Protein
	Updated 2021-07-09

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Expression of Protein Values

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Preferred method of protein measurement

In the official recommendations, FAO Food and Nutrition Paper 77, the preferred method for measuring protein is stated as
 
     "the sum of individual amino acid residues (the molecular weight of each amino acid less the molecular weight of water)";
 
this is the most precise method of calculating the protein content of foods.

However, the currently available amino acid data do not cover all the foods needed in a comprehensive food composition database. Therefore, using the traditional Kjeldahl method (or comparable methods) is acceptable, see below.

For more information on protein calculated from amino acids, see Compilers' ToolBox™'s page on Amino Acids).

Acceptable method of protein measurement

The acceptable method of protein detrmination is therefore the most commonly used calculation of the so-called "crude protein" from the amount of total nitrogen (N) analysed in the food by the Kjeldahl or comparable method (Dumas, Kjel-Foss (automated Kjeldahl using antimony-based catalyst), Kjeltec, etc).

By multiplying the total nitrogen content with a food matrix specific factor, the nitrogen-to-protein conversion factor (NCF) or Jones factor:

protein content =

total nitrogen content x specific conversion factor

the "crude protein" content is found.

It should be noted that calculating the protein content this way most often overestimates the "true" protein content as the resulting protein content also includes a contribution from non-protein nitrogen (NPN). Total nitrogen is the sum of that derived from amino acids, which generally represent the vast majority, and that from non-protein nitrogen (NPN) sources, generally smaller in quantity, existing in foods.

This means that the more correct and acceptable way of calculating "true" protein values is:

protein content =

(total nitrogen content - non-protein nitrogen) x specific conversion factor

The most common non-protein nitrogen comprises compounds like free amino acids, nucleotides, nucleic acids, urea, creatine/creatinine, ammonium nitrogen, urea, etc. as well nitrate/nitrite and purines. The non-protein nitrogen content is low in most foods, generally 5-10% of total nitrogen or less; but there are foods, like human milk or seafood in which the non-protein nitrogen part can be as high as about 20% of the total nitrogen,, e.g. in crustaceans and molluscs - in sharks and rays even higher. For more information on non-protein nitrogen, see the page on Non-Protein Nitrogen in Foods.

Furthermore, there may be important differences in "crude nitrogen yield" as determined by the different analytical nitrogen determination procedures. More information in Analytical methods for nitrogen and their impact on protein values below.

The total nitrogen content is traditionally determined by the dominating Kjeldahl or Dumas methods, but several other methods are available for specific applications. Greenfield and Southgate (FAO Rome, 2003), page 100, presents a list of methods of analysis for nitrogen and protein.

 

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Nitrogen-to-Protein Conversion Factors

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The development of nitrogen-to-protein conversion factors

G. J. Mulder

As far back as in 1838, the Dutch chemist G. J. Mulder publshed an article (in French) about "the composition of animal substances" in which he describes the precipitation of a highly purified substance in sulphuric acid from  from "albumine" in animal tissue. He named the substance "proteïnsulphuric acid". He presented a common elemental composition C₄₀H₆₂N₁₀O₁₂ for the substance (not including the suphuric acid), the "proteïn" - a name the Swedish chemist J. J. Berzelius had suggested to Mulder in correspondance the previous year.
In 1839, G. J. Mulder published his findings in German.
Mulder found that the nitrogen content of the isolated chemical substance, "proteïn" was 16%, i. e. the conversion from nitrogen to protein is 100/16, which equals 6.25 - the nitrogen-to-protein conversion factor was "born".
The factor 6.25 was used as nitrogen-to-protein factor from that time - and has been used as the generic nitrogen-to-protein factor ever since.   

W. O. Atwater and A. P. Bryant

In 1883, W. O. Atwater publishes his fish data in German. Atwater reports that proteïn is calculated from total nitrogen multiplied by the factor 6.25 according to "normal practice", although he has some doubts about the results' validity: "Nach meiner persönlichen Meinung wäre es richtiger, die Eiweissstöffe und die stickstoffhaltenden Extraktivstoffe direkt zn bestimmen, was in der That in dieser Untersuchung bei vielen Proben geschehen ist" [en. in my personal opinion, it would actually be more correct to determine the albuminoids and nitrogen containing extracts (non-protein nitrogen) separately, which in fact has been done for many samples in this study].

