Counting Nitrogen Atoms – Part 7: Connective Tissues and Gelatin

Determining Total Meat Content (Part 7):  Connective Tissues and Gelatin
By Eben van Tonder
7 January 2019

Previous Installments in Counting Nitrogen Atoms

Part 1:  From the start of the Chemical Revolution to Boussingault

Part 2:  Von Liebig and Gerard Mulder’s theory of proteins

Part 3:  Understanding of Protein Metabolism Coming of Age

Part 4:  The Background of the History of Nutrition

Part 5: The Proximate Analysis, Kjeldahl and Jones (6.25)

Part 6:  The Codex

an ancient style of chinese bronze tripod called a ding


When calculating meat content, it is customary to make a distinction between fat, lean meat, and connective tissue.  In modern meat formulations, it becomes even more important since gelatin and connective proteins can be bought from spice companies with specific functional benefits and are often added to meat formulations.  It provides structure, firmness, improved gelling and water holding capacity, to mention a few.  Maximum fat and connective proteins and minimum amount of lean meat are therefore customarily prescribed in food legislation around the world.  This article is not intended to be an overview of the history of connective tissue determination; nor is it intended to summarise the various ways it is dealt with by different countries in either its food laws or monitoring programs to police compliance. It is intended to introduce the topic and show how it came about that connective tissue is dealt with separately from fat and lean meat in many countries and show the complexity of the issue by looking at the variety of ways that different governments try and deal with it.

Related to the EU, we have seen that regional rules related to meat content continue to be relevance, albeit it not being on the same legal standing as the EU rules and legislation.  We learned in the last article that due to consumer habits and expectations which differ within the EU member states, “raw material descriptions and customary usage in the meat trade are described on a national basis. In Germany, guidelines for meat and processed meats (Leitsätze für Fleisch und Fleischerzeugnisse, 2015) are part of the “Deutsche Lebensmittelbuch” (German Food Book), which is a collection of guidelines describing the manufacture, composition, and the characteristic properties of food.”  (Lautenschlaeger  and  Matthias,  2017)

“Within the “Deutsche Lebensmittelbuch-Kommission” (German Food Book Commission), there are food-specific expert committees working out the details of the guidelines, taking into account the European food law as well as international food standards such as the FAO “Codex Alimentarius.” Commissioners are representatives of different stakeholders within the food area: scientists, trade and manufacturer associations, consumer groups, surveillance authorities, Federal Ministry of Food and Agriculture, etc. The latter is also publishing the guidelines agreed on.”  (Lautenschlaeger and Matthias,  2017)

“For the commercial manufacture of processed meats, meat only means skeletal muscles with adherent or embedded fat and connective tissue as well as lymph nodes, nerves, vessels, and pork salivary glands. Phrenic and chewing muscles are included. Partly, meat may contain a distinct portion of bones and cartilages, if the products are usually prepared in the consumer household (e.g., chops) and if meat products are processed from whole muscles (e.g., bone-in ham). Rind may be part of pork meat for cuts from the hind leg, from shoulder, breast and belly, and back fat.”  (Lautenschlaeger and Matthias,  2017)

“Apart from these general settings, the Guidelines (Leitsätze für Fleisch und Fleischerzeugnisse, 2015) characterize in detail meat raw material used for the manufacture of processed meat products in terms of the content of fat and connective tissue.”  (Lautenschlaeger and Matthias,  2017)  This introduces the subject of connective protein (Stromal Proteins) which features important in the meat content determination and limits in much of the world. It is therefore important to understand what connective tissue is.

