1、 g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58Determination of total milk-clotting activityICS 67.100.01Milk and milk products Ovine and caprine
2、rennets BRITISH STANDARDBS ISO 23058:2006BS ISO 23058:2006This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 November 2006 BSI 2006ISBN 0 580 49763 1Amendments issued since publicationAmd. No. Date Commentscontract. Users are responsible for
3、its correct application.Compliance with a British Standard cannot confer immunity from legal obligations.National forewordThis British Standard was published by BSI. It is the UK implementation of ISO 23058:2006.The UK participation in its preparation was entrusted to Technical Committee AW/5, Chemi
4、cal analysis of milk and milk products.A list of organizations represented on AW/5 can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a Reference numbersISO 23058:2006(E)IDF 199:2006(E)INTERNATIONAL STANDARD ISO23058IDF199First ed
5、ition2006-09-15Milk and milk products Ovine and caprine rennets Determination of total milk-clotting activity Lait et produits laitiers Prsures ovines et caprines Dtermination de lactivit totale de coagulation du lait BS ISO 23058:2006ii iiiContents Page Foreword iv Introduction . vi 1 Scope . 1 2 N
6、ormative references . 1 3 Terms and definitions. 1 4 Principle. 2 5 Reagents and materials . 2 6 Apparatus 3 7 Sampling 4 8 Preparation of test sample. 4 8.1 Liquid ovine (or caprine) rennet 4 8.2 Ovine (or caprine) rennet paste. 4 8.3 Powdered ovine (or caprine) rennet . 5 9 Procedure 5 9.1 Prepara
7、tion of substrate 5 9.2 Preparation of the calf and adult bovine rennet reference solutions 5 9.3 Preparation of the calf and adult bovine rennet reference working solutions. 6 9.4 Preparation of the ovine (or caprine) rennet test solution . 6 9.5 Clotting 6 10 Calculation and expression of results
8、7 10.1 Calculation. 7 10.2 Expression of results . 8 11 Precision 8 11.1 Interlaboratory test . 8 11.2 Repeatability 9 11.3 Reproducibility 9 12 Test report . 9 Annex A (informative) Results of interlaboratory trial 10 Bibliography . 13 BS ISO 23058:2006iv Foreword ISO (the International Organizatio
9、n 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 interested in a subject for which a technical committee has been established has
10、 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 Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. Inter
11、national 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 the technical committees are circulated to the member bodies for voting. Publicat
12、ion 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 rights. ISO shall not be held responsible for identifying any or all such patent righ
13、ts. ISO 23058IDF 199 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 ISO 23058:2006vForeword IDF (the International Dairy Federation) is a worldwide
14、 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 with ISO in the development of standard methods of analysis and sampling for mi
15、lk 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 at least 50 % of the IDF National Committees casting a vote. Attention is drawn
16、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 23058IDF 199 was prepared by the International Dairy Federation (IDF) and Technical Committee ISO/TC 34, Food produc
17、ts, Subcommittee SC 5, Milk and milk products. It is being published jointly by IDF and ISO. All work was carried out by the Joint ISO-IDF Action Team on Enzymes in cheesemaking, of the Standing Committee on Milk components and characterization of physical properties, under the aegis of the project
18、leaders Mrs M. Harboe (DK) and Mr C. Repelius (NL). BS ISO 23058:2006vi Introduction Animal rennets comprise all milk-coagulating products derived from ruminants, but until now only bovine rennets have been characterized according to the ISO/IDF standards on composition and milk-clotting activity. O
19、vine and caprine rennets are also available in the market and usually follow the same specifications as for bovine rennets. Each of these enzymes has its own characteristics as far as milk-clotting activity and cheese-making properties are concerned. There are differences in temperature and pH sensi
20、tivity and especially in the organoleptical properties of the cheese produced. A limited number of manufacturers produce these specific rennets and in different presentations (liquid, powder or rennet paste). No internationally recognized reference methods specific to the characterization and isolat
21、ion of samples of these products have been available until now. BS ISO 23058:20061Milk and milk products Ovine and caprine rennets Determination of total milk-clotting activity 1 Scope This International Standard specifies a method for the determination of the total milk-clotting activity of an ovin
22、e or caprine rennet, including rennet paste, containing only chymosin and pepsin as the active coagulating enzymes on a standard milk substrate prepared using a calcium chloride solution of 0,5 g per litre (pH 6,5). The method allows analysis of ovine and caprine rennets in accordance with the relat
23、ive milk-clotting activity test (REMCAT) for bovine rennets given in ISO 11815IDF 157. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of
24、 the referenced document (including any amendments) applies. ISO 648, Laboratory glassware One-mark pipettes ISO 1042, Laboratory glassware One-mark volumetric flasks IDF 110B, Calf rennet and adult bovine rennet Determination of chymosin and bovine pepsin contents (chromatographic method) 3 Terms a
