protein determination; casein; milk; Kjeldahl method

Chapter 12

The detectives milk their method for protein determination in milk for all it’s worth

📁 Case overview: The detectives get a bloody good case this Halloween when a client asks them for help with protein determination in milk. Lieutenant Cornlumbo leads the team in using the Kjedahl technique to measure casein content in two different milk samples. But will the results bewitch the client or spook her into looking for another agency for help? Read on to find out.

It’s a beautiful morning, the colored leaves are shimmering golden in the low autumn sun. The detectives are a buzz, as they pile up in the trunk of their cargo bike and Shallot Holmes pedals them off to a customer visit. They chatter happily as they pull up into a dairy farm. The client is happy to show them her cows and to give them a nice tasting of fresh milk. They sit in her garden, eating fresh pie, surrounded by baskets full of pumpkins and freshly picked apples and begin discussing the case.

The farmer is growing her business and would like to start selling her milk to several local grocery stores. But she needs to follow strict regulations, including protein determination in milk. This is precisely what she needs the detectives to help her with. She would like to contact a local testing lab to analyze her samples, but she wants to instruct them exactly what methods to follow, as they are no experts in food analytics.

The detectives assure her they will demonstrate the best possible technique for her needs. They get back on their bike to head back to the office. Shallot Holmes complains it is much harder to bike with them after all the pie they’ve consumed and the detectives laugh and cheer him on.

What ensues was hard for Shallot Holmes to foresee. All five detectives want to be in charge of the case of protein determination in milk and a screaming fight follows. Eggcule Poirot insists he knows the most about milk, as eggs are white and so is milk. Miss Mapple fights him off saying nothing goes better than milk and apple pie. And Nancy Beef wants a turn, because it is in fact from her relatives that most milk comes from.

Shallot Holmes decides to settle the argument with a quiz. The quiz goes as follows:

1. What is the main protein ingredient in milk?
Lieutenant Cornlumbo gets it right first by saying casein.

2. How much of the protein content of milk does casein make up?
Nancy Beef is the closest by stating that casein accounts for nearly 80% of the protein content of milk.

3. What are other uses of casein?
Lieutenant Cornlumbo is the fastest again by listing uses of casein as additive in medicine or technical uses as a binder, cosmetics, paint and adhesives.

So Lieutenant Cornlumbo is awarded the case! Despite being more of an NIR analysis expert, he is happy to dip into classical reference methods for protein determination in milk.

After a quick dig through his textbooks and one Kjeldahl Knowledge Base, he begins by giving an overall view of the method. They will precipitate casein from a test portion by adding acetic acid solution. They will digest the filtrate with sulfuric acid to convert the nitrogen to ammonium sulfate. After conversion to ammonia through the alkalinization with sodium hydroxide, they will distill the ammonia of the sample into boric acrid receiver by steam distillation, then titrate with hydrochloric acid solution. Finally, the casein content will be the difference between the total nitrogen content and the non-casein nitrogen content. The latter will be calculated using the titrant volume and the sample weight.

Miss Mapple, as the Kjeldahl expert, nods approvingly and the five set out to carry out the plan for protein determination in milk. Firstly they purchase two sample types: Whole milk UHT, and Partial skimmed milk UHT. Then they start the digestion process with the following protocol:

1. They place 20.0 g of the sample in a volumetric flask.
2. They add 250 ml of water at 40⁰C and 2 ml of acetic acid solution (10%), then swirl the flask to mix and let it stand for about 10 minutes.
3. They add 2 ml of sodium acetate solution (1 mol/L) and swirl to mix.
4. They let the mixture cool down to ambient temperature and fill the flask up with water to the calibration mark.
5. They filter through a filter paper and collect the entire filtrate.
6. They place 50 mL of the filtrate in a 300 mL sample tube.
7. They add 2 Kjeldahl tablets, a portion of 20 mL of sulfuric acid (98%) and a spatula tip of stearinic acid.
8. They prepare additional blanks, chemicals without samples.
9. They carefully suspend the sample by gently swirling the tube.
10. They connect their scrubber to their digestion unit for absorbing the acid fumes created during digestion.
11. They insert the rack containing the samples into the preheated unit.
12. They digest the samples according to “npn / ncn in milk” method with the parameters listed below:

StepTemperature (⁰C)Time (min)

After the elapsed time, they notice that the sample tube not clear and blue-green. Miss Mapple recommends digesting for additional 30 minutes as in step 2. This solves the problem. They let their samples cool down to ambient temperature and proceed to distillation and titration in the process of protein determination in milk.

