Determining Total Meat Content (Part 5): The Proximate Analysis, Kjeldahl and Jones (6.25)
By Eben van Tonder
5 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
Summary
We have progressed in our study of the historical development of the concept of using Nitrogen to determine meat content to the Proximate analysis, and its accompanying use of the Kjeldahl method, the Jones factors and a review of the nutritional importance of the Proximate system. Our study leads us to unexpected places as we are challenged in our views on managing a complex operation like a large food factory.
Food Analysis
The history of the development of food analysis goes back to the people we met in our introductory articles in the persons of Baussingault, and Liebig. To the list, we should probably add Sir Humphrey Davey, but Davey held a fundamentally different view of nutrition compared to Liebig and Baussingault. Where these two men held the basis for plant nutrition to be mineral, Davey was in the camp of Albrecht Daniel von Thaer (1752-1828) on the subjected who believed humus to be the foundational principle of plant nutrition. “According to this theory, humus is the main source of plant nutrients, next to the previously recognized role of water, obviously. In Thaer’s opinion, minerals played only a supporting role in providing plants with humic compounds. Therefore, the whole soil fertility depends only on the amount of humus present in it. He presented his views in his work “The Principles of Agriculture”.” (Antonkiewicz and Łabetowicz, 2016)
“The humus theory for plant nutrition was the dominant concept explaining the essence of plant nutrition for tens of years. Von Liebig was the first to explain, through his experimental works, the basics of the problem of mineral plant nutrition. In 1841, a publication entitled “Die Organische Chemie in ihrer Anwendung auf Agrikultur und Physiologie” – “Organic Chemistry in Its Applications to Agriculture and Physiology” was released, with a new theory of mineral plant nutrition. This book opens a new chapter in the development of the science of plant nutrition. It attracted great interest not only in scientific world but also among a lot of farmers. Liebig wrote that not humus but mineral salts (are taken up with water by roots from soil) and carbon dioxide assimilated from air in the photosynthesis process are the direct food for plants. For stable plant yields, soil should be supplied with mineral fertilizers in order to replenish the deficiency of nutrients caused by their removal from the field along with plant yield. Liebig formulated his theory about mineral plant nutrition based on other scientists’ studies, and also through deduction from a chemical analysis of plants. As a chemist and analyst, he conducted many studies on the chemical composition of plants. He determined that plants release carbon, hydrogen, oxygen and nitrogen (which are present in them) during combustion, and composition of the generated ash always includes phosphorus, sulfur, calcium, potassium, magnesium, silicon, and many times sodium.” (Antonkiewicz and Łabetowicz, 2016)
In the end, it was the scientific rigour of Liebig that won the day. Not just his new techniques opened up new discoveries, but also the question of whether science and practice presented a duality. These two concepts were juxtaposed in the mind of the landowners who saw the views of Liebig and Albrecht Daniel von Thaer as much more than a debate about the essence of plant nutrition. Liebig’s word was scientific and based in a laboratory. Of course, it had to have practical application, but he wrestled with solving fundamental questions first before he moved on to the practical and in many instances, as we know from our own experiences, even the best scientific work doesn’t always work in practice at the first attempt. Such is the nature of the beast.
Von Thaer, on the other hand, was a consummate pragmatist and by all accounts, a skilled manager. I am impressed by the reported neatness of the workshops on the estates that he was in charge of. Reports have it that they were open for inspection by the public and everything had its place. Thaer’s work, “Principles of Agriculture” contain the result of his experience through a series of years. We can feel his passion and approach in his work. It embraces the theory of the soil, the clearing of land, ploughing, manuring, and irrigation, hedges and fences, management of meadow and pasture lands; the cultivation of wheat, rye, corn, oats, barley, buckwheat, hops, tobacco, clover, and all the varieties of grasses; the economy of kine stock, breeding and feeding; the management of the dairy, and the use of manures, and the various systems of cultivation, keeping journals and farm records. In brief, it is a complete cyclopaedia or circle of practical agriculture. (Homans, 1857)
These issues that we don’t necessarily see as opposing views today would take on a life of its own in Germany which impacted (determined?) the course of history related to the nutritional sciences and the evaluation of foods.
As Liebig’s approach of rigorous science and experimentation started to dominate, tools were being developed on which more discoveries were predicated. Better techniques were developed, gradually, to separate the food fragments which played a role in nutrition including protein, fat, and fibre. The Liebig/ Dumas method for example, for determining the Nitrogen content in food was developed in 1830 and 1840. The famous Kjeldahl method was published in 1883 and much later the use of the 6.25 conversion number of N to protein would become the more complete Jones numbers. At the dawn of the 20th century, food chemistry was firmly established. (Dryden, 2008) Along with improved techniques and tools, the philosophical wars raged on.