Connective Proteins

“Connective tissue is composed of a watery substance into which is dispersed, a matrix of stromal protein fibrils; these stromal proteins are collagen, elastin, and reticulin.  Collagen is the single most abundant protein found in the intact body of mammalian species, being present in horns, hooves, bone, skin, tendons, ligaments, fascia, cartilage and muscle. Collagen is a unique and specialised protein which serves a variety of functions. The primary functions of collagen are to provide strength and support and to help form an impervious membrane (as in skin). In meat, collagen is a major factor influencing the tenderness of the muscle after cooking.  Collagen is not broken down easily by cooking except with moist—heat cookery methods. Collagen is white, thin and transparent. Microscopically, it appears in a coiled formation which softens and contracts to a short, thick mass when it is heated and helping give cooked meat a plump appearance. Collagen itself is tough; however, heating (to the appropriate temperature) converts collagen to gelatin which is tender.

Elastin (often yellow in colour) is found in the walls of the circulatory system as well as in connective tissues throughout the animal body and provide elasticity to those tissues.  Reticulin is present in much smaller amounts than either collagen or elastin. It is speculated that reticulin may be a precursor to either collagen and/or elastin as it is more prevalent in younger animals.”  (

From Ancient Usage to Questions About its Nutritional Value

Connective tissue is something that ancient people undoubtedly noticed as part of meat constituent right from the first animal that was butchered.  From before the dawn of recorded history, connective tissues would have been used in a variety of applications.  We have indeed very old references to its use. We know that collagen, for example, was used for centuries to create strings to bind things and for strings on musical instruments. Catstring or catgut is made by twisting together strands of purified collagen taken from the serosal or submucosal layer of the small intestine of healthy ruminants (cattle, sheep, goats) or from beef tendon and has been in use for a long time 900’s AD.  (Wray, 2006)  Gut strings were being used as medical sutures as early as the 3rd century AD as Galen, a prominent Greek physician from the Roman Empire is known to have used them.   (Nutton, 2012)

Abū al-Qāsim Khalaf ibn al-‘Abbās al-Zahrāwī al-Ansari (Hamarneh, et al., 1963)(Arabic: أبو القاسم خلف بن العباس الزهراوي‎;‎ 936–1013), popularly known as Al-Zahrawi (الزهراوي), Latinised as Abulcasis (from Arabic Abū al-Qāsim), was an Arab Muslim physician, surgeon and chemist who lived in Al-Andalus in the early 900’s CE. He is considered as the greatest surgeon of the Middle Ages (Meri, 2005), and has been described as the father of surgery.  (Krebs, 2004).  He became the first person to have used Catgut to stitch up a wound. He discovered the natural dissolvability of the Catgut when his monkey ate the strings of his musical instrument called an Oud. (Rooney, 2009)

Later, in 1818, the modern founder of surgery, Joseph Lister, and his former student William Macewen independently and quite remarkably, almost at the exact same time, reported on the advantages of a biodegradable stitch using “catgut”, prepared from the small intestine of a sheep.  Over the ensuing years, countless innovations have extended the reach of collagen in the engineering and repair of soft tissue in medicine and numerous other industrial applications. (Chattopadhyay, 2014)

There must have been questions of the value of connective tissue as a food source from as early as the concept of nutrition entered the human mental ether.  Ancient chefs wrestled with its place in food dishes since the time when culinary arts were developed on account of its toughness.  When bronze cooking pots made their appearance in the bronze age, mothers and chefs alike would have discovered gelatin (cooked collagen).  These bronze age cooks would have encountered it as what was left the next morning after meat was cooked in these bronze container and left overnight in the bowl.  The fat “gelling” in the pot stalled as gelatin. Ancient  Rome and Egypt knew it and used it in a variety of industrial applications, but the first recent historical mention comes to us from the work of Denis Papin (1647 – 1712).  Educated at the University of Angers in western France with a degree in medicine, he was appointed as an assistant to Huygens in his laboratory at the Academies des Sciences.  Here is did work on air pumps.  This prepared him ideally for his later work in extracting gelatin from bones through steam cooking.  (Robinson, 1947)

Denis visited London, probably with letters from  Huygens where he was immediately appointed by Boyle. In 1679 he showed the Royal Society his famous digester. This was an apparatus designed to cook food in and soften bones under pressure. He described it in a book published in 1681 published by the Royal Society. The device used the same basic principles still in use today to extract gelatin from bones. In personal notes, he mentions that he participated in a supper of the Royal Society and all the food was prepared with his device.  (Robinson, 1947)

Old illustration of steam digester invented by French physicist Denis Papin in 1679.jpg

Old illustration of steam digester invented by Denis Papin in 1679.

