🍎 Lactose content of all types of dried milk, dried ice-mixes (also in the presence of other carbohydrates and reducing substances) and processed cheese — test portion of at least 1 g dissolved in water at 40–50 °C, deproteination with potassium hexacyanoferrate(II), zinc sulfate and NaOH, filtration, hydrolysis of lactose with β-galactosidase and oxidation of galactose with β-galactose dehydrogenase and NAD+, absorbance of NADH at 340 nm in a 1 cm cell, correction for galactose present in the sample; recovery check with lactose standard solution 100 % ± 2 %; result in % by mass; PN-ISO 5765-2:2015-09

Food chemistry PN-ISO 5765-2

Overview

WHAT THE STANDARD COVERS. We read the text of the standard in the iTeh sample of ISO 5765-2:2002 | IDF 79-2:2002 (first edition of 01.09.2002, English text) — PN-ISO 5765-2:2015-09 adopts ISO 5765-2:2002 as identical (PKN search): ISO and IDF forewords, introduction, clauses 1–6 and clause 7 up to 7.5.2 — the sample ends on page 5. We do not read the determination itself (7.6, including the pipetting scheme), calculation (8), precision (9), the test report (10) or normative Annex A (good laboratory practice rules for enzymatic analyses); we know their titles from the table of contents.

ACCORDING TO THE TEXT OF ISO 5765-2:2002. The standard was prepared by ISO/TC 34 "Food products", Subcommittee SC 5 "Milk and milk products", jointly with IDF and in collaboration with AOAC International (joint ISO/IDF/AOAC action team on lactose and lactate determination). Introduction: the method uses the galactose moiety of lactose and complements ISO 5765-1, which uses glucose; the choice depends on the glucose or galactose content of the sample — with glucose considerably higher than lactose Part 2 is recommended, with galactose considerably higher — Part 1; with low contents of both either method may be used, with high contents of both the accuracy of both methods is considerably reduced; in heat-treated milk and products part of the lactose may have been converted to lactulose — Part 1 does not determine it, while Part 2 partially determines it as lactose; in intensively heat-treated products (e.g. sterilized milk) part of the lactose may be bound to protein by the Maillard reaction and is determined by neither method; reliable results only when GLP rules for enzymatic analyses are applied strictly (Annex A). Scope (1): determination of lactose in all types of dried milk, in ice-mixes in dry form in the presence of other carbohydrates and reducing substances, and in processed cheese. Definition (2.1): lactose content — mass fraction of substances determined by the procedure of this part, expressed as a percentage by mass. Principle (3): a solution or suspension of the test portion is deproteinated; the purified extract is treated with β-galactosidase (splitting lactose into glucose and galactose) and β-galactose dehydrogenase in the presence of NAD+ (oxidation of galactose to galactonic acid with reduction of NAD+ to NADH); the amount of NADH is determined from the absorbance at 340 nm; the lactose content is proportional to the amount of NADH after correction for galactose present in the sample at the start. Reagents (4): water for enzyme solutions at least double glass-distilled; with an enzyme suspension of different activity the volume in the pipetting scheme is changed proportionally; reagents 4.4 and 4.6–4.8 are available as a commercial kit (example in a footnote); potassium hexacyanoferrate(II) — 3.6 g trihydrate in 100 ml; zinc sulfate — 7.2 g heptahydrate in 100 ml; NaOH 0.1 mol/l; citrate buffer pH 6.6 ± 0.1 (trisodium citrate, citric acid, magnesium sulfate; 3 months at 0–5 °C); phosphate (KH2PO4) buffer pH 8.6 ± 0.1 (8 weeks); NAD+ in citrate buffer — 25 mg in 5 ml (3 weeks); β-galactosidase suspension (from Escherichia coli, EC 3.2.1.23) in 3.2 mol/l ammonium sulfate, at least 60 U/ml, about 12 months at 0–5 °C, kept in ice when in use; β-galactose dehydrogenase suspension (from Pseudomonas fluorescens, EC 1.1.1.48), at least 8 U/ml, at least 6 months, kept in ice when in use; lactose monohydrate standard solution 0.8 mg/ml — 400 mg lactose dried to constant mass at 87 °C in 500 ml water, 2 days at 0–5 °C. Apparatus (5): analytical balance (1 mg, readability 0.1 mg), pipettes, 100 ml volumetric flasks, medium filter paper about 15 cm, spectrometer for 340 nm with 1 cm cells, plastic paddles for cells, water bath 20–25 °C (needed only below 20 °C room temperature), blending apparatus for cheese suspensions, drying oven (87 ± 2) °C, grinding or grating device for cheese. Sampling (6) is not part of the method — ISO 707 recommended. Procedure check (7.1): lactose recovery test with a new batch of NAD+, β-galactosidase or dehydrogenase, after storing these reagents for more than 2 weeks unused, after a break in work or when circumstances justify it — 5.0 and 10.0 ml of standard solution into 100 ml flasks and further as a sample; recovery for both dilutions 100 % ± 2 % (taking into account the purity of the lactose); outside this range reagents, technique, pipettes and spectrometer are checked and the test repeated. Test sample preparation (7.2): dried milk and ice-mixes — in an airtight container of about twice the sample volume, mixing by shaking and inverting; processed cheese — removing rind, smear and mouldy layer, grinding or grating (preferably twice), mixing; suspension with an equal amount of water. Test portion (7.3): 1 g or more to 1 mg, dissolved in at least 20 ml of water at 40–50 °C, transferred to a 100 ml flask and diluted to about 60 ml; lactose in the cell preferably 5–100 µg; absorbance A2 (galactose in the sample) 0.1–0.4; with lactose below 0.2 % a test portion above 1 g is needed — then the volume of precipitated fat, protein and other substances may significantly influence the volume of the solution. Reagent blank in duplicate (7.4). Deproteination (7.5): successively 5.0 ml hexacyanoferrate(II), 5.0 ml zinc sulfate and 10.0 ml NaOH with swirling after each addition, made up to 100 ml, 30 min standing without remixing, filtration discarding the first portion of filtrate.