The Proximate Analysis
Background
The German Agriculture Research Stations was a driving force in the development of German farming from the mid-1800s and a model for similar developments around the world. Wilhelm Crusius has it that the first German Agricultural Research Station was created on 28 September 1850 during a banquet honouring Leipzig’s new marble statue of Albrecht Daniel von Thaer. He recalls that several agricultural leaders from the kingdom of Saxony agreed to terms that saw the Möckern estate near Leipzig transformed into an institution to investigate the application of scientific knowledge to agriculture. The Möckern facility is widely believed to be the words first state-supported agriculture research station. It was believed that the Möckern facility represented a fulfilment of Thaer’s vision. He propagated a system of “rational” agriculture. Landowners loved him as they looked for ways to increase their yields through comparative investigations, but was sceptical of what we call research. (Finlay, 1988)
It is generally believed to be Liebig who founded the agriculture research stations, through his 1840 work Chemistry and its application to Agriculture and Physiology. Many suggested that it was the excitement created by this publication around the world, that Germanys research stations were founded upon. It is believed by many that the agricultural research station became a haven for the agricultural chemists. The line of thinking then continues that the American Agricultural stations were created based on the German model in the 1870’s and 80’s. Even the notion of research is also linked to Liebig through his famous research laboratory in Giesen. (Finlay, 1988)
A study of the Möckern facility challenge these notions. Fundamental science and agriculture chemistry were not, in fact, initial driving forces behind this first agriculture station. The Saxon officials had praise for Liebig’s recognition of the importance of chemical compounds in plants and animal growth but scorned his insistence on laboratory research. Liebig created plant manure in his laboratory. It was practically insoluble. A white chemical crust formed over fields in practical demonstrations and his invention was a disaster. Many believe that the creation of the agriculture stations would provide an opportunity to verify such work. They saw a division between science and practice and the stations would be a place to unite these two polar opposites. In the early days of the Möckern facility, scientists and what was called “practitioners” had equal authority. As in America, in Germany, the scientists did not win control over these stations for some time and not after a long and hard battle. (Finlay, 1988)
In a 1988 article, The German Agricultural Experiment Stations and the Beginnings of American Agricultural Research, Finlay brilliantly examines the leading forces behind the creation of the agricultural stations and the duel between those who relied on science and those who held practice to be superior.
The German Agriculture Experiment stations became the model for similar stations set up in America. Wilbur Atwater of Connecticut had great admiration for the Möckern station. He got involved in work at another station, the one at Weende. Here they were involved in calorimeter research in the 1860s and Atwater expanded on the research. (Marcus, 2015)
Weende Experiment Station
The physiological chemistry work of Liebig had only an indirect application in agriculture. Wilhelm Henneberg was one of his students who applied his theories and methods directly in agriculture research. This would be one example of the triumph of science and laboratory research and fundamental to our understanding of the current methods for determining meat content. Scientists at these research stations directed the priority of work away from the achievement of immediate practical goals and towards the examination of basic scientific questions. Henneberg became the director at the Agriculture Experiment Station at Weende near Göttingen in 1857. He made a huge contribution to this shift and introduced a program using livestock as experimental organisms, incorporating the methods and instruments that he was introduced to in Munich. Precision and quantification were very important to him. He used instruments like the Petterkoffer respiration apparatus. (Phillips and Kingsland, 2015)
He stressed the importance of controlling environmental variables in laboratory settings and focused on fundamental questions in animal metabolism. His assistant was Frederich Stohmann who helped with the findings of his experiments. They directed the findings at farmers and physiologists, but in reality, made no effort to practically apply the results of their work. (Phillips and Kingsland, 2015)
Henneberg challenged the view of Thaer who emphasised close interaction of science and practice and the integration of plant and animal agriculture. Agriculture sciences rose to great prominence in Germany during this time and animal nutrition was one of its most successful branches. (Phillips and Kingsland, 2015)
Henneberg and Stohmann (1860, 1864) developed a top-level, very broad, classification of food components for routine analysis which they devised for animal feed. It is a “partitioning of compounds in feed into six categories based on the chemical properties of the compounds. This analysis was an attempt to duplicate animal digestion. (Artemia)
