1、BRITISH STANDARD BS 5766-15: 1997 ISO 6655:1997 Methods for analysis of animal feeding stuffs Part 15: Determination of soluble nitrogen content after treatment with pepsin in dilute hydrochloric acid ICS 65.120BS5766-15:1997 This British Standard, having been prepared under the directionof the Cons
2、umer Products and Services Sector Board, was published under theauthority of the Standards Board and comes into effect on 15October1997 BSI 09-1999 ISBN 0 580 28466 2 National foreword This British Standard reproduces verbatim ISO6655:1997 and implements it as the UK national standard. The UK partic
3、ipation in its preparation was entrusted to Technical Committee AW/10, Animal feeding stuffs, which has the responsibility to: aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the
4、UK interests informed; monitor related international and European developments and promulgate them in the UK. A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publicatio
5、ns referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provision
6、s 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. Summary of pages This document comprises a front cover, an inside front cover, pagesi andii, theISO title page, pag
7、es ii to iv, pages1 to6 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 the inside front cover. Amendments issued since publication Amd. No. Date CommentsBS5766-15:1997 BSI 09-1999 i Cont
8、ents Page National foreword Inside front cover Foreword iii Text of ISO 6655 1ii blankBS5766-15:1997 ii BSI 09-1999 Contents Page Foreword iii 1 Scope 1 2 Normative references 1 3 Principle 1 4 Reagents 1 5 Apparatus 1 6 Sampling 1 7 Preparation of test sample 1 8 Procedure 1 9 Expression of results
9、 2 10 Precision 3 11 Test report 3 Annex A (normative) Determination of pepsin activity 4 Annex B (informative) Results of interlaboratory test 6 Annex C (informative) Bibliography 6 Table 1 Repeatability limit (r) and reproducibility limit (R) 3 Descriptors: Agricultural products, food products, an
10、imal feeding products, chemical analysis, determination of content, nitrogen.BS5766-15:1997 BSI 09-1999 iii 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 n
11、ormally carried out through ISO technical committees. Each member body 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 i
12、n the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard
13、 requires approval by at least75% of the member bodies casting a vote. International Standard ISO6655 was prepared by Technical Committee ISO/TC34, Agricultural food products, Subcommittee SC10, Animal feeding stuffs. Annex A forms an integral part of this International Standard. Annex B andAnnex C
14、are for information only.iv blankBS5766-15:1997 BSI 09-1999 1 1 Scope This International Standard specifies a method for the determination of the soluble nitrogen content of animal feeding stuffs after treatment with pepsin in dilute hydrochloric acid. This method does not distinguish between protei
15、n nitrogen and non-protein nitrogen. NOTE 1The values obtained on using this method have no direct connection with digestibility in vivo. NOTE 2If non-protein nitrogen is to be excluded from the test result, its content should be determined using an appropriate method. 2 Normative references The fol
16、lowing standards contain provisions which, through reference in this text, constitute provisions of this International Standard. At the time of publication, the editions indicated were valid. All standards are subject to revision, and parties to agreements based on this International Standard are en
17、couraged to investigate the possibility of applying the most recent editions of the standards indicated below. Members of IEC and ISO maintain registers of currently valid International Standards. ISO 5983:1997, Animal feeding stuffs Determination of nitrogen content and calculation ofcrude protein
18、content Kjeldahl method 1) . ISO 6498:1983, Animal feeding stuffs Preparation of test samples. 3 Principle Incubation of the sample for48h at40 C in a solution of pepsin in dilute hydrochloric acid. FILTRATION OF THE SUSPENSION AND DETERMINATION OF THE NITROGEN CONTENT OF EITHER THE FLITRATE OR THE
19、RESIDUE REMAINING IN THE FILTER IN ACCORDANCE WITH THE KJELDAHL METHOD SPECIFIED IN ISO5983. IN THE LATTER CASE, DETERMINATION OF THE NITROGEN CONTENT OF THE SAMPLE AS WELL, IN ACCORDANCE WITH ISO5983. 4 Reagents Use only reagents of recognized analytical grade, and distilled or demineralized water