The food detectives use a steam distillation unit for Kjeldahl nitrogen analysis and the following parameters for distillation:

Water80 ml
Sodium hydroxide80 ml
Boric acid (4%)50 ml
Reaction time5 s
Steam power100%
Distillation Time240 s
Titration Start240 s
Titration TypeBoric Acid
Stirrer Speed Dist.5
Stirrer Speed Titr.7

Finally they are ready to calculate their results for their protein determination in milk, first as a percentage of nitrogen with the following equations:

Kjeldahl method; protein content; milk


wNCN : weight fraction of non casein nitrogen [g/g]

VSample : amount of titrant for the sample [ml]

VBlank : mean amount of titrant for the blank [ml]

VFlask : Volume volumetric flask (200 ml)

z : molar valence factor (1 for HCl, 2 for H2SO4)

c : titrant concentration [mol/l]

f : titrant factor (for commercial solutions normally 1.000)

MN : molecular weight of nitrogen (14.007 g/mol)

F : Factor for the volume of the precipitate (0.994 for whole milk and

0.998 for skimmed milk)

For partial skimmed milk the factor was calculated taking into

account the fat content. The factor for partial skimmed milk = 0.995.

mSample : sample weight [g]

%N : percentage of weight of nitrogen [%]

%NCN : percentage of weight of non casein nitrogen [%]

%Casein : Casein content [%]

6.38 : sample-specific protein factor for milk

Lieutenant Cornlumbo claps his hands in pleasure. It is time to look at the results! First, they look at casein protein determination in milk:

First they look at their whole milk sample:

Whole milkMass of sample (g)Volume of sample (ml)%NCNCasein (%)
Sample 120.33664.100.1042.65
Sample 220.18074.010.1022.66
Sample 320.23564.020.1022.66
Sample 420.26554.050.1032.65
Rsd (%)0.770.19

Where the mean blank volume for the sample was 0.298 ml (n=4) and casein was calculated taking into account the measured total nitrogen content of 0.519 %.

Then they examine the data for their partially skimmed milk sample:

Partially skimmed milkMass of sample (g)Volume of sample (ml)%NCNCasein (%)
Sample 120.18074.100.1032.63
Sample 220.18353.980.1022.64
Sample 320.21923.960.1012.64
Sample 420.27793.990.1022.64
Rsd (%)0.640.16

Where the mean blank volume for the sample was 0.298 ml (n=4) and casein was calculated taking into account the measured total nitrogen content of 0.515 %.

Quickly, they compare their results in the protein determination in milk to standard method VDLUFA VI C30.4:

Kjeldahl method for protein determinationStandard method VDLUFA VI C30.4
Sample tube10 g Tablets con.
- 47.7% K2SO4
- 47.7% Na2SO4
- 2.8% TiO2
- 1.8% CuSO4
Water80 ml3.98

They notice no impact on the choice of catalyst, and note they need less water to digest the sample with their method than the standard method. They did double the volumes and weights up to the filtration step compared to the standard method, but this was only done to obtain enough filtrate to digest the sample in duplicate if necessary. The ratio between sample and chemicals used is equal to the official method.

All in all, protein determination in milk using a digestion unit and a Kjeldahl system provided the team with reliable and reproducible results with low relative standard deviations that correspond to literature values (Souci Fachmann Kraut, CRC Press, 7th edition, 2008).

The detectives happily bike back to the dairy farm to inform their client about the results of their experiments. She is very happy she has found a good method for protein determination in milk that she can pass on to the local testing lab. The detectives bid their farewells and climb back onto the cargo bike. Night has fallen and as Shallot Holmes pedals along, the four investigators cuddle under a warm blanket, look up at the night sky and gaze at the Milky Way in what was a very milky successfully day.