After extracting the fat, the sample is subjected to an acid digestion, simulating the acid present in the stomach, followed by an alkaline digestion, simulating the alkaline environment in the small intestine. The crude fibre remaining after digestion is the portion of the sample assumed not digestible by monogastric animals. In the proximate analysis of feedstuffs, Kjeldahl nitrogen, ether extract, crude fibre, and ash are determined chemically. The determination of nitrogen allows the calculation of the protein content of the sample. It is important to remember that proximate analysis is not a nutrient analysis, rather it is a partitioning of both nutrients and non-nutrients into categories based on common chemical properties.” (Artemia)
“At that time the nutritionally important components of protein had not been recognized, all neutral fats were considered to be nonspecific sources of energy, and vitamins were unknown. However, the multiplicity of the carbohydrates and the practical difficulties of their separate chemical determination were clearly recognized. These workers believed that for nutritional description the carbohydrates could be grouped into (1) the starches and the sugars, and (2) a coarse fibrous fraction. The latter they isolated as an insoluble residue after boiling the food sample first with dilute acid and then with dilute alkali. These procedures were intended to simulate the acidic gastric digestion and the subsequent alkaline intestinal digestion of ingested food. The insoluble residue they called crude fibre. With analytical figures for ether extract, ash, nitrogen, and crude fibre of a moisture-free food sample, they needed only to convert the value for nitrogen to its equivalent in terms of protein (i.e., N x 6.25), add to this the other three group values, and subtract the total from the original weight of dry sample, thus by difference to arrive at an estimate of the “soluble carbohydrates.” This fraction they called nitrogen-free extract (NFE).” (Loyed, et al. 1960)
“The majority of foods in human diets, as well as those in the diets of some laboratory animals used in nutrition studies, are so low in crude fibre that this fraction can often be disregarded, and the custom has gradually become general, especially in human nutrition, to omit the crude fibre determination. When this is done it is the total carbohydrate that is estimated “by difference.” (Loyed, et al. 1960)
“Probably because the chief components of nitrogen-free extract (NFE) are sugars and starches, we are prone to forget that this fraction includes all the nonfibrous, ether–insoluble, water-soluble organic materials of the food (or other material analyzed). Thus all water-soluble vitamins must be included in this fraction. Quantitatively, the vitamins are an insignificant part of the NFE, but in any broad charting of the makeup of foods in terms of the Weende partition these vitamins are part of the NFE in the same way that the fat-soluble vitamins are part of ether extract.” (Loyed, et al. 1960)
“Being determined by difference, the figure for NFE is also subject to an appreciable but variable error that may be as large as the algebraic sum of any analytical and/or sampling errors of each of those fractions determined by direct analysis.
Variability of average
Weende analysis values
It is appropriate at this point to comment on the errors to be expected in numerical values obtained from the several parts of the Weende analysis. These arise from several sources. Errors in the chemical manipulations-that are, analysts’ errors are usually negligible. Sampling errors, however, are often large because foods and the residues of animal digestion are not usually homogeneous. In addition, different lots of foods called by the same name are seldom identical in “proximate” makeup. Consequently, average composition figures found in tables of food composition are not necessarily applicable to a particular lot of a foodstuff. Nevertheless, it is often more feasible to estimate the protein, or the fat, or the carbohydrate, of some particular lot of a foodstuff by referring to tables of average composition than to obtain specific values by analysis. When average values are used in this way it should be remembered that the composition figures given for natural foods may be subject to coefficients of variation such as those listed in the table below.” (Loyed, et al. 1960)
“For example, if the average crude protein content of cornmeal is given in a table as 10%, it is probable that two samples out of three purchased at random would on analysis give values between 9.2 [10 minus 10 (8%)] and 10.8 [10 plus 10(8%)] percent protein (see figure above). Similarly, cornmeal may average 72% NFE, and hence two samples out of three might be expected to give values between 69.8% and 74.2% (that is, 72 ± 3%).” (Loyed, et al. 1960)
This current application of this system of analysis can be summarised as follows:
- moisture,
The moisture content is determined as the loss in weight that results from drying a known weight of food to constant weight at 100 degrees C. This method is satisfactory for most foods, but with a few, such as silage, significant losses of volatile material may take place.
