`
`INTERNATIONAL STANDARD
`
`
`
`OO
`INTERNATIONAL ORGANIZATION FOR STANDARDIZATION -MEXIYHAPOJHAA OPTAHH3AUHA 10 CTAHJAPTHIALLHMM ORGANISATION INTERNATIONALE DE NORMALISATION
`
`Meat and meat products — Determination of nitrate content
`(Reference method)
`
`Viandes et produits 4 base de viande — Détermination de fa teneur en nitrates (Méthode de référence)
`
`First edition — 1975-09-01
`
`Descriptors : meat, meat products, chemical analysis, determination of content, nitrates.
`
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`UDC 637.5: 546.175
`Ref. No. 1SO 3091-1975 (E)
`
`
`
`ISO3091-1975(E)
`
`Human Power of N Company
`EX1059
`Page 1 of 8
`
`

`

`
`
`INTERNATIONAL STANDARD
`
`
`
`aI
`
`NTERNATIONAL ORGANIZATION FOR STANDARDIZATION -MEXIYHAPOJHAA OPTAHH3AUMA 10 CLAHJAPTHIALIMM ORGANISATION INTERNATIONALE DE NORMALISATION
`
`Meat and meat products — Determination of nitrate content
`(Reference method)
`
`Viandes et produits 4 base de viande — Détermination de la teneur en nitrates (Méthode de référence)
`
`First edition — 1975-09-01
`
`Descriptors : meat, meat products, chemical analysis, determination of content, nitrates.
`
`Price based on 5 pages
`Page | of 8
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`eeeoo—ESESESEeEeEeeeee
`UDC 637.5: 546.175
`Ref. No. 1SO 3091-1975 (E)
`
`
`
`ISO3091-1975(E)
`
`Page 1 of 8
`
`

`

`FOREWORD
`
`ISO (the International Organization for Standardization) is a worldwide federation
`of national standards institutes (ISO Member Bodies). The work of developing
`International Standards is carried out through ISO Technical Committees. Every
`Member Body interested in a subject for which a Technical Committee has been set
`up 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.
`
`Draft International Standards adopted by the Technical Committees are circulated
`to the Member Bodies for approval before their acceptance as
`International
`Standards by the ISO Council.
`
`!1S0 3091 was drawn up by Technical Committee
`International Standard
`1SO/TC 34, Agricultural food products, and circulated to the Member Bodies in
`May 1974.
`
`It has been approved by the Member Bodies of the following countries :
`
`Australia
`Austria
`Bulgaria
`Czechoslovakia
`Denmark
`Egypt, Arab Rep.of
`Ethiopia
`France
`
`Germany
`Hungary
`India
`Ireland
`Israel
`Netherlands
`Poland
`Romania
`
`South Africa, Rep. of
`Spain
`Thailand
`Turkey
`United Kingdom
`U.S.S.R.
`Yugoslavia
`
`The Member Body of the following country expressed disapproval of the document
`on technical grounds:
`
`Canada
`
`© International Organization for Standardization, 1975 «
`
`Printed in Switzerland
`
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`

