtained o
4、n request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application.Reference numbersISO 11815:2007(E)IDF 157:2007(E)INTERNATIONAL STANDARD ISO11815IDF157First edition2007-07-01Milk Determination of tot
5、al milk-clotting activity of bovine rennets Lait Dtermination de lactivit totale de coagulation du lait dans la prsure de bovins BS ISO 11815:2007ii iiiContents Page Foreword iv Foreword. v Introduction . vi 1 Scope . 1 2 Normative references . 1 3 Terms and definitions. 1 4 Principle. 2 5 Reagents
6、2 6 Apparatus 3 7 Sampling 4 8 Preparation of test sample. 4 8.1 Liquid rennet . 4 8.2 Powdered rennet. 4 9 Procedure 5 9.1 Preparation of substrate 5 9.2 Preparation of rennet reference standard solutions. 5 9.3 Preparation of the calf and adult bovine rennet reference working solutions. 5 9.4 Prep
7、aration of rennet test solution. 6 9.5 Clotting 6 10 Calculation and expression of results 6 10.1 Calculation. 6 10.2 Expression of results . 8 11 Precision 8 11.1 Interlaboratory test . 8 11.2 Repeatability 8 11.3 Reproducibility 8 12 Test report . 9 Annex A (informative) Interlaboratory trial 10 B
8、ibliography . 11 BS ISO 11815:2007iv Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body
9、interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotech
10、nical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by t
11、he technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent right
12、s. ISO shall not be held responsible for identifying any or all such patent rights. ISO 11815IDF 157 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF. BS
13、 ISO 11815:2007vForeword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member country. Every National Committee has the right to be represented on the IDF Standing Committees carrying out the technical work. IDF collaborates
14、 with ISO in the development of standard methods of analysis and sampling for milk and milk products. Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the National Committees for voting. Publication as an International Standard requires approval by
15、at least 50 % of the IDF National Committees casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO 11815IDF 157 was prepared by the Int
16、ernational Dairy Federation (IDF) and Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published jointly by IDF and ISO. All work was carried out by the Joint IDF-ISO Action Team on Enzymes in cheese making, of the Standing Committee on Minor compo
17、unds and characterization of physical properties, under the aegis of its project leader Mr A. van Boven (NL). This edition of ISO 11815|IDF 157 cancels and replaces IDF 157A: 1997, which has been technically revised. BS ISO 11815:2007vi Introduction Bovine rennets (calf and adult) contain, in variou
18、s amounts, both chymosin and bovine pepsin as main milk-clotting enzymes. Each of these enzymes has its own characteristics as far as milk-clotting activity and cheese-making properties are concerned. The most obvious difference between these enzymes is the stronger pH dependence of the milk-clottin
19、g activity of pepsin. For economic reasons, therefore, it is very important to know the total milk-clotting activity of a certain rennet type and to have that characterized relative to an internationally recognized reference standard with known composition and milk-clotting activity. The method is c
20、ommonly known as the relative milk-clotting activity test (acronym: REMCAT). A qualitative determination of the six most common milk-clotting enzymes in a sample is performed according to IDF 110B. In the case of mixtures of milk-clotting enzymes other than bovine chymosin and pepsin, no correct det
21、ermination of the total milk-clotting activity for the sample can be obtained. BS ISO 11815:20071Milk Determination of total milk-clotting activity of bovine rennets 1 Scope This International Standard describes a method for the determination of the total milk-clotting activity of bovine rennet cont
22、aining only chymosin and bovine pepsin as the active coagulating enzymes on a standard milk substrate at pH 6,5. To produce accurate results with this method, test samples of unknown origin are to be checked for the absence of main milk-clotting enzymes of non-bovine origin by using an appropriate m
23、ethod (e.g. IDF 110B). This International Standard can also be applied to determine the total milk-clotting activity of fermentation-produced chymosin. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edi
24、tion cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 648, Laboratory glassware One-mark pipettes ISO 1042, Laboratory glassware One-mark volumetric flasks IDF 110B:1997 Calf rennet and adult bovine rennet Determination of c
25、hymosin and bovine pepsin contents (Chromatographic method) 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 total milk-clotting activity amount of activity set relative to the first batch of international calf rennet reference standard powder
26、 and the first batch of adult bovine rennet reference standard powder, respectively NOTE 1 For the first batch of both the calf and the adult bovine rennet reference standard powder, the activity was defined at 1 000 International Milk-Clotting Units per gram (IMCU/g). Future preparations of referen
27、ce standards will be set relative to the previous reference standards. NOTE 2 The total milk clotting activity of the calf and adult bovine rennet reference standard powders is approximately 1 000 IMCU/g, but their exact activity is stated on the certificate of analysis. BS ISO 11815:20072 NOTE 3 Th
28、e total proteolytic (milk-clotting) activity of these calf (or adult bovine) rennet reference standard powders is checked every second year by an alternative method, for example on a synthetic hexapeptide substrate by NIZO1). 4 Principle The time needed for a visible flocculation of a standard milk
29、substrate prepared with a 0,5 g/l calcium chloride solution (pH 6,5) is determined. The clotting time of a rennet sample is compared under identical chemical and physical conditions to that of a reference standard with known milk-clotting activity and having the same enzyme composition as the sample