STATUS (as of 03.10.2026). The PKN search engine (query "PN-ISO 5765-2", 03.10.2026) returns one result: PN-ISO 5765-2:2015-09 — Polish version, "Introduces: ISO 5765-2:2002 [IDT]", without a withdrawal note.

HOW MANY LABORATORIES AND IN WHAT FORM (copy of the accreditation scope database, load up to 17.09.2026, read 03.10.2026). The number 5765-2 appears in 2 records at 2 laboratories: AB 285 and AB 1447. In the current PCA documents (read 03.10.2026; AB 285 issue no. 28 of 13.01.2026, AB 1447 issue no. 16 of 01.06.2026) the number appears at the same 2 laboratories; in each document the number of page markers equals the number of PDF pages, without duplicates; searching all PCA documents downloaded from the BIP on 03.10.2026 found no laboratories outside the database copy. Forms: "PN-ISO 5765-2:2015-09" (AB 285) and "PN-ISO 5765-2:2015-09 IDF 79-2:2002" (AB 1447). None of these items is suspended. The "Laboratories" tab (query "PN-ISO 5765-2") shows both laboratories — checked by a tab query on the labcoda.pl production server on 03.10.2026.

WHAT THE LABORATORIES TEST (PCA items). AB 285: milk and milk products — lactose content (0.09–80.96) %, enzymatic-spectrophotometric method. AB 1447: milk and milk products — lactose content (0.10–85.00) %, enzymatic method.

WHERE A SEEMINGLY CORRECT RESULT IS EASY TO GET. Matrix: the scope of the standard is dried milk, dry ice-mixes and processed cheese (1), while both PCA items refer generally to "milk and milk products" — for other dairy products the report should show on what basis the method was applied. Choice of part: with high glucose — Part 2, with high galactose — Part 1; with high contents of both the accuracy of both methods is considerably reduced (introduction). Heat-treated products: lactulose is partially determined as lactose, and lactose bound in the Maillard reaction is not detected (introduction). Reagents: enzyme suspensions are kept in ice, and after a new batch or longer storage recovery of 100 % ± 2 % is checked (4.7, 4.8, 7.1). Test portion: lactose in the cell preferably 5–100 µg, A2 0.1–0.4; below 0.2 % lactose a larger test portion and influence of the precipitate volume on the solution volume (7.3) — the lower ends of both laboratories' ranges (0.09 and 0.10 %) lie in this zone. Deproteination: order of reagents and 30 min without remixing before filtration (7.5.1). Standard: lactose monohydrate is dried at 87 °C, and the solution is kept for at most 2 days (4.9).

WHAT WE DO NOT GIVE. We do not reproduce the pipetting scheme and measurement conditions (7.6), calculation formulas (8), precision data (9) or the GLP rules of Annex A — they are outside the sample. We do not summarize ISO 5765-1 or ISO 707 in this entry.

Method principle

The sample solution is deproteinated with potassium hexacyanoferrate(II), zinc sulfate and sodium hydroxide, and in the filtrate lactose is split by the enzyme β-galactosidase into glucose and galactose. Galactose is then oxidized by β-galactose dehydrogenase, reducing NAD+ to NADH, the amount of which is measured by absorbance at 340 nm. Lactose is calculated from the increase in NADH after subtracting the galactose present in the sample before hydrolysis.

Applications

• All types of dried milk (1)
• Ice-mixes in dry form, also with other carbohydrates and reducing substances (1)
• Processed cheese (1, 7.2.2)
• Milk and milk products — lactose content — 2 accreditation scope records of 2 laboratories in PCA

Key parameters

Standard

Standard number

PN-ISO 5765-2:2015-09

Title (PL)

Mleko w proszku, mieszanki lodziarskie w proszku i sery topione — Oznaczanie zawartości laktozy — Część 2: Metoda enzymatyczna wykorzystująca galaktozową część laktozy

Title (EN)

Dried milk, dried ice-mixes and processed cheese — Determination of lactose content — Part 2: Enzymatic method utilizing the galactose moiety of the lactose

Step-by-step procedure

1. Procedure check

   Recovery of lactose standard 100 % ± 2 % with new reagents or after a break (7.1). PN-ISO 5765-2:2015-09 — no withdrawal note (PKN, 03.10.2026).

2. Test sample preparation

   Powder — mixing in an airtight container; processed cheese — grinding and suspension with water (7.2).

3. Test portion

   ≥ 1 g, water at 40–50 °C, 100 ml flask, about 60 ml (7.3); reagent blank in duplicate (7.4).

4. Deproteination

   Potassium hexacyanoferrate(II), zinc sulfate, NaOH, to 100 ml, 30 min, filtration discarding the first portion (7.5).

5. Enzymatic determination

   β-galactosidase and dehydrogenase with NAD+, absorbance at 340 nm, correction for free galactose (3, 7.6 — outside the sample).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Enzyme suspensions are stored at 0–5 °C and, when in use, in a vessel immersed in crushed ice (4.7, 4.8)

Frequently asked questions