Gelatine’s value as a food source was revived by food scarcity during the French Revolution, at which time Louis Proust (1754-1826) improved the methods of gelatine manufacture. “The famous physician and physiologist Frangois Magendie (1783-1855) served as chairman of the French commission for examining the nutritive value of gelatine in 1842.” (Sahyun, M. (Editor). 1948)  (Dawson, 1908)

In the early 1800s, it was believed that as much bouillon (French for broth, sold as stock cube in Australia, Ireland, New Zealand, South Africa, and the UK or broth cube in the Philippines), in the form of dehydrated bouillon,  could be obtained from one pound of bones as from six pounds of meat.  Its commercial development was, therefore, viewed with great interest. “In 1817 Jean d’Arcet, Jr. or Jean Darcet (7 September 1724 – 12 February 1801), a French chemist, devised a new method of extracting gelatin from bones in order to provide food for the poor.   A device which soon supplanted the older methods in which used papain (a proteolytic enzyme extracted from the raw fruit of the papaya plant, being a proteolytic enzyme which helps to break proteins down into smaller protein fragments called peptides and amino acids which forms the basis of its use as a meat tenderizer) or boiling with acids was developed.  The statement of d’Arcet that he was now able to make five beeves (plural for beef) out of four, coupled with the approval with which the College de France looked upon his results, led to the quite general introduction of d’Arcet’s extract of bones into the hospitals and almshouses in Paris, where 60 grams of this gelatin were regarded as equivalent to 1,500 grams of meat. But soon criticisms and complaints began to arise in various quarters.”  (Dawson, 1908)

“On June 30, 182 1, M. Donne read a paper before the Academy of Sciences. He had experimented on himself, he reported that he had promptly lost two pounds in weight, while animals which he had fed with gelatin soon showed such a distaste for it that they preferred to die of starvation rather than eat it.  Moreover, on November 8th of the same year appeared a report of the physicians and surgeons of the Hotel-Dieu. Their six conclusions may be summarized by saying that in comparison with the bouillon made with meat, that in which gelatin was employed was more distasteful, more putrescible, less digestible, less nutritious and that, moreover, it often brought on diarrhoea. This report was signed by amongst others, Magendie.  (Dawson, 1908)

The Academy of Sciences now took steps in the matter and appointed a committee to investigate known as the “Gelatin Commission.”  (Dawson, 1908)

Dawson (1908) summarises the commission’s work.  He states that the members of the commission characterized their findings as “very conservative” and could give the following conslusions:

“1. By no known process can there be extracted from bones a substance which, either when taken alone or when mixed with other substances, can replace meat.”  (Dawson, 1908)

“2. Gelatin, fibrin, albumen, taken alone, support animals for a very limited time. In general, these substances soon excite an intolerable distaste to a degree which renders starvation preferable.”  (Dawson, 1908)

“3. The same immediate principles artificially united and rendered of an agreeable sapidity by seasoning, are accepted with more resignation and for a longer time than when they are isolated ; but finally they have no better effect upon the nutrition, for animals which eat them, even in considerable quantities, die with the symptoms of complete inanition.”  (Dawson, 1908)

“4. Meat (muscle) in which gelatin, albumen, and fibrin are united by the laws of organic nature and are associated with other materials as fats, salts, etc., suffice even in very small quantities for a complete and prolonged nutrition.” (Dawson, 1908) This conclusion is in very much in line with our current food legislation.

Dawson (1908) states that “the remaining five conclusions, though interesting, are perhaps of somewhat less importance than those which have already been quoted.”