30、, which was determined using IDF 110B. 5 Reagents Use only reagents of recognized analytical grade, and distilled or demineralized water or water of equivalent purity. 5.1 Buffer solution, pH 5,5. Add, using a pipette (6.1), 10,0 ml of 1 mol/l acetic acid (CH3COOH) to 10,0 g of sodium acetate trihyd
31、rate (CH3COONa3H2O) and mix. Dilute with water to 1 000 ml. If necessary, adjust the pH to 5,5 with acetic acid or sodium acetate trihydrate. 5.2 Calcium chloride stock solution, c(CaCl2) = 500 g/l. Calcium chloride solutions with the required accurate concentration of 500 g/l calcium chloride and t
32、he actual density stated are commercially available2). Store the solution as specified by the manufacturer. Prior to use, bring the calcium chloride stock solution to room temperature (18 C to 22 C). Check the concentration of the solution by titration with EDTA (ethylenediaminetetraacetic acid) eve
33、ry year. 5.3 Calcium chloride working solution, c(CaCl2) = 0,5 g/l. Use the density of the stock solution (5.2) to calculate the mass needed to obtain a final amount of 0,5 g/l calcium chloride in the working solution. The mass of the solution should be equivalent to the addition of 2,00 ml of the s
34、tock solution with the exact concentration required, c(CaCl2) = 500 g/l; in that case, the solution mass is 2,70 g. Weigh, to the nearest 0,01 g, about 2,70 g of calcium chloride stock solution (5.2) of exactly known concentration at room temperature (18 C to 22 C) in a 2 000 ml one-mark volumetric
35、flask. Dilute to the mark with water and mix. The calcium chloride solution shall be freshly prepared on the day of use. Weighing of the calcium chloride stock solution (5.2) is recommended in order to prepare correctly the calcium chloride working solution as the viscous solution is difficult to pi
36、pette. Alternatively, an intermediate calcium chloride solution of 50 g/l may be prepared and further diluted before use. 1) Netherlands Institute for Dairy Research (NIZO), PO Box 20, 6710 BA Ede, The Netherlands. This information is given for the convenience of users of this International Standard
37、 and does not constitute an endorsement by ISO or IDF of these products. 2) Ordering address: Chr. Hansen A/S, 1-27 Jernholmen, 2650 Hvidovre, Denmark (Fax: +45 36 86 77 76). This information is given for the convenience of users of this International Standard and does not constitute an endorsement
38、by ISO or IDF of these products. BS ISO 11815:200735.4 Low-heat, low-fat, spray-dried milk powders, of good renneting and bacteriological quality. NOTE Low-heat, low-fat, spray-dried milk powders meeting the requirements are commercially available 2),3). 5.5 Calf rennet reference standard powder 4),
39、 in pouches of approx. 2,7 g powder, containing 98 % chymosin and 98 % bovine pepsin in terms of enzyme activity as determined according to IDF 110B. The exact total milk-clotting activity is indicated on the certificate of analysis and should be about 1 000 IMCU/g. The adult bovine rennet reference
40、 standard powder is a primary reference standard; a secondary liquid standard may be made and used if it has been determined that the same result is obtained. Store the adult bovine reference standard powder in the dark at 18 C, protected against moisture. For short periods, for example during trans
41、port, it may be kept at ambient temperatures. 6 Apparatus Usual laboratory equipment and, in particular, the following. 6.1 Micropipette or any other pipette, capable of delivering 0,5 ml in less than 1 s with a repeatability of 0,2 % or better. 6.2 One-mark pipettes, in accordance with ISO 648, to
42、deliver appropriate amounts. Alternatively, a diluter (e.g. a Hamilton diluter) with the same high precision may be used for diluting the rennets. For measuring the substrate, a syringe or a dispenser delivering the appropriate amount with a repeatability of 0,4 % may also be used. 6.3 One-mark volu
43、metric flasks, as specified in ISO 1042, of the required capacities. 6.4 Thermometer, calibrated, graduated between 20 C and 45 C, with a precision of 0,1 C. 6.5 pH meter, capable of measuring the pH in 0,01 units. 6.6 Analytical balance, capable of weighing to the nearest 1 mg. 3) Institut national
44、 de la Recherche agronomique, France, which is dependent on the Station exprimentale laitire, BP 94, 39800 Poligny, France. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO or IDF of these products. 4) AMAFE. Ordering
45、 address: Chr. Hansen A/S, 1-27 Jernholmen, 2650 Hvidovre, Denmark (Fax: +45 36 86 77 76). This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO or IDF of these products. BS ISO 11815:20074 6.7 Stopwatch, capable of readin
46、g in seconds. 6.8 Flasks or test tubes, for milk-clotting testing, with a suitable capacity (see 6.9.1 and 9.5.1). 6.9 Water bath, capable of maintaining a temperature of 32 C 1 C, but also capable of maintaining the temperature constant to within 0,2 C throughout the bath. The water bath should hav
47、e following attachments. 6.9.1 Electric motor, provided with a rotating spindle to which the flask or test tube (6.8) can be attached, capable of rotating at a suitable angle of about 30 with the water surface of the water bath. NOTE The rotation speed is not important for this method; a speed of 2
48、r/min to 4 r/min is suitable. 6.9.2 Electric lamp, placed in such a position as to illuminate the flask or test tube (6.8) effectively. A screen with a dark background, placed in the water bath, may be used to improve the determination of the milk-clotting flocculation point in the flask or test tub
49、e. 7 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 the method specified in this International Standard. Recommended sampling methods are given: for liquid rennet (8.1) in Clause 9 of ISO 707|IDF 50:19971; and for powdered rennet (8.2) in Clause 13 of ISO 707|IDF 50:19971. Store the test samples in the dark at a temperature of between 0 C and 5 C. 8 Preparation of test sample 8.1 Li
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