🥩 Nitrogen and Crude Protein in Milk and Milk Products — Kjeldahl Method

Food Chemistry PN-EN ISO 8968-1

Overview

STATUS (as of 28.10.2026). PN-EN ISO 8968-1:2014-03 — Polish version, published 10.12.2015, implements EN ISO 8968-1:2014 and ISO 8968-1:2014 [IDT]; it replaced PN-EN ISO 8968-1:2004 and PN-EN ISO 8968-2:2004. The standard is current — no withdrawal note.

SCOPE (clause 1). The standard (ISO 8968-1 | IDF 20-1) specifies the determination of nitrogen content and the calculation of crude protein in milk and milk products by the Kjeldahl principle, with traditional or block digestion. It applies to: liquid cow's milk (whole, partially skimmed, skimmed) and whole goat's and sheep's milk; hard, semi-hard and processed cheese; dried milk and dried milk products, including milk-based infant formulae, milk protein and whey protein concentrates, casein and caseinates. It does not apply to samples containing ammonium caseinate. If non-milk nitrogen is present in the product, the crude protein result will be inaccurate. Other foods and feeds are tested according to other standards.

PRINCIPLE (clause 4). The test portion is digested with a mixture of concentrated sulfuric acid and potassium sulfate, with copper(II) sulfate as catalyst — organic nitrogen is converted to ammonium sulfate. Potassium sulfate is not a catalyst: it raises the boiling point of the acid and provides a stronger oxidizing mixture. Excess sodium hydroxide is added to the cooled digest, and the liberated ammonia is steam distilled into an excess of boric acid solution and titrated with a hydrochloric acid standard volumetric solution. The nitrogen content is calculated from the amount of ammonia.

REAGENTS (clause 5). Nitrogen-free potassium sulfate; copper(II) sulfate pentahydrate solution 5.0 g/100 ml; sulfuric acid 95–98 % (about 1.84 g/ml), nitrogen-free; sodium hydroxide solution 50 g/100 g (in automatic distillation units e.g. 40 % is allowed when the more concentrated one plugs the system — provided it is in excess); boric acid 40.0 g/l with 3 ml of indicator per litre (light orange solution, protected from light and ammonia fumes); indicator — methyl red and bromocresol green in ethanol in the ratio 1 : 5; hydrochloric acid (0.1 ± 0.0005) mol/l, preferably bought standardized — not more concentrated than 0.1 mol/l, because that degrades repeatability and reproducibility (alternatively sulfuric acid 0.05 mol/l). For checks: ammonium sulfate (at least 99.9 %, dried 2 h at (102 ± 2) °C), tryptophan or lysine hydrochloride (at least 99 %, not dried) and sucrose with a nitrogen content of at most 0.002 %.

APPARATUS (clause 6). Analytical balance 0.1 mg; water bath 38–40 °C; automatic burette (e.g. 20 ml, resolution at least 0.004 ml, ISO 8655-3) or — for milk only — a 50 ml class A burette graduated every 0.1 ml (ISO 385); Kjeldahl flasks of 500 or 800 ml or 250 ml tubes for the block; boiling aids (e.g. alundum granules, not reused). The traditional digestion apparatus holds the flasks at about 45°, does not heat them above the level of their contents and has fume extraction; the maximum heater setting is set so that 250 ml of water at 25 °C with 5–10 boiling aids boils after 5–6 min (after preheating: gas burner 10 min, electric heater 30 min). For block digestion: a block with temperature control and measurement, an exhaust manifold and a scrubber or filter pump of acid-resistant material, a steam distillation unit. For pH end-point titration — an automatic titrator with a pH meter calibrated in the range pH 4–7.

PREPARATION OF THE SAMPLE (clauses 7–8). Sampling is not part of the standard (ISO 707 | IDF 50 recommended). Liquid milk is warmed in a water bath at 38–40 °C, mixed gently by inversion (without frothing or churning) and cooled to room temperature. From cheese the rind, smear or mouldy surface layer is removed, the sample is ground, mixed quickly and analysed as soon as possible; the test portion is wrapped in nitrogen-free filter paper (in the block it can be omitted and weighed by difference, because the paper promotes foaming). Dried milk is brought to 20–25 °C, transferred to an airtight container of about twice its volume and mixed by rotating and inverting.