The conclusions of the commission were not all that unanimous, but however you look at it, the nutritional value and later, the nitrogen content was questioned.  Its nutritional qualities were disputed since the 1600s. In 1907 the German Hygienist and Bacteriologist, K. B. Leighman became the first scientist to study the toughness of meat and this became the first step towards identifying the amount of connective tissue in different muscles. This, in turn allowed a proper investigation into the nutritional value of connective tissue.

Measuring Connective Tissue in Meat and Determining its Nutritional Value

K. B. Lehmann (1907) showed that the toughness of raw meat depended largely upon its content of connecting fibre.  Together with some of his students, they were also able to show that a decrease in toughness resulting from cooking was related to the collagen of connective tissue rather than to the elastin.  Under the influence of moist heat, the collagen is readily changed to gelatin, thus losing its toughness.  He employed two devices in his analysis.  One measured the force needed to bite through a meat sample and the second, measuring the breaking strength of the muscle.  Evaluation of meat texture grew into a distinct field of study (Chichester, et al. (Editors), 1965), and he put the matter of the percentage of connective tissues to lean meat firmly on the agenda of scientists.

The nutritional value of connective tissue and its occurrence in different muscles was taken up by Mitchell in the 1920s along with a number of coworkers.  In a published article from 1927, they state that before their work was undertaken there was “evidence of a circumstantial character that the more fibrous a cut of meat the lower the biological value of its nitrogen would be. Thus, it was found that a cut of veal, evidently very fibrous when dried, ground, and sieved, gave a biological value of only 62, considerably lower than the values obtained with other meat samples; i.e., 69 for a sample of beef and 74 for a sample of pork. Similarly, in unpublished experiments, the biological value of the total nitrogen of a particularly tough and fibrous piece of beef, the lower round (heel) cut from a bull, was found to be only 56, not much higher than the value for white flour; i.e., 52.”  (Mitchell, et al, 1927)  When Mitchell started his investigations, what was lacking in scientific literature was quantitative data relating to the connective tissue content of the various samples of meat used.  Along with others at this time, they remedied this through their work.

In terms of nutrition, they found that the biological value of the nitrogen of pork tenderloin which was one of the pork muscle they investigated, containing a minimal amount of connective tissue, was found to be 79.  In comparison, they created pork cracklings where they removed the fat and consisting largely of connective tissue and on the same scale as used for the tenderloin, found the biological value of the nitrogen to be 25.

“When the two materials were mixed in the proportion of 3 parts of tenderloin nitrogen to 1 part of cracklings nitrogen, a distinct depression of the biological value of the tenderloin nitrogen was observed, the mixture possessing a value of 72.”

The way that they tested for connective tissue in meat is of interest.  Mitchell states that K. B. “Lehmann found that results of the mechanical test were so variable that a series of ten to twenty individual determinations should be run in order to obtain a representative average value. When only a limited amount of meat is available, therefore, it may be impossible to use the mechanical test to advantage” and he suggested the use of chemical measured for toughness.

From Mitchell’s 1926 article, The Determination of the Amount of Connective Tissue in Meat, they used two methods in determining the connective tissue content in different muscles, one being mechanical and the other being chemical.

They were able, mainly through chemical processes to separate collagen and elastin out sufficiently and to use it in trails to show through animal feeding trails that the nitrogen from these are of a lesser nutritional value than nitrogen from the muscle protein. (Mitchell, 1927). It is this determination that is at the heart of the limits that is placed on the inclusion of collagen protein in processed meat products. Their findings corroborated work done by Hoagland and Snider in the USA in 1926 where they showed that there is a wide difference in the nutritive value of the proteins of various organs and tissues of the animal. As early as 1932, Curtis, et al, speculated as to the reason for the findings of Mitchell and suggested that it was “probably due to the fact that the proteins of connective tissues are high in collagen which yields gelatin, which is recognized as being deficient in tyrosine, cystine and tryptophane.” (Curtis, 1932)

It can be seen from this, why some countries opted to deduct the nitrogen from connective tissue when determining protein and lean meat from the total nitrogen found in the meat product. Nutritionally, the proteins referenced by the N in connective tissues and that from other muscle protein is not be the same. It becomes complicated and many countries don’t bother make this distinction.