20、or water of at least equivalent purity. The reagents (except the standard materials) shall be practically free from nitrogenous compounds. 4.1 Dilute hydrochloric acid, c(HCl) = 0,075 mol/l. 4.2 Pepsin, having an activity of2,0 units per milligram in accordance with the definition given inAnnex A. C
21、heck the pepsin activity in accordance with the method specified inAnnex A. 4.3 Pepsin solution in hydrochloric acid, with a pepsin activity of about400 units per litre. Dissolve 0,2 g 0,001g of pepsin (4.2) in1l of dilute hydrochloric acid (4.1). Prepare this solution immediately before use. If the
22、 activity of the pepsin deviates from2,0 units per milligram, adjust the mass of pepsin to obtain a solution with a pepsin activity of400 units per litre. 4.4 Hydrochloric acid, c(HCl) = 7,5 mol/l ( 20= 1,125g/ml). 5 Apparatus Usual laboratory apparatus and, in particular, the following. 5.1 Water b
23、ath or incubator, capable of being maintained at40 C 1 C. 5.2 Kjeldahl digestion flasks, of suitable capacity. 5.3 Filter paper, fast filtration rate, acid-resistant. 5.4 Distillation and titration equipment 6 Sampling Sampling is not part of the method specified in this International Standard. A re
24、commended sampling method is given in ISO6497 3. It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during transport or storage. 7 Preparation of test sample Prepare the test sample in accordance with ISO6498. If the fat content of
25、the test sample exceeds10% (m/m), extract the fat in accordancewith ISO6498, and take the contentofextracted fat into account in the calculation (clause9). 8 Procedure 8.1 Test portion Weigh, to the nearest0,001g, about2g of the prepared test sample. 1) To be published. (Revision of ISO5983:1979)BS5
26、766-15:1997 2 BSI 09-1999 8.2 Incubation Place the test portion in a500ml volumetric flask and add450ml of the pepsin solution (4.3) previously heated to40 C. Shake so as to avoid agglomeration. Check that the pH of the suspension is lower than1,7. Place the flask in the water bath or incubator (5.1
27、) set at40 C and leave it there for48h. Shake after8h,24h and32h. After 48 h, add 15 ml of hydrochloric acid (4.4) and cool to20 C. Dilute to the mark with water, shake and filter through filter paper (5.3). Proceed in accordance with8.3 or8.4. 8.3 Determination of the nitrogen content of the flitra
28、te 8.3.1 Digestion of organic matter Take 250 ml of the flitrate (8.2) and transfer it into a Kjeldahl digestion flask (5.2). Add the reagents necessary for digestion as specified in ISO5983:1997, 8.2.1. Mix and boil. NOTEIt may be advisable to add an antifoaming agent. Keep the solution boiling vig
29、orously until the water has almost completely evaporated. Eliminate the last traces of water with care, reducing the rate of heating. After the liquid has become clear, continue heating for1h, then leave to cool. 8.3.2 Distillation and titration Proceed as specified in ISO5983:1997, 8.2.2 and8.2.3.
30、8.3.3 Blank test Carry out a blank test using the same procedure but omitting the test portion. Proceed in accordance with clause9. 8.4 Determination of the nitrogen content of the residue and of the test sample 8.4.1 Digestion of organic matter in the residue Wash the filter paper and residue (8.2)
31、 with warm water until they are free from acid. Transfer the filter paper with residue to a Kjeldahl digestion flask (5.2). Add the reagents necessary for digestion as specified in ISO5983:1997, 8.2.1. Mix and boil. NOTEIt may be advisable to add an antifoaming agent. Remove the water by boiling vig
32、orously initially and next by reducing the rate of heating to eliminate the last traces of water. After the liquid has become clear, continue heating for1h, then leave to cool. 8.4.2 Distillation and titration Proceed as specified in ISO5983:1997, 8.2.2 and8.2.3. 8.4.3 Determination of the nitrogen
33、content of the test sample Determine the nitrogen content of the prepared test sample (see clause7) in accordance with ISO5983. 8.4.4 Blank test Carry out a blank test using the same procedure but omitting the test portion. 9 Expression of results 9.1 Calculation of the soluble nitrogen content obta
34、ined using the procedure specified in8.3 Provided that the quantities of sulfuric acid used to collect the ammonia for the determination and for the blank test are equal (see ISO5983), calculate the soluble nitrogen content of the test sample by the equation where Report the result to the nearest0,1