CRUDE PROTEIN AND THE FACTOR. In the dairy industry crude protein is usually calculated as nitrogen content × 6.38. For nutrition labelling of food, Regulation (EU) No 1169/2011 (Annex I point 10) defines protein as total nitrogen determined by the Kjeldahl method × 6.25.

WHAT WE DO NOT GIVE HERE. The test portion masses for individual products (Table A.1), amounts of reagents, temperatures and times of traditional and block digestion, distillation conditions, recovery criteria in the checks, calculation formulae and precision data (clauses 9–11) — we only know the titles of these clauses.

Method principle

A test portion of milk or a milk product is digested in concentrated sulfuric acid with potassium sulfate (raises the boiling point and increases the oxidizing power) and copper(II) sulfate as catalyst; organic nitrogen is converted to ammonium sulfate. Digestion may be traditional — in Kjeldahl flasks — or in a block, in tubes in a heating block.

Excess sodium hydroxide is added to the cooled digest; the liberated ammonia is steam distilled into boric acid solution with indicator (methyl red and bromocresol green) and titrated with 0.1 mol/l standardized hydrochloric acid — with an indicator or pH end point. The nitrogen content is calculated from the amount of ammonia after subtracting the blank, and crude protein — by multiplying nitrogen by a conversion factor.

Applications

• Liquid cow's milk (whole, partially skimmed, skimmed) and whole goat's and sheep's milk
• Hard, semi-hard and processed cheese
• Dried milk and dried milk products — including milk-based infant formulae, milk protein and whey protein concentrates, casein and caseinates (not ammonium caseinate)
• Calculation of protein for nutrition labelling — according to Regulation (EU) No 1169/2011 Kjeldahl nitrogen × 6.25

Key parameters

Standard

Standard number

PN-EN ISO 8968-1:2014-03

Title (PL)

Mleko i przetwory mleczne — Oznaczanie zawartości azotu — Część 1: Zasada Kjeldahla i obliczanie białka surowego

Title (EN)

Milk and milk products — Determination of nitrogen content — Part 1: Kjeldahl principle and crude protein calculation

Step-by-step procedure

1. Laboratory sample

   Sampling is not part of the standard (ISO 707 | IDF 50 recommended); the laboratory should receive a representative sample, undamaged and unchanged during transport and storage (clause 7).

2. Preparation of the test sample

   Milk: water bath 38–40 °C, gentle inversion without frothing, cooling. Cheese: remove the rind and surface layer, grind, mix, analyse as soon as possible; wrap the test portion in nitrogen-free paper. Dried milk: bring to 20–25 °C, transfer to a container about twice as large and mix by rotating and inverting (clause 8).

3. Digestion

   The test portion is digested with sulfuric acid, potassium sulfate and copper(II) sulfate solution — traditionally in Kjeldahl flasks (maximum heater setting from the 250 ml water test: boiling after 5–6 min) or in a heating block with exhaust manifold (clause 4, 6.10, 6.12). Amounts and conditions — 9.1 and 9.2 of the standard.

4. Distillation

   Add excess sodium hydroxide to the cooled digest; steam distil the liberated ammonia into a flask with boric acid solution and indicator (clause 4, 5.6).

5. Titration

   Titrate the distillate with (0.1 ± 0.0005) mol/l hydrochloric acid using an automatic burette or titrator to an indicator or pH end point; the colour change of the indicator also serves to check the pH titration (5.6–5.7, 6.6, 6.18).

6. Blank and checks

   Carry out a blank and checks of the course of the determination with the substances specified in the standard — ammonium sulfate, tryptophan or lysine hydrochloride and sucrose (5.8–5.10); the procedure and criteria of these checks are described in the part of the standard outside the available text.

7. Calculation

   Nitrogen from the volume of titrant used for the sample minus the blank: wN = 1.4007 · (Vs − Vb) · c / m (in %, with V in ml, c in mol/l, m in g). Crude protein = wN × factor — in the dairy industry usually 6.38; for food labelling in the EU 6.25.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Concentrated sulfuric acid and 50 % sodium hydroxide solution are strongly corrosive — follow the safety data sheets
• Carry out digestion with fume extraction (traditional apparatus) or an exhaust manifold with scrubber (block) — the standard requires this (6.10, 6.14–6.15)
• Adding sodium hydroxide to the acid digest releases heat — add it to the cooled digest (clause 4)

Frequently asked questions