`

`
`
`ISO 3091-1975 (E)
`INTERNATIONAL STANDARD
`
`
`Meat and meat products — Determination of nitrate content
`(Reference method)
`
`1 SCOPE AND FIELD OF APPLICATION
`
`5.2.2 Reagent Il
`
`This International Standard specifies a reference method
`for the determination of the nitrate content of meat and
`meat products.
`
`dihydrate
`acetate
`zinc
`of
`220g
`Dissolve
`[Zn(CH3COO)3-2H20] and 30 ml of glacial acetic acid in
`water and dilute to 1 000 mi.
`
`2 REFERENCES
`
`5.2.3 Borax solution, saturated
`
`ISO 2918, Meat and meat products — Determination of
`nitrite content (Reference method).
`
`ISO 3100, Meat and meat products — Sampling.
`
`decahydrate
`disodium tetraborate
`of
`50g
`Dissolve
`(Na>B,407-10H 20) in 1000 ml of tepid water and cool to
`room temperature.
`
`3 DEFINITION
`
`nitrate content of meat and meat products: The nitrate
`content determined according to the procedure described in
`this International Standard and expressed as milligrams of
`potassium nitrate per kilogram (parts per million).
`
`4 PRINCIPLE
`
`Extraction of a test portion with hot water, precipitation of
`the proteins and filtration.
`
`Reduction of the extracted nitrates to nitrite by metallic
`cadmium, Development of
`a red colour by addition of
`sulphanilamide and N-1-naphthylethylenediamine dihydro-
`chloride to the filtrate and photometric measurement at
`a wavelength of 538 nm.
`
`5 REAGENTS
`
`reagents shall be of analytical quality. The water used
`All
`shall be distilled water or water of at
`least equivalent
`purity.
`
`5.1 Zinc rods,
`7mm.
`
`length about 15cm and diameter 5 to
`
`5.2 Solutions for precipitation of proteins
`
`5.2.1 Reagent|
`
`trihydrate
`of potassium ferrocyanide
`106g
`Dissolve
`[K4Fe(CN)g5-3H»O] in water and dilute to 1 000 mi.
`
`5.3 Cadmium sulphate solution, 30 g/l.
`
`Dissolve 37g of cadmium sulphate (3CdSO4-8H20O)
`water and dilute to 1 000 ml.
`
`in
`
`5.4 Hydrochloric acid solution, about 0,1 N.
`
`Dilute 8ml of concentrated hydrochloric acid solution
`(P29 1,19 g/ml) to 1 000 mI with water.
`
`5.5 Ammonia buffer solution, pH 9,6 to 9,7.
`
`acid
`hydrochloric
`concentrated
`of
`20ml
`Dilute
`(P29 1,19 g/ml) with 500 ml of water. After mixing, add
`10g of ethylenediamine tetra-acetic acid disodium-salt
`dihydrate,
`[CH2N(CHzCOOH)CH2COONa]2-2H20,
`and
`55 ml of concentrated ammonia (p29 0,88 g/ml). Dilute to
`1 000 m! with water and mix. Check the pH.
`
`5.6 Sodium nitrite standard solutions.
`
`Dissolve 1,000 g of sodium nitrite (NaNO) in water and
`dilute to 100 ml
`in a one-mark volumetric flask. Pipette
`5 ml of the solution into a 1000 mi one-mark volumetric
`flask. Dilute to the mark.
`
`Prepare a series of standard solutions by pipetting 5 ml,
`10m! and 20 ml of this solution into 100m! one-mark
`volumetric flasks and diluting to the mark with water.
`These
`standard solutions contain respectively 2,5 yg,
`5,0 pg and 10,0 pg of sodium nitrite per millilitre.
`
`The standard solutions and the dilute (0,05 g/l) sodium
`nitrite solution from which they are prepared shall be made
`up on the day of use.
`
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`

`ISO 3091-1975 (E)
`
`5.7 Solutions necessary for colour development
`
`6.9 Conical flask, 300 mi.
`
`5.7.1 Solution |
`
`Dissolve, by heating on a water bath, 2 g of sulphanilamide
`(NHz3CgH4SO2NH3)
`in 800 ml of water. Cool, filter,
`if
`necessary, and add 100ml of concentrated hydrochloric
`acid solution (P29 1,19 g/ml), while stirring. Dilute to
`1 000 mI! with water.
`
`5.7.2 Solution Il
`
`Dissolve 0,25 g of N-i-naphthylethylenediamine dihydro-
`chloride (C;gHz;NHCHzCH2NH>-2HCl)
`in water. Dilute
`to 250 ml with water.
`
`7 SAMPLE
`
`7.1 Proceed from a representative sample of at least 200 g.
`See ISO 3100.
`
`if this
`immediately or,
`7.2 Prepare the test sample (8.1)
`cannot be done, store the sample at a temperature of 0 to
`5 °C,for not longer than 4 days.
`
`Store the solution in a well-stoppered brown bottle. !t shall
`be kept in a refrigerator, for not longer than one week.
`
`8 PROCEDURE
`
`5.7.3 Solution III
`
`8.1 Preparation of test sample
`
`Dilute 445 ml of concentrated hydrochloric acid solution
`(P29 1,19 g/ml) to 1 000 mi with water.
`
`5.8 Potassium nitrate standard solution.
`
`Dissolve 1,465 g of potassium nitrate (KNOg) in water and
`dilute to 100m!
`in a one-mark volumetric flask. Pipette
`5 ml of the solution into a 1000 ml volumetric flask and
`dilute to the mark.
`
`This solution contains 73,25 yg/ml of potassium nitrate.
`
`This standard solution shall be prepared on the day of use.
`
`6 APPARATUS
`
`Usual laboratory equipment and the following items :
`
`Make the sample homogeneous by passing it at least twice
`through the meat mincer (6.1) and mixing. Keep it
`in a
`completely
`filled,
`air-tight,
`closed
`container
`under
`refrigeration.
`
`Analyse the test sample as soon as possible, but always
`within 24 h.
`
`NOTE — In the case of uncooked products, analyse immediately
`after homogenization.
`
`8.2 Preparation of the cadmium column
`
`8.2.1 Place 3 to 5 zinc rods (5.1) in the cadmium sulphate
`solution (5.3) contained in a beaker
`(11 of cadmium
`sulphate solution is sufficient for preparing one cadmium
`column).
`
`6.1 Mechanical meat mincer, laboratory size, fitted with a
`perforated plate with holes not greater
`than 4mm in
`diameter.
`
`8.2.2 Remove the spongy metallic cadmium deposit from
`the zinc rods every 1 or 2 h by swirling them in the solution
`or rubbing them against each other.
`
`6.2 Analytical balance.
`
`6.3 One-mark volumetric flasks of 100ml, 200m! and
`1 000 ml, complying with ISO/R 1042, Class B.
`
`if
`and,
`and 20mI
`6.4 One-mark pipettes of 10ml
`necessary, with another capacity, according to the aliquot
`of filtrate (8.8.1), complying with ISO/R 648, Class A.
`
`6.5 Boiling water bath.
`
`6.6 Fluted filter paper, diameter about 15cm,
`nitrite and nitrate.
`
`free of
`
`8.2.3 Finally, after 6 to 8h, decant the solution and wash
`the deposit
`twice with 11 of water, taking care that the
`cadmium is continuously covered with a layer of liquid.
`
`the cadmium deposit with 400ml of
`8.2.4 Transfer
`hydrochloric acid solution (5.4) to a laboratory mixer and
`blend for 10s.
`
`Return the contents of the mixer to the beaker.
`
`8.2.5 Occasionally stir up the cadmium deposit with a
`glass rod. After leaving it for a night under hydrochloric
`acid solution, stir once more to remove all bubbles of gas
`from the cadmium.
`
`6.7 Glass equipment for the reduction of the nitrate (see
`figure).
`
`the solution and wash the cadmium slurry
`8.2.6 Decant
`twice, each time with 1
`| of water.
`
`6.8 Photoelectric colorimeter or spectrophotometer with
`cells of 1 cm optical path length.
`
`8.2.7. Fit a glass wool plug to the bottom of the glass
`column intended to contain the cadmium (see figure).
`
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`