Even 40 years before D. B. Jones published his famous work on nitrogen-to-protein conversion factors, now called Jones' factors, Atwater and Bryant indicated that specific nitrogen-to-protein conversion factors were necessary not only to estimate protein content right, but also carbohydrate content when using "total carbohydrate by difference" calculation - if protein is overestimated, carbohydrate is underestimated and vice versa.
Here is a table from Atwater and Bryants report from 1900:
 

 

It is worth noting that Atwater and Bryant actually estimated nitrogen-to-protein conversion factors for vegetables and fruits more than a hundred years ago. Jones did not forget these, but said that "There is not sufficient knowledge regarding the proteins of fruits and vegetables to justify the calculation of e special factor". However, more recent findings seems to support the low factors of 5.65 and 5.80 for vegetables and fruits, respectively, mentioned by Atwater and Bryant (Fujihara et al (2001), Salo-Väänänen and Koivistoinen (1996) and Yeoh and Wee (1994)). These new findings actually indicate that the nitrogen-to-protein conversion factors for vegetables and fruits should be even lower.

D. Breese Jones

Factors for Converting Percentages of Nitrogen in Foods and Feeds into Percentages of Proteins, United States Department of Agriculture, Circular 183, 1931 (slightly revised August, 1941).

Jones determined the specific nitrogen to protein factors on the basis of the nitrogen content of more than 121 different proteins isolated from plant and animal sources. It is remarkable that no really serious attempt has been made since 1941 to enlarge this list. For the remaining foods the factor 6.25 is applied ‘until more is known regarding their protein’.
It should also be noted that Jones' work is based on the Kjeldahl method for determining the nitrogen content. This means that other methods of analysis for nitrogen giving slightly different results compared to the Kjeldahl method, may require adjusted nitrogen-to-protein conversion factors (see below).

Jones is in his report strongly emphasizing how uncertain the values he presents are. Although the scientific literature shows some attempts to question of Jones’ findings, it is remarkable that relatively little work has been done in this field – especially regarding the long timespan of more than ninety years. 

In the following Nitrogen-to-Protein conversion factors are abbreviated NCF. However, there are many other acronyms are used for NFC (Nitrogen-to-Protein Conversion factor), like N-Prot, N2P, k_P', N:P, NPCF, etc.

The original Jones' factors for calculating protein from the nitrogen content of food (from Circular 183, 1941):
 

 

 

As Greenfield and Southgate state, the Jones' factors "are being fragmentally questioned", but until now, no work has been done on establishing a new full set of NCFs, and Jones' NCFs are still very much "in power".

There is however some evidence that the Jones' NCFs are somewhat too high and authors of scientific articles have tried to point out their findings for many years, but it is still the old Jones' factors that are prevailing. The newer research into NCFs is sometimes also contradictory.

In References below a list of publications on the subject of NCFs is given.

Be Careful with the Source of Nitrogen-to-Protein Conversion factors - one authoritative source

It is important to be careful when selecting nitrogen-to-protein conversion factors (NCFs) as some sources contain errors (or may not have documented their references properly).

Greenfield and Southgate (FAO Rome, 2003), page 103, refer to Food and Agriculture Organization /World Health Organization. Energy and protein requirements. Report of a Joint FAO/WHO Ad Hoc Expert Committee. FAO Nutrition Meetings No. 52. FAO, Rome 1973 , as their source.
When you read this publication and its reference to the nitrogen-to-protein conversion factors given in its Annex 3 , it turns out that the specific NCFs are given in the annex in order to be able to calculate back to protein (NCF: 6.25), because the protein requirements are based on protein (NCF: 6.25) and most food composition tables are using specific NFCs (or Jones' factors).

The FAO/WHO report indicates that the specific NCFs are taken from FAO amino acid table: Amino acids of foods and biological data on proteins, Rome 1970 (FAO Nutritional Studies, No. 24) .
In the Foreword of this publication, it is stated that the NCFs are taken from Jones (1941).

Many food composition tables and databases refer to the NCF tables given in the preface of the printed versions McCance and Widdowson's The Composition of foods, which from 4th edition (1978) up to the 6th edition contains some discrepancies compared to Jones' establshed specific factors.

Therefore, use the original source:

D. Breese Jones
Factors for Converting Percentages of Nitrogen in Foods and Feeds into Percentages of Proteins, United States Department of Agriculture, Circular 183, 1931 (slightly revised August, 1941)

or more recent scientific work on nitrogen-to-protein conversion factors.

 

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Nitrogen-to-protein conversion factors in international legislation

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The European Directive on Food Information to the Consumer

The Council Directive 90/496/EEC of 24 September 1990 on nutrition labelling for foodstuffs and more recent amendments () defines protein as follows

'protein' means the protein content calculated using the formula: protein = total Kjeldahl nitrogen × 6,25

i.e. only the general Jones factor, 6.25, is used.

