FOOD ANALYSIS : PROTEIN METHODS
Total nitrogen times the factor
Counts protein nitrogen and non-protein nitrogen
Protein nitrogen only, times the factor
Non-protein nitrogen is taken out first
In milk, non-protein nitrogen is about 6 percent of the total nitrogen.
Figure 1: Crude protein counts all the nitrogen in the food. True protein counts only the nitrogen that is part of protein.
Introduction
Crude protein and true protein differ by one thing: non-protein nitrogen. Crude protein is not a direct measure of protein. It is nitrogen multiplied by a factor: the Kjeldahl method measures the nitrogen in a food, and the analyst multiplies that nitrogen by 6.25, because most food proteins hold about 16 percent nitrogen. That nitrogen includes non-protein nitrogen, such as urea, which has no food value as protein. True protein takes the non-protein nitrogen out first: the protein is precipitated, the non-protein nitrogen stays in solution, and only the protein nitrogen is multiplied by the factor.
Quick comparison
Table 1: Crude protein compared with true protein
| Aspect | Crude protein | True protein |
|---|---|---|
| What it means | Total nitrogen times the factor | Protein nitrogen only, times the factor |
| Counts | Protein nitrogen plus non-protein nitrogen | Protein nitrogen only |
| How it is found | Kjeldahl or Dumas nitrogen, times the factor | Precipitate the protein with trichloroacetic acid, then measure the nitrogen in the precipitate |
| Factor | 6.25 for most foods, 6.38 for milk, 5.70 for wheat | Same factors: 6.38 for milk |
| Value | Higher, because it counts all nitrogen | Lower, by the non-protein nitrogen share |
| Milk example | Milk with 0.50 percent nitrogen: 0.50 times 6.38 = 3.19 percent | The same milk: 3.00 percent after the non-protein nitrogen is taken out |
| Non-protein nitrogen | Included, and it inflates the value | Removed before the calculation |
| Use | Labels, trade, and routine testing | Research and checks where the real protein matters |
| Extra step | None | Trichloroacetic acid precipitation and filtration |
Scientific explanation
Definition
Crude protein: nitrogen in a food multiplied by a factor, used as the protein value. True protein: the protein in a food after the non-protein nitrogen has been taken out, worked out from the nitrogen in the precipitated protein. Non-protein nitrogen, NPN: the nitrogen in a food that is not part of protein, found in low molecular weight compounds that are not precipitated with the milk proteins by 12 percent trichloroacetic acid, TCA.
Working principle
Crude protein: the Kjeldahl method measures all the nitrogen in the sample, and the factor turns it into protein. Because the method measures nitrogen, not protein itself, a nitrogen-rich chemical with no food value would count as protein in the calculation. True protein: the protein in the milk sample is precipitated by adding TCA so that the final concentration of TCA in the mixture is about 12 percent. The precipitated proteins are filtered, and the filtrate holds the non-protein nitrogen. Then the nitrogen content of the precipitated proteins is determined by the macro Kjeldahl method, and that nitrogen is the true protein nitrogen of the milk.
Apparatus
Crude protein: Kjeldahl digestion and distillation set, or Dumas combustion instrument. True protein: the same Kjeldahl set, plus trichloroacetic acid solution, filter, and glassware for the precipitation step.
Measurement procedure
Crude protein: digest, distil, and titrate the sample, work out the nitrogen percent, and multiply by the factor. True protein: add TCA to the milk sample to about 12 percent, filter off the precipitated protein, measure the nitrogen in the precipitate by macro Kjeldahl, and multiply that nitrogen by the factor. The non-protein nitrogen is found from the filtrate: total nitrogen and non-protein nitrogen content of the test sample are measured, and the true protein nitrogen is found by subtracting the non-protein nitrogen from the total nitrogen.
Factors affecting readings
Crude protein: the wrong factor gives a wrong value even when the nitrogen measurement is perfect, and any non-protein nitrogen inflates the result. True protein: the level of non-protein nitrogen in freshly drawn milk is fairly constant, but it increases on ageing, above all if bacteria grow, so an old sample will show a bigger gap between crude and true protein. The non-protein nitrogen content of milk is 250 to 350 mg per kg, or about 6 percent of the total nitrogen present in milk. Urea, the principal non-protein nitrogen component in milk at 6 mmol per litre, is strongly linked with the heat stability of milk.
Formulas and calculations
Crude protein percent = total nitrogen percent times the factor. True protein percent = true protein nitrogen percent times the factor, where true protein nitrogen percent = total nitrogen percent minus non-protein nitrogen percent. The non-protein nitrogen content, expressed as a percentage by mass, is found by multiplying its nitrogen content by 6.38 for milk.
Worked example from the FTN Food Analysis notes and the milk manual: a milk sample gives 0.50 percent nitrogen. Crude protein is 0.50 times 6.38 = 3.19 percent. Non-protein nitrogen is about 6 percent of the total nitrogen, so it is 0.06 times 0.50 = 0.03 percent nitrogen. True protein nitrogen is 0.50 minus 0.03 = 0.47 percent. True protein is 0.47 times 6.38 = 3.00 percent. The gap between crude and true protein is 0.19 percent, and it is the non-protein nitrogen counted as protein.
Industrial applications
- Milk buyers pay by protein, and the routine payment value is crude protein from the Kjeldahl method.
- Dairy research uses true protein where the real protein matters, such as cheese yield and heat stability work.
- Urea in milk is watched because it is linked with heat stability: milk from cows on pasture holds about twice the urea of milk from cows on dry feed.
- Feed trials use true protein to judge how much of the nitrogen in a ration the animal turns into milk protein.
Advantages and limitations
Crude protein: it is fast, it is the standard for labels and trade, and it needs no extra step. Its limit is that it counts non-protein nitrogen as protein, so it reads high wherever non-protein nitrogen is present, and a nitrogen-rich chemical with no food value would count as protein.
True protein: it gives the real protein, free of non-protein nitrogen. Its limit is the extra precipitation and filtration step, which takes time and care, and it is not the value printed on labels or used for payment.
FAQ
Question: Why is crude protein called crude?
Because it is not a direct measure of protein. It is nitrogen multiplied by a factor, and any nitrogen that is not protein is counted too.
Question: What is non-protein nitrogen in milk?
Low molecular weight nitrogen compounds that are not precipitated with the milk proteins by 12 percent trichloroacetic acid. Urea is the principal one, at 6 mmol per litre.
Question: How much of the nitrogen in milk is non-protein nitrogen?
About 6 percent of the total nitrogen, or 250 to 350 mg per kg of milk.
Question: Does non-protein nitrogen change as milk ages?
Yes. The level in freshly drawn milk is fairly constant, but it increases on ageing, above all if bacteria grow.
Question: Which factor do I use for milk protein?
Milk uses 6.38. A milk sample with 0.50 percent nitrogen gives crude protein of 0.50 times 6.38 = 3.19 percent.
References and further reading
- FTN Food Analysis, Unit 4: Protein and fat analysis (notes on disk): crude protein, factors 6.25, 6.38, 5.70, worked example 0.50 percent nitrogen, 3.19 percent crude protein.
- Manual for Analysis of Milk and Milk Products, FSSAI, 2012 (book extract on disk, milk-analysis-manual.txt): sections 19.2 and 19.3, non-protein nitrogen 250 to 350 mg per kg, about 6 percent of total nitrogen, urea 6 mmol per litre, true protein by TCA precipitation.
- Nielsen, S. S., Food Analysis, Springer: protein analysis (consulted through Google Books).