`

`ISO 3091-1975 (E)
`
`8.2.8 Wash the cadmium into the glass column with water
`until the height of the cadmium bedis about 17 cm. Drain
`the column occasionally during filling, taking care not to
`allow the level of the liquid to fall below the top of the
`cadmium bed. Eliminate inclusions of gas (for example with
`a knitting needle). The liquid should flow outat a rate not
`exceeding 3 ml/min.
`
`8.3 Test portion
`
`Weigh, to the nearest 0,001 g, 10 g of the test sample.
`
`8.4 Deproteination
`
`the test portion quantitatively into the
`8.4.1 Transfer
`conical flask (6.9) and add successively 5 ml of saturated
`borax
`solution
`(5.2.3)
`and 100ml
`of water
`at
`a
`temperature not below 70 °C.
`
`the flask and its contents for 15 min on the
`8.4.2 Heat
`boiling water bath (6.5) and shake repeatedly.
`
`8.4.3 Allow the flask and its contents to cool to room
`temperature and add successively 2 ml of reagent | (5.2.1)
`and 2? ml of reagent II
`(5.2.2). Mix thoroughly after each
`addition.
`
`one-mark
`to a 200ml
`contents
`the
`8.4.4 Transfer
`volumetric flask (6.3). Dilute to the mark with water and
`mix. Allow the flask to stand for 30min at
`room
`temperature.
`
`8.4.5 Carefully decant the supernatant liquid and filter it
`through the fluted filter paper (6.6) so as to obtain a clear
`solution.
`required to determine both the nitrate and the
`is
`it
`NOTE — If
`nitrite content on the same sample, the same deproteinatedfiltrate
`can be used for both.
`
`8.6.3 After nearly 100 ml of effluent has been collected,
`remove the flask from under the column and dilute to the
`mark with water.
`
`8.6.4 Pipette 10 ml of the eluate into a 100 ml one-mark
`volumetric flask (6.3) and proceed as specified in 8.8.2 to
`8.8.4.
`
`as
`the eluate,
`nitrite concentration of
`the
`8.6.5 If
`determined from the calibration curve (see 8.10), is below
`0,.9uqg of
`sodium nitrite per millilitre
`(ie. 90% of
`theoretical value), the cadmium column should be rejected.
`
`8.7 Reduction of nitrate to nitrite
`
`8.7.1 Pipette into the reservoir on top of the column
`20 ml of the filtrate (8.4.5) and simultaneously add 5 ml of
`ammonia buffer solution (5.5).
`Collect the effluent from the column in a 100 ml one-mark
`volumetric flask (6.3).
`
`8.7.2. Proceed as specified in 8.6.2 and 8.6.3.
`
`8.8 Colour measurement
`
`8.8.1 Pipette an aliquot portion of the eluate (V ml), but
`not more than 25 ml,
`into a 100 ml one-mark volumetric
`flask (6.3) and add water to obtain a volume of about
`60 ml.
`
`8.8.2 Add 10 ml of solution | (5.7.1), followed by 6 ml of
`solution III] (5.7.3), mix and leave the solution for 5 min at
`room temperaturein the dark.
`
`8.8.3 Add 2ml of solution II (5.7.2), mix and leave the
`solution for 3 to 10 min at room temperature in the dark.
`Dilute to the mark with water.
`
`8.5 Pre-treatment of the cadmium column
`
`8.8.4 Measure the absorbance of the solution in a 1cm
`
`Wash the cadmium column successively with 25 ml of
`hydrochloric acid solution (5.4), 50 ml of water, and 25 ml
`of the 1 + 9 diluted ammonia buffer solution (5.5). Do not
`permit the level of the liquid in the funnel to fall below the
`top of the capillary inlet tube of the cadmium column.
`
`a spectro-
`colorimeter or
`using a photoelectric
`cell
`photometer (6.8) at a wavelength of about 538 nm.
`NOTE — If the absorbance of the coloured solution obtained from
`the test portion exceeds that obtained for the standard solution
`with the highest concentration, repeat
`the operations described in
`8.8, reducing the quantity of eluate pipetted in 8.8.1.
`
`8.6 Checking the reducing capacity of
`column
`
`the cadmium
`
`8.9 Number of determinations
`
`8.6.1 Pipette 20 ml of potassium nitrate standard solution
`(5.8) and simultaneously add 5ml of ammonia buffer
`solution (5.5),
`into the reservoir on top of the cadmium
`column. Collect
`the
`effluent
`in
`a 100ml one-mark
`volumetric flask (6.3).
`
`8.6.2 When the reservoir is nearly empty, wash the walls
`with about 15 ml of water; repeat the same treatment with
`another 15 ml portion of water.
`After
`this portion has
`run into the column as well,
`completely fill the reservoir with water.
`
`Carry out two independent determinations, beginning with
`different test portions taken from the same test sample.
`
`8.10 Calibration curve
`
`8.10.1 Pipette respectively into four 100 ml one-mark
`volumetric flasks (6.3) 10 ml of water and 10 ml of each of
`the three sodium nitrite standard solutions (5.6), containing
`2,5 ug, 5,0 ug and 10,0 wg of nitrite per millilitre.
`
`8.10.2. To each flask add water to obtain a volume of
`about 60 ml and proceed as described in 8.8.2 to 8.8.4.
`
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`