25、nd definitions For the purposes of this document, the following terms and definitions apply. 3.1 total milk-clotting activity of a standard milk substrate at pH 6,5 amount of activity set relative to the international calf rennet reference standard powder and the adult bovine rennet reference standa
26、rd powder NOTE 1 For the first batch of both the calf rennet reference standard powder and the adult bovine rennet reference standard powder (also used for the analysis of bovine and caprine rennets), this was defined as 1 000 International Milk-Clotting Units per gram (IMCU/g). Future preparations
27、of reference standards will be set relative to the previous reference standards. NOTE 2 The total milk-clotting activity of the microbial coagulant reference standard powder is approximately 1 000 IMCU/g, but the real activity with respect to the international calf rennet control powder is indicated
28、 on the glass ampoules. NOTE 3 The total proteolytic (milk-clotting) activity of the microbial coagulant reference standard powder is checked on a synthetic hexapeptide substrate every second year by NIZO 1). 1) Netherlands Institute for Dairy Research (NIZO), P.O. Box 20, 6710 BA Ede, The Netherlan
29、ds. 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 23058:20062 4 Principle The time needed for visual flocculation of a standard milk substrate prepared with a calcium chloride solut
30、ion of 0,5 g per litre (pH 6,5) is determined. The clotting time of the ovine (or caprine) rennet sample is compared to that of a bovine rennet reference standard with a defined enzyme composition of 75:25 and with known milk-clotting activity. It is measured relative to a reference standard having
31、75 % calf chymosin and 25 % bovine pepsin because the composition of the test samples is unknown and a reference standard with a ratio 75/25 should cover most samples with enough accuracy. It is not worthwhile to measure the true chymosin/pepsin composition of ovine and caprine rennets as their comp
32、osition is generally close to the above ratio. NOTE The composition of ovine and caprine liquid or powder rennets may be determined using IDF 110B, but the accuracy for these rennet types has not been validated. 5 Reagents and materials Use only reagents of recognized analytical grade, unless otherw
33、ise specified, and distilled water 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 trihydrate (CH3COONa3H20) and mix. Dilute with water to 1 000 ml. Adjust the pH to 5,
34、5 if necessary. 5.2 Calcium chloride stock solution, c(CaCl2) = 500 g/l. Calcium chloride solutions with the required accurate concentration of 500 g/l of calcium chloride and the actual density stated are commercially available2). Store the solution as described by the manufacturer. Prior to use, b
35、ring 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) every year. 5.3 Calcium chloride working solution, c(CaCl2) = 0,5 g/l. Use the density of the calcium chloride stock solution
36、(5.2) to calculate the mass of calcium chloride needed to obtain a final amount of 0,5 g/l of calcium chloride in the calcium chloride working solution. The mass of the solution should be equivalent to the addition of 2,00 ml of the stock solution with the exact concentration required, c(CaCl2) = 50
37、0 g/l; in that case, the solution mass is 2,70 g. Weighing the calcium chloride stock solution (5.2) is recommended in order to be able to prepare the calcium chloride working solution, as the viscous solution is difficult to pipette. Weigh, to the nearest 0,01 g, about 2,70 g of the calcium chlorid
38、e stock solution (5.2) of exactly known concentration at room temperature (18 C to 22 C) in a 2 000 ml one-mark volumetric flask. Dilute to the mark with water and mix. The calcium chloride solution shall be freshly prepared on the day of use. Alternatively, an intermediate calcium chloride solution
39、 of 50 g/l may be prepared and further diluted before use. 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 by ISO or IDF
40、of these products. BS ISO 23058:200635.4 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 available2), 3). 5.5 Calf rennet reference standard powder4), in pouches con
41、taining 2,7 g of 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 standard p
42、owder is a primary reference standard; a secondary liquid standard may be made and used if it has been ensured 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 transport, it may b
43、e 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 deliver approp
44、riate amounts. Alternatively, a dilutor (e.g. a Hamilton diluter) with the same high precision may be used for diluting the coagulants. 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 volumetric flas
45、ks, in accordance with 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 de la R
46、echerche agronomique, France, which is dependent on the Station exprimentale laitire, P.P. No. 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 a
47、ddress: 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 23058:20064 6.7 Stopwatch, capable of reading
48、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 keeping the maintained temperature constant within limits of 0,2 C throughout the bath (see also 9.5.1
49、), with the 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 very important for this method; a speed of between 2 r/min and 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