The consultations (2003) with the EU Member States concerning Directive 90/496/EEC on Nutrition Labelling for Foodstuffs are reported in the Discussion Paper on Revision of Technical Issues [DG Health and Consumer Protection, Directorate E - Safety of the food chain, May 2006 ].
There is no change of the definition of protein suggested in the report.

The European Advisory Services has prepared an interesting report on Impact Assessment of Mandatory Nutrition Labelling in the European Union for DG Sanco, European Commission, November 2004 [].

The new European law on food information to the consumer, Regulation (EU) No 1169/2011 on the provision of food information to consumers , entered into application on 13 December 2014. The obligation to provide nutrition information will apply from 13 December 2016. In the new regulation , protein still has the same definition: protein = total Kjeldahl nitrogen × 6,25.

The Codex Committee on Nutrition and Foods for Special Dietary Uses

In the discussions in the Codex Committee on Nutrition and Foods for Special Dietary Uses concerning the Draft Revised Standard for Infant Formula, it has emphasized that the specific factors (i.e. the Jones' factors) are not based on current scientific evidence, and it has been suggested that for infant formula a factor of 6.25 should be used for calculating protein from Kjeldahl nitrogen - unless "a scientific justification is provided for the use of a different conversion factor for a particular nitrogen source [Comment from EC 2006-10-13, ].

In this context, the International Dairy Foundation has prepared a comprehensive review of scientific literature on nitrogen conversion factors - especially for milk, milk products and soy products: IDF Bulletin 405-2006 - Comprehensive review of scientific literature pertaining to nitrogen protein conversion factors []. The review concludes that there is substantial scientific evidence to support a specific nitrogen-to-protein conversion factor for specific sources of protein (e.g. milk and milk products, soy and soy products, etc.) rather than the introduction of a single inaccurate nitrogen conversion factor, as has been envisaged in the revision of the Codex Standard for Infant Formula.

In their investigations, Tomé et al. (2019 ) give a very comprehensive investigation of the current literature on nitrogen-to-protein conversion factors and indicate that "it has been known for decades that using total nitrogen content with a conversion factor of 6.25 to quantify total protein is imperfect and can lead to a 15–20% error in the actual protein content" - and ""Because both the protein and the nitrogen component can be determined by different approaches, the resulting values of the conversion factor can vary. In the literature, different terminologies have been used for the conversion factors, depending on the method by which they have been calculated". Tomé et al. find slightly lower results for milk and milk products than those found in the study above.

 

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Analytical methods for nitrogen and their impact on protein values

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The Kjeldahl and Dumas methods

Protein is in practice, i.e. regulations and food composition work, generally accepted and defined as Kjeldahl nitrogen multiplied by a factor, the so-called nitrogen-to-protein conversion factor.
This means that the Kjeldahl method is the generic analytical method in nitrogen analysis and protein determination.

The nitrogen-to-protein conversion factors or Jones' factors mentioned above relate to total nitrogen determined by the Kjeldahl method. The Kjeldahl method has some drawbacks - it is rather polluting and time-consuming - which makes alternative methods like the Dumas method preferable. The Dumas procedure involves combustion for the determination of nitrogen in organic substances.

AOAC approved the Dumas method for determination of nitrogen in meat and meat products in 1993 (AOAC Method No. 992.15), and it is widely used in the meat sector. The AOAC Method specification mentions that the Dumas method gives higher nitrogen results (1%) than the common Kjeldahl method. The AOAC specifications recommend a nitrogen-to-protein conversion factor of 6.25 being used with the Dumas method, also.
However, you may see the use of a nitrogen-to-protein conversion factor of 6.12 (instead of the general Jones factor 6.25) to calculate protein from total nitrogen determined by the Dumas method for meat and meat products (Danish Meat Research Institute, 1993). 

More literature on comparison of Kjeldahl, Dumas and NIR methods:

Etheridge et al. - A comparison of nitrogen values obtained utilizing the Kjeldahl nitrogen and Dumas combustion methodologies (Leco CNS 2000)
Thomson et al. - Testing for bias between the Kjeldahl and Dumas methods for the determination of nitrogen in meat mixtures, by using data from a designed interlaboratory experiment
Thomson et al.  -  A comparison of the Kjeldahl and Dumas methods for the determination of protein in foods, using data from a proficiency testing scheme
Mihaljev et al.  -  Comparison of the Kjeldahl method, Dumas method and NIR method for total nitrogen determination in meat and meat products
Müller  -  Dumas or Kjeldahl for reference analysis?

