ISO 26462:2010 download free.Milk Determination of lactose content – Enzymatic method using difference in pH.
I Scope
ISO 26462 specifies an enzymabc method For the determination of the lactose content of rTslk
and reconstituted milk by measurement of the difference in p11 (differential pH measurement)
2 Terms and definitions
For the purpose of ISO 26462, the folling terms and definitions apply.
2.1
lactose content In milk
amount of substance concentration of compounds de(emned by the procedure specified in ISO 26462
NOTE The lecose concent of rni. C expressed in milllmoies per litre. For wwerecn & the result Into other isins, see Table 1.
2.2
unit of enzyme activity International unit standard unit
U
amount of enzyme which catalyses the transformation of one micromole of substrate per minute tinder standard conditions
3 Principle
-Galactoeidase is added to deave lactose into ucose and galaciose. At pH 7,8. glucose is phosphorylated by glucokinase, ttieretiy releasing protons that induce a change in pH. The pH change vafles as a function of the lactose content of the sample and is measured by using a differential pH analyser.
4 Reagents
During the analysis, unless otherwise stated, use only reagents of recogrxzed analytical grade and distilled or demineralized water or water of equivalent puniIy
4,1 Butler solutIon, p11 7.8
Dissolve 0242 got tris4hydroxymethyl)melhytamine (IrIs). 0.787 gof adenosine 5’-tniphosphate disodium salt (ATP). 0.304 g of trtsoditm’i phosphate (Na3PO*’12H20), 0,009 g of sodium hydroxide (NaOH), 0,2039 of
4.6 Strong regenerating solution
DANGER — The us. of sodium fluoride (NsF) alone and in combination with IICI may cause health problems due to inhalation and:or skin absorption. This international Standard does not purport to address all the safety problems ii any, associated with Its use. Ills th responsibIlity of the user to establish appropriate safely and health practices and to ensure compliance with any national regulatory conditions.
Dissolve 30 g of nitric acid (NNO3) with a mass traction, w(HNO’3) 69%. 309 of hydroctdonc acid (HCI) with
a mass traction, w(HCI) • 37%, 309 of sodium fluonde (NaF), and 1 g of octyiphenoxypolyettioxyethanol in a
1 000 ml one-mark volumetric flask (5.8). Make up to the mark with water and mix.
The strong regeneration solution can be kept for 1 year if stored in non-corroding material at room temperature
5 Apparatus
UsuaJ laboratory ecipment and, in particular, the following.
5.1 Analytical balance, capable of weighing to the nearest I mg.
5.2 Mlcroplpettse. capacity 20 d. ISO 7550151. with positive displacement.
53 Water bath, capable of maintaining a temperature of 38 C ± 1 C.
5.4 One-mark volumetric flasks capacilies 100 ml and 1 000 ml. ISO l042) class A.
5.5 Differential p11 apparatus. shown schematically in Figure Al.
The differential pH apparatus consists of penslaltic pumps to circulate Iuids, a mixing chamber, two glass capillary flow-through electrodes (El and E2), and an electronic system for measurement.
5.6 One-mark volumetric flasks, capacity 1 001) ml and of mateiial capable of storing the extremely corrosive strong regenerating solution (4.6).
6 SamplIng
Sampling is not parj of the method specified in ISO 26462. A recommended sampling method is given in ISO 707 IIDF 50111.
It is important that the laboratory receive a truly representative sample which has not been damaged or changed during lranspotl or stOrage.
7 Preparation of test sample
Warm the tesi sample to 38 C in the water bath (5.3) whIle mixing. Cool the sample to 20 C, before preparing the test portion.
8 Procedure
8.1 General
Since me vanous types of differential pH apparatus (5,5) avadable differ in design and handbng, the operator shall carefully follow the instrument manufacturei’s instructions for setting up, calibration, and operation of the instrument. Swllch me Instrument on and allow Its operating conditions to stabilize.
where ci Is the concentration, In mIlmoIes per litre, 01 the lactose standard solution (4.3).
8.4 Checking the calibration
Chedi the calibration by analysing 20 lii of lactose standard solution (4.3) m accordance with the procedure in 8.5, The results oblained shall be between 148,5 mmol/l and 151.5 mmolIl for the lactose determination. If these values are not met, repeat the calibration procedure.
8.5 DeterminatIon
Operate the instrument and introduce the test portion in accordance with the manufacturers instructions.
Add, with one mlcropqette (5.2), 20 p1 of test sample (Clause 7) and, with another, 20 p1 of glucokinase enzyme solution (4.2.1) to the mixing chamber of the differential pH apparatus (5.5). Dilute with buffer solution (4.1) to a total volume of 1 200 p1. FIll El and E2 with the mixture of buffer, test portion, and glucokinase obtained- Measure the offset differential pH, D5. between the two electrodes.
Using another mlcropipetie, add 20 p1 of 13’galactosadase enzyme solution (4.2.2) to the mixture of buffer, test portion, and gluockinase in the mixing chanter and mix. Pu E2 with the mixture of buffer, test portion. glucokmase. and (-galac1osidase.
Alter completion of the enzymatic reaction, measure the ,flset differential pH, D. between the two electrodes NOTE Completion Is reached Mien the variation Ias not eiceeded 1 mpH unit over the last i mth.
8.6 CheckIng the stability
Alter analysing r.o mere than 30 test pardons and at the end of each analytical series, analyse two blank solutions to chedi the zero point and 20 il 01 the lactose standard solution (4.3) using the determination procedure (8,5) Ia check the calibration.
The second zero value shall be within 0 mmoi4 ± 1,5 mmoIll and the standard value In the range 148,5 mmoiil to 151,5 mmoVl. If the values obtained are out of range, repeat both the offset blank determination (8.2) and the calibration procedure (8.3).
8.7 Cleaning procedure
Wash the electrodes and the mixing chamber of the differential pH apparatus (5.5), replacing the buffer solution (see also Annex A) With the cleaning solutIon (4.4). If the equipment Is in hi operation, leave the electrodes in contact with the deamrig solution until the next use while renewing the cleaning solution every 120 mm. When not In full operation, treat the electrodes according to the manufacturer’s instructions.
11 PrecIsion
11.1 Interlaboralory test
The values for repeatability and reproducibility hmils are expressed for the 95% probability level and may not be applicable to concentration ranges and matrices other than those given.
11.2 RepeatabIlity
The absolute difference between two individual single test results, obtained with the same method on identical lest material in the same laboratory by the same operator using the same equipment within a short Interval of lime. will In not more than 5% of cases be greater than 2,96 mmol of lactose monohydrale per litre.
11.3 Reproducibility
The absolute difference between two individual single test results, obtained with the same method on identical lest material in different laboratories with dilferent operators using different equipment, wl In not more than 5% of cases be greater than 3.13 mmol of lactose monohydrate per litre
12 Test report
The lest report shall contain at least the following information:
a) all information necessary for the complete identification of the sample:
b) the samptirig method used. II known;
C) the test method used. with reference to ISO 26462 (ISO 264621 IDF 2142010);
d) all operating details not specifIed en ISO 26462, or regarded as optional, together with details of any incidents which may have influenced the test result(s):
e) the test result(s) obtained:
I) it the repeatability has been checked, the final quoted result obtained.