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Protein


 Updated 2016-02-06

 Expression of Protein Values

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. Furthermore, there may be an important difference in "crude protein yield" as determined by the different nitrogen determination procedures.
More information 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), part 1, page 100, gives a list of methods of analysis for nitrogen and protein.

 


 Nitrogen-to-Protein Conversion Factors

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 he 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:
 

Atwater W.O. and Bryant A.P., 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).

Although the scientific literature shows attempts to question some of Jones’ findings, it is remarkable that relatively little work has been done in this field – especially regarding the long timespan of almost eighty years. 

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

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

 Jones factors 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 http://toolbox.foodcomp.info/References/Protein/FAO%20NCF%201973.pdf, as their source.
When you read this publication and its reference to the nitrogen-to-protein conversion factors given in its Annex 3 http://toolbox.foodcomp.info/References/Protein/FAO%20NCF%201973.pdf, 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) http://www.fao.org/docrep/005/ac854t/ac854t00.htm.
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.

 


 Nitrogen-to-protein conversion factors in international legislation

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 http://ec.europa.eu/food/safety/labelling_nutrition/labelling_legislation/index_en.htm, entered into application on 13 December 2014. The obligation to provide nutrition information will apply from 13 December 2016. In the new regulation http://eur-lex.europa.eu/legal-content/en/ALL/?uri=CELEX:32011R1169, 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.

 


 Analytical methods for nitrogen and their impact on protein values

The Kjeldahl and Dumas methods

Protein is generally defined as Kjeldahl nitrogen multiplied by a factor, the socalled 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 althernative 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 nitrogen determined by the Dumas method for meat and meat products (Danish Meat Research Institute, 1993). 

More literature on comparison of Kjeldahl and Dumas 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

Never 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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 News
New release of Brazilian Food Composition Table.

2017-10-17
Version 6.0 of the Brazilian Food Composition Table is now available.
See the TBCA website.
 
Release of the Nigerian Food Composition Database.

2017-10-01
Version 1.0 of the Nigerian Food Composition Database is now available.
See the Nigeria Food Data website.
 
New release of the NZ Food Composition Database

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The 2017 release of the NZFCD products are now available on the NZFCD website.
 
FoodOn
A farm to fork ontology.

2017-02-02
FoodOn is a new ontology built to represent entities which bear a “food role”, currently based largely on LanguaL.
For more information,
see the FoodOn site.