35、g/kg. 9.2 Calculation of the soluble nitrogen content obtained using the procedure specified in8.4 Provided that the quantities of sulfuric acid used to collect the ammonia for the determination and for the blank test are equal (see ISO5983), calculate the soluble nitrogen content of the test sample
36、 by the equation where W 1 is the soluble nitrogen content, in grams per kilogram, of the test sample obtained, by the procedure specified in8.3; c is the concentration, in moles per litre, of the sodium hydroxide solution (seeISO5983:1997, 4.9.1) used for the titrations; m is the mass, in grams, of
37、 the test portion; M is the molar mass, in grams per mole, of nitrogen (M = 14g/mol); V 0 is the volume, in millilitres, of the sodium hydroxide solution (see ISO5983:1997, 4.9.1) used for the blank test; V 1 is the volume, in millilitres, of the sodiumhydroxide solution (seeISO5983:1997, 4.9.1) use
38、d for thedetermination (8.3.2). W 2 is the soluble nitrogen content, in grams per kilogram, of the test sample obtained by the procedure specified in8.4; W 1 2 V 0 V 1 () c M m - = W 2 W N V 0 V 1 () c M m - =BS5766-15:1997 BSI 09-1999 3 Report the result to the nearest0,1g/kg. 9.3 Calculation of so
39、luble crude protein content If it is desired to express the result as soluble crude protein, multiply the soluble nitrogen content determined by the factor6,25. 10 Precision 10.1 Interlaboratory test Details of an interlaboratory test on the precision of the method are given inAnnex B. The values de
40、rived 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 identical test material in the same laboratory by the same
41、 operator using the same equipment within a short interval of time, will in not more than5% of cases exceed the value of r shown inTable 1. 10.3 Reproducibility The absolute difference between two single test results, obtained using the same method on identical test material in different laboratorie
42、s by different operators using different equipment, will in not more than5% of cases exceed the value of R shown inTable 1. 11 Test report The test report shall specify: all information necessary for the complete identification of the sample; the method with which sampling was carried out, if known;
43、 the method used; the test result(s) obtained, expressed as soluble nitrogen or soluble crude protein and, in the latter case the conversion factor used (i.e.6,25); if the repeatability has been checked, the final quoted result obtained; all operating details not specified in this International Stan
44、dard, or regarded as optional, together with details of any incidents occurred when performing the method, which may have influenced the test result(s). Table 1 Repeatability limit (r) and reproducibility limit (R) c is the concentration, in moles per litre, of thesodium hydroxide solution (seeISO59
45、83:1997, 4.9.1) used for thetitrations; m is the mass, in grams, of the test portion; M is the molar mass, in grams per mole, of nitrogen (M =14g/mol); V 0 is the volume, in millilitres, of the sodiumhydroxide solution (seeISO5983:1997, 4.9.1) used for theblank test; V 1 is the volume, in millilitre
46、s, of the sodiumhydroxide solution (seeISO5983:1997, 4.9.1) used for the determination (8.4.2); W N is the nitrogen content, in grams perkilogram, of the test sample determinedin8.4.3. Sample Mean soluble crude protein content g/kg a r g/kg R g/kg Alfalfa meal Maize gluten feed Coconut meal Grass si
47、lage Bone meal Feather meal 136,6 141,5 149,5 192,9 512,0 574,3 7,2 8,9 7,4 7,9 8,9 22,1 26,5 28,9 31,6 36,3 63,1 166,3 a Based on dry matter.BS5766-15:1997 4 BSI 09-1999 Annex A (normative) Determination of pepsin activity A.1 Scope This annex specifies a method for the determination of the activit
48、y of the pepsin used in the determination of soluble nitrogen content after treatment with pepsin in dilute hydrochloric acid. 2 Definition For the purposes of this annex, the following definition applies. A.2.1 unit of pepsin activity that quantity of pepsin which liberates per minute, under the co
49、nditions specified, a quantity of hydroxyaryl groups whose celoration by Folin-Ciocalteus reagent has an absorbance equivalent to that of14mol of tyrosine under the same conditions A.3 Principle Treatment of haemoglobin with pepsin in dilute hydrochloric acid. Precipitation of the non-hydrolysed protein fraction by trichloroacetic acid. Filtration and addition of sodium hydroxide and Folin-Ciocalteus reagent. Measuremen
