BS ISO 27105:2016 pdf free download.Milk and cheese — Determination of hen’s egg white lysozyme content by high performance liquid chromatography.
BS ISO 27105 specifies a method for the quantitative determination of hen’s egg white lysozyme content in milk and cheese.
The method is suitable for measuring low levels of hen’s egg white lysozyme with a quantification limit ollO mg/kg.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
hen’s egg white lysozyme content
mass fraction of substance determined by the procedure specified
Note ito entry: The lysozyme content is expressed as milligram per kilogram.
3 Pilnciple
Casein and denatured whey proteins from milk and cheese are precipitated isoelectrically at pH 4,3 (cheese) or at pH 2,2 (milk). Acid-soluble hen’s egg white lysozyme is then determined by reversed- phase high-performance liquid chromatography (HPLC) and fluorescence detection. The lysozyme peak can be verified by LC/MS (see Annex A).
4 Reagents and reference substances
Use only reagents of recognized analytical grade, unless otherwise sp.ecifled, and only dLstilled water or water of equivalent purity.
4.1 Reagents and materials
4.1.1 SodIum chloride solution. c(NaCl) = I molfl. Dissolve 58,44 g of sodium chloride in II water,
4.1.2 Hydrochloric acid. c(HCI) = I mcI/I.
Dissolve 4,0 ml or hydrochloric acid with mass fractIon 37 % in a 50 ml one-mark volumetric flask. Dilute to the mark with water.
4.1.3 Sodium hydroxide, c(NaOH) I mol/l.
Dissolve 2,6 ml of sodium hydroxide with a mass fraction olSO % In a SD ml one-mark volumetric flask.
Dilute to the mark with water.
4.1.4 Trifluoracetic acid (ChCOOH), analytical grade.
5.8.5 Fluorescence detector, capable of operating at 280 nm excitation and at 340 nm emission.
6 SamplIng
Sampling is not part of the method specified in BS ISO 27105. A recommended sampling method is given in ISO 707110F 50411
It is important that the laboratory receives a sample, which is representative and has not been damaged or changed during transport or storage.
7 Procedure
7.1 PreparatIon ollysozyme standard solution
7.1.1 Lysozyme standard stock solution
Weigh to the nearest 0.01 mg, 10mg ol lysozyme (j) lnto a 10 ml one-mark volumetric flask and wait For complete dissolution. Dilute to the mark with sodium chloride solution (iLl).
Prepare fresh the standard stock solutions daily.
7,1.2 Lysozyme standard working solution
Pipette 80 p1 oIthe lysozyme standard stock solution (see 2.11) into a 10 ml one-mark volumetric flask. Dilute to the mark with sodium chloride solution (4.1.1).
The obtained lysozyme standard working solution contains 8.0mg oFlysozyme per litre.
7.2 Test portion
7.2.1 Milk
Weight to the nearest 0,01 g, 10.00 g of test sample into a 100 ml beaker.
7.2.2 Cheese
flefore weighing, grate the test samples of cheese, Weight to the nearest 0,01 g.. 2.00 g of test sample into a 100 ml beaker.
NOTE Soft cheese can be grated after Freezing.
72.3 Preparation of test solution
Add 20 ml of sodium chloride solution (iLl) to the test portion (see Z2J, or 2.22) and mix. Ad,usl the pH of the obtained solution drop wise with sodium hydroxide solution (iLl) to pH 6.0.
Homogenize the test solution For 30 s by using the homogenizer (51) at a speed rate oF 2 500 rpm to
3 000 rpm. Rinse the homogenizer in a separate 100 ml beaker using 10 ml olsodium chloride solution
(411). Add the rinsing to the test solution.
Stir the beaker containing the test solution on a magnetic stirrer at room temperature for 1 h. Adjust the pH of the test solution obtained from the test portion of milk (see 221) to pH 2.2 or that obtained From the test portion of cheese to pH 4,3 by using hydrochloric acid (jLZ).
Transfer the test solution from the 100 ml beaker to a 50 ml one-mark volumetric flask. Use sodium chloride solution (411) to rinse the 100 ml beaker and add the rinsing to the test solution. Dilute to the mark with the sodium chloride solution (4.1.1) and mix.
C5 is the concentration, in milligrams per litre, of the standard working solution (see 2.12); H1 is the numerical value of the peak height or area of the test solution (see 22). In counts; H1 Is the numerical value of the peak height or area of the standard working solution (see 2.12].
in counts;
mt Is the mass, in grams, of the test portion (see Z21 or 222):
V1 Is the volume, in millilitres, of the test solution (see Z2.).
Check equipment linearity and reagent blank regularly. This may be needed in case of lysozyme contents of more than 100 mg/kg of cheese which can lead to a non-linear response. In such cases, the use of a multiple point calibration is necessary.
8.2 ExpressIon of results
Express the results to one decimal place. Express test results ol below 10 mg/kg as: less than
10 mg/kg, or equivalent expression.
9 PrecIsion
9.1 Intertaboratory test
The values for repeatability and reproducibility derived from an interlaboratory testtzj were determined in accordance with ISO 5725’1l and ISO 5725-2431
The values for the repeatability and the reproducibility limit are expressed for the 95 % probability level and may nat be applicable to concentration ranges and matrices other than those given. Details of the interlaboratory test on the precision of the method are given in Annex B.
9.2 Repeatability
The absolute difference between two individual single test results, obtained with the same method on identical test material in the same laboratory by the same operator using the same equipment within a short interval of time, shall in not more than S % of the cases be greater than 7,7 % (95 % confidence intervals 6.2 % to 8,5 %).
9.3 Reproducibility
The absolute difference between two individual single test results, obtained with the same method on
Identical test material in different laboratories with dIfferent operators using different equipment, shall
In not more than 5% of the cases be greater than 33,8% (95 % conFidence intervals 20,3 % to 57,5 %).
Running the chromatographic system on another temperature, using a different column type, or other dIffering conditions do not result automatically In false or less sensitive results but need thoroughly to be validated.
10 Test report
The test report shall contain at least the following information:
a) all information necessary for the complete identification of the sample:
b) the sampling method used. ilknown;
c) the test method used, with reference to BS ISO 27105, i.e. ISO 27IOSIIDF 216;
d) aft operational details not specffied in BS ISO 27105, or regarded as optional. together with details olany incident wbich may have influenced the test resuLt(s);
e) the test result(s) obtained, and, if the repeatability has been checked, the final quoted results obtained.