`ISO 3091-1975 (E)
`
`by plotting the
`calibration curve
`8.10.3 Draw the
`concentrations,
`in
`measured
`absorbances
`against
`the
`micrograms per millilitre, of the standard sodium nitrite
`solutions.
`
`9 EXPRESSION OF RESULTS
`
`9.1 Methodof calculation and formula
`
`is the nitrite content of the sample, expressed
`NaNO,
`as milligrams of
`sodium nitrite per kilogram and
`determined according to ISO 2918.
`
`Take
`
`as
`
`the
`
`result
`
`the arithmetic mean of
`
`the two
`
`for
`requirement
`the
`that
`provided
`determinations,
`repeatability (see 9.2) is satisfied. Express the result to the
`nearest 1 mg per kilogram of product.
`
`9.2 Repeatability
`
`Calculate the nitrate content of the sample, expressed as
`milligrams of potassium nitrate per kilogram, using the
`formula :
`
`The difference between the results of two determinations
`carried out simultaneously or in rapid succession, by the
`same analyst, shall not be greater than 10 % of the mean
`
`10 000
`value.
`
`KNOz = 1,465|c x —NaNO,
`mxVv
`
`where
`
`10 TEST REPORT
`
`mis the mass, in grams, of the test portion;
`
`Vis the volume, in millilitres, of the aliquot portion of
`the eluate (see 8.8.1);
`
`is the concentration of sodium nitrite, in micrograms
`e
`per millilitre,
`read from the calibration curve,
`that
`corresponds with the
`absorbance
`of
`the
`solution
`prepared from the test portion (see 8.8.4);
`
`The test report shall show the method used and the result
`obtained; it shall also mention all operating conditions not
`specified in this
`International Standard, or
`regarded as
`optional,
`as well
`as any circumstances that may have
`influenced the result.
`
`include all details necessary for complete
`The report shall
`identification of the sample.
`
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`

`

`ISO 3091-1975 (E)
`
`Dimensions in millimetres
`
`Reservoir (capacity 40 to 50 ml)
`
`Solution
`
`Rubber stopper
`
`Cadmium column
`
`Glass wool
`
`Rubber tubing
`
`Effluent
`
`int. 12to 14
`
`
`
`
`
`g@ext.7 to 8
`
`¢ int. 0,4 to 0,6
`
`NOTE — A flexible connection may be used between the bottom of the column and the effluent capillary tube, in order to allow adjustment of
`the height of the capillary tube and thus of the flow rate.
`
`FIGURE — Apparatus fornitrate reduction
`
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