Newer methods include the automated CHN elemental analyzer (Perkin-Elmer 2400 CHN elemental analyzer), which gives rapid and reliable results of the elements C, H and N, but the equipment is expensive.

 

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General Remarks

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Although many scientific findings during the last centuries reveal that protein values are overestimated when calculated from total nitrogen (Kjeldahl nitrogen), its seems that for ease of calculations, the general method for calculating "crude" protein using a fixed/general nitrogen-to-protein factor of 6.25 g protein/g nitrogen is still prevailing, e.g. in legislation and thereby also in food composition tables/databases.
The general factor of 6.25 g protein/g nitrogen in many cases grossly overestimates the protein content of many foods.

Many attempts have been made to refine the original specific nitrogen-to-protein conversion factors (see References below) defined by D.B. Jones back in 1931, but until now no consensus has been reached to suggest a new and more comprehensive set of factors. This may be caused by conflicting results between the different studies, but may also be a result of legislative, financial or economical issues.  

The use of the recommended method for calculating protein as the sum of (anhydrous) amino acid residues is sparsely used due to the lack of good amino acid data and cost of amino acid analysis. As more and more data on the amino acid content in foods become available, the recommended method hopefully gains more attention. It should be remarked in this context that there are several examples in the literature making incorrect use of the "sum of amino acids" instead of the "sum of amino acid residues" in connection with protein calculation. This will evidently lead to wrongly calculated protein results.

As both the recommended and accepted methods for calculating protein content rely on the nitrogen content of foods. It is therefore evident that the nitrogen content must be determined with a reliable method, and that non-protein nitrogen is subtracted from total nitrogen ("crude" nitrogen) before the calculation of the protein content. It is furthermore important to know, how the nitrogen-to-protein conversion factors have been derived, i.e. if the factors have been derived from "crude" nitrogen values or nitrogen values corrected for non-protein nitrogen, which is seen in a few cases.

In order to establish a new, detailed set of nitrogen-to-protein conversion factors, it is important to investigate and collect as many studies as possible. Different studies yield different results often due to different methods for the same food. Differences are also due to different production of the same type of foods.
 

 

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References

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  Journal of the American Oil Chemists' Society, Vol. 96, No. 4, pp. 339-364, April 2019.
  DOI: 10.1002/aocs.12196
  
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• López C.V.G, del Carmen M., García C., Fernández F.G.A., Bustos C.S., Chisti Y., Sevilla J.M.F.:
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  Bioresource Technology, Vol. 101, pp. 7587–7591, 2010 - DOI: 10.1016/j.biortech.2010.04.077
   
• Lourenço S.O., Barbarino E., De-Paula J.C., Pereira O.C., Marquez U.M.L.:
  Amino acid composition, protein content and calculation of nitrogen-to-protein conversion factors for 19 tropical seaweeds.
  Phycological Research, 50: 233–241, 2002 - DOI: 10.1046/j.1440-1835.2002.00278.x - pdf available from ResearchGate.net
   
• Lourenço S.O., Barbarino E., Lavin P.L., Marquez U.M.L., Aidar E.:
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  European Journal of Phycology, Volume 39, Issue 1, 2004. pages 17-32 - DOI: 10.1080/0967026032000157156
   
• Magomya A.M., Kubmarawa D., Ndahi J. A., Yebpella G. G.:
  Determination Of Plant Proteins Via The Kjeldahl Method And Amino Acid Analysis: A Comparative Study.
  International Journal of Scientific & Technology Research, Volume 3, Issue 4, pp. April 2014.
   
• Mariotti F., Tomé D., Mirand P.P.:
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  Critical Reviews in Food Science and Nutrition, 48:177–184 (2008) - DOI: 10.1080/10408390701279749
   
• Mattila P., Salo-Väänänen P.P., Könkö K., Aro H., Jalava T.:
  Basic Composition and Amino Acid Contents of Mushrooms Cultivated in Finland.
  J. Agric. Food Chem. 2002, 50, pages 6419-6422 - DOI: 10.1021/jf020608m
   
• Milton K. and Dintzis F.R.:
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  Biotropica, Vol. 13, No. 3 (Sep.), 1981, pp. 177-181
   
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• Morr C.V.:
  Nitrogen Conversion Factors for Several Soybean Protein Products.
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• Morr C.V.:
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• Mossé J.:
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• Notheisz K.:
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  World Health Organization and Food and Agriculture Organization of the United Nations, 2019 .
  ISBN 978-92-5-132544-5 (FAO)/ISBN 978-92-4-151698-3 (WHO)
   
  
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