Let’s take a look at how some handled the issue in the past and today to get an appreciation for the complexity of the issue.

Converting Proteins to Meat

South Africa

I South Africa we use 6.25 to effect nitrogen to protein conversion and then use a factor of 30 to convert protein to lean meat (or we use 4.8 to convert directly from nitrogen to lean meat, being 30/6.25 = 4.8)  From the South African Food, Drugs and Disinfectants Act No. 13 of 1929, 4 (iv) which reads as follows: “In all cases where it is necessary to calculate total meat under regulations 14 (1), (2), (3) and (4), the formula used shall be:—

Percentage Lean Meat = (Percentage Protein Nitrogen × 30)

Two ratios we are therefore familiar with are used to move from protein content in a substance to lean meat.  These ratios are, the ratio of percentage protein nitrogen to lean meat %, being % N x 30 = lean meat % and the nitrogen to protein factor which is 6.25 meaning N x 6.25 = total protein.

Direct conversion factor between % protein and lean meat will, therefore, be 30 / 6.25 = 4.8.  This is used as %N in a substance x 4.8/100 = Lean Meat Content (eqw).  Soya that contains 50% protein, therefore, is equal to 50 x 4.8 = 240/ 100 = 2.4 eqw Lean Meat Equivalent. No mention is made of nitrogen from connective tissues.

United Kingdom

In the UK, including connective tissues in meat content calculations have been used for many years. They use different ratios to convert between nitrogen content and lean meat directly without the factor 30 conversion between protein and lean meat, used in South Africa.  “Meat content in sausages is done using average nitrogen factors for lean meat, including the portion of connective tissue and fat, normally associated with lean meat.  The following percentages nitrogen on a fat-free basis have been agreed upon between the Society for Analytical Chemistry, the Royal Society of Chemists and others and are used for control purposes in the UK.”  (Ranken, et al., 1997)

Breast of Chicken3.9
Dark meat of chicken3.6
Whole carcass chicken3.7
Ox liver3.45
Pig liver3.65
Liver of unknown origin3.55

“The pork and beef factors are average values for all cuts of meat from the animals in question and may be incorrect for particular cuts whose composition (proportion of connective tissue in intermuscular fat) differs markedly from the average, as is the case with many of the cuts used for manufacturing.”  (Ranken, et al., 1997)

“The factors recommended by the Analytical Methods Committee of the Royal Society of Chemistry have been changed from time to time, the most recent recommended values for pork meat (with interstitial fat but without rind and subcutaneous fat (1991) include:

whole side:  3.5
leg:  3.49
neck or collar:  3.38
hand: 3.42
loin: 3.66
belly:  3.5

They dealt with the matter of connective tissue. “The analysis may estimate the connective tissue from a determination of hydroxyproline, deduct the connective tissue nitrogen content from the total nitrogen and calculate a connective tissue free-meat content.  On the assumption that pork meat with the rind on, or beef flank meat, contains no more than 10% of connective tissue, a value for added connective tissue can be calculated.”  (Ranken, et al., 1997)

“Of course, meat products may contain nitrogenous substances other than meat protein and the detection and estimation of these may present difficulties.  The Stubbs and More calculation applied to the analysis of British sausages assumes that the non-meat solids present consist of rusk with a nitrogen content of 2% and the appropriate deduction is made from the total nitrogen content before calculating and “apparent” meat content.  Soya, milk or other proteins may be estimated spectroscopically or by other means, provided that the sample has not been strongly heated and the appropriate corrections made.  ELISA (enzyme-linked immunosorbent assay) methods can be used for cooked samples.” (Ranken, et al., 1997)

“Attempts have been made to estimate meat content directly by measurement of the content of 3-methylhistidine, an amino acid which is characteristic of meat protein, but this process is not reliable unless the species of meat is known, the 3-methylhistidine content of muscle being rather variable.” (Ranken, et al., 1997)

In other countries (besides the UK), as is the case in South Africa, reference is made to the composition of meat products by referring to the nitrogen or protein content of dry, fat-free products.  Some countries refer to the water : protein or similar ratios.  (Ranken, et al., 1997)

“From the figures above the water : protein ratio in muscle meat is close to 77% water : 23% protein = 3.35.  The ratio is a pure number, independent of the fat ratio of the meat.”  (Ranken, et al., 1997)

Germany: The Feder Number

“The Feder number, used in Germany, is the ratio of water to organic, non-fat in a sample, or,

Feder Number.png

Organic non-fat consists of the protein plus other substances which are almost all nitrogenous; in practice, this is close to the protein content as estimated from the nitrogen content.”  (Ranken, et al., 1997)


“In France, the relationship HPD (humidité du produit degraissé or ‘moisture of the defatted product’) is used.  For pure muscle from the date provided, the value is 77%.”  (Ranken, et al., 1997)


“In USA regulations and in the FAO/ Codex recommendation a related ratio is used protein on fat-free (PRF) or,

US Calculation.png

The limiting value of the expression is the protein content of the fat-free muscle or 23%.

The expression (100 x fat %) in both HPD and PRF  is not the protein plus water content of the sample but water plus protein plus ash.  It may, therefore, be affected by differences in ash content as will be found for instance in cured meats.”  (Ranken, et al., 1997)

In the USA, protein of ingredients that are derived by “hydrolysis, extraction, concentration or drying”  (CFR, 2007c; USDA-FSIS, 1995b) are considered as non-meat protein for formulation purposes.  (Tarté, R. (Editor), 2009)


Likewise, in the EU, “meat-related ingredients derived from meat protein, fat or connective tissue and which have undergone a treatment such as purification (e.g. gelatin, collagen fibre, refined fats, etc. . . .), hydrolysation (e.g. protein hydrolysates, etc. . . .), extraction (e.g. meat extract, bouillons, etc.c. . . .)” (CLITRAVI, 2002) are all excluded from the definition of meat as is mechanically deboned or recovered meat.  These must be listed separately on product labelling.  (Tarté, R. (Editor), 2009)


Calculating lean meat content in a formulation is a complicated task and legislation should be studied carefully to ensure compliance. Many countries limit the amount of connective tissue or proteins derived from connective tissue such as gelatin and handle what must be declared on product labelling differently.

Further Reading

On Gelatin, chapter 4, Protein Gelation.  Damodaran, S. (Editor).  1997.  Food Proteins and Their Applications.  Marcel Dekker.

How much meat is in your sausage?

Understanding the theory and practice of meat content calculations

From the Food Safety Authority of IrelandMeat_Content_Calculation

Historical, mathematical and nutritional bases of Pearson Square as a fit method for ruminant rations

Simple Ration Formulation- Pearsons’s Square

Chapter 7 of Tarté, R. (Editor), Ingredients in Meat Products: Properties, Functionality, and Applications, published by Springer. Chapter 7 is titled “Meat-derived protein Ingredients” and was written by Rodrigo Tarté.

Return to Index page.

(c) eben van tonder

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Robinson, H. W..  1947. Denis Papin (1647-1712). Notes and Records of the Royal Society of London, Vol. 5, No. 1 (Oct., 1947), pp. 47-50 (4 pages), Published by: Royal Society

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Tarté, R. (Editor). 2009.  Ingredients in Meat Products: Properties, Functionality and Applications.  Springer

Photo Credit:

An ancient style of Chinese bronze tripod called a Ding

Old illustration of steam digester invented by French physicist Denis Papin in 1679

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