- ash,
The ash content is determined by ignition of a known weight of the food at 550°C until all carbon has been removed. The residue is the ash and is taken to represent the inorganic constituents of the food. The ash may, however, contain material of organic origin such as sulphur and phosphorus from proteins, and some loss of volatile material in the form of sodium, chloride, potassium, phosphorus, and sulphur will take place during ignition. The ash content is thus not truly representative of the inorganic material in the food either qualitatively or quantitatively. There is incomplete recovery of individual minerals. It is one of the aspects of the proximate analysis, less used in modern food analysis. (hmhub.me) “Ash is a mixture of food minerals. The food Organic Matter (OM) content is (OM = DM – ash) is frequently used as a way of correcting data for mineral contamination, as will happen when measurements are made with grazing animals.” (Dryden, 2008)
- crude protein,
The crude protein (CP) content is calculated from the nitrogen content of the food, determined by a modification of a technique originally devised by Kjeldahl over 100 years ago. In this method, the food is digested with sulphuric acid, which converts to ammonia all nitrogen present except that in the form of nitrate and nitrite. This ammonia is liberated by adding sodium hydroxide to the digest, distilled off and collected in standard acid, the quantity so collected being determined by titration or by an automated colourimetric method. It is assumed that the nitrogen is derived from protein containing 16 percent nitrogen, and by multiplying the nitrogen figure by 6.25 (i.e. 100/16) an approximate protein value is obtained. This is not ‘true protein’ since the method determines nitrogen from sources other than protein, such as free amino acids, amines and nucleic acids, and the fraction is therefore designated crude protein. (hmhub.me)
The Dumas method in which a sample is burnt and the N gas released is measured, was developed before the Kjeldahl method but has become popular only following the development of automated methods of carrying out the analysis. The method recovers all the sample N and so may give slightly higher values than the Kjeldahl method, depending on the sample analysed.” (Dryden, 2008)
- ether extract,
The ether extract (EE) fraction is determined by subjecting the food to a continuous extraction with petroleum ether for a defined period. The residue, after evaporation of the solvent, is the ether extract. As well as lipids it contains organic acids, alcohol, pigments, fat-soluble vitamins, waxes, as well as fats. (hmhub.me) It is used to isolate lipids for more detailed fractionation into fatty acids and waxes. The EE is, nevertheless, still reported as a measure for total lipid. In the current official method, the extraction with ether is preceded by hydrolysis of the sample with sulphuric acid and the resultant residue is the acid ether extract. (Dryden, 2008)
- crude fibre, and
Einhof extracted the fibrous part of food in 1805. His concept of fibre is very distant from our present-day understanding. To him, it was what was obtained after rubbing and washing the food to obtain the residue that is “resistant.” He may have thought that it was not nutritious. Boussingault and Davy specifically stated that it is not since it could not be digested. They had no experimental proof of this. (Dryden, 2008) Henneberg and Stohmann developed a method for analyzing crude fiber in 1859. The carbohydrate of the food is contained in two fractions, the crude fibre (CF) and the nitrogen-free extractives (NFE). The former is determined by subjecting the residual food from ether extraction to successive treatments with boiling acid and alkali of defined concentration; the organic residue is the crude fibre. (hmhub.me)
- nitrogen-free extractives.
When the sum of the amounts of moisture, ash, crude protein, ether extract and crude fibre (expressed in g/kg) is subtracted from 1000, the difference is designated the nitrogen-free extractives. The crude fibre fraction contains cellulose, lignin, and hemicelluloses, but not necessarily the whole amounts of these that are present in the food: a variable proportion, depending upon the species and stage of growth of the plant material, is contained in the nitrogen-free extractives. Nitrogen Fee Extracts (NFE) was originally assumed to be mainly soluble carbohydrate and so was expected to be highly digestible. The nitrogen-free extractives fraction is a heterogeneous mixture of all those components not determined in the other fractions. It includes sugars, fructans, starch, pectins, organic acids, and pigments, in addition to those components mentioned above. (hmhub.me)
“Unfortunately, the reagents used to measure crude fibre (CF), may remove up to 60% of the cellulose, about 80% of the hemicellulose and a highly variable (10 – 95%) proportion of the lignin.The NFE contains some of the plant cell wall material an can be less digestible than CF. Besides this, NFE contains all those chemical entities which are not measured by the other methods. NFE is not now used in food analysis.” (Dryden, 2008)
(Source: hmhub.me)
Schematically, it can be represented as follows: (chart by (Dryden, 2008))
This system had a long-lasting effect on our approach to food analysis. We still use Dry Matter as the basis for expressing analytical data in calculating food intake of formulating diets. (Dryden, 2008)
N is important
Nitrogen is important for meat curers due to the role of NO formation from NO2- in the curing reaction. Have a look at my 2016 article, Mechanisms of meat curing – the important nitrogen compounds, and Reaction Sequence: From nitrite (NO2-) to nitric oxide (NO) and the cooked cured colour. Nitrogen in organic material is present in the form of amine groups (-NH2) as constituents of amino acids (proteins) and amino sugars and measuring it is important to a study of proteins and nutrition in general.
“The chemistry of nitrogen is complex due to the fact that nitrogen assumes several oxidation states (Sawyer et al., 2003). Nitrogen is one of the most important elements for plant nutrition. The compounds of nitrogen are of great worth in water resources, in the atmosphere, and in the life process of all plants and animals. Four forms of dissolved nitrogen are of greater interest: organic, ammonia, nitrite, and nitrate, ordered in an increasing state of oxidation. All these forms of nitrogen, as well as nitrogen gas (N2), are mutually convertible, being components of the biological nitrogen cycle (Pehlivanoglou-Mantas and Sedlak, 2006; Worsfold et al., 2008). It is very important to ascertain the contribution (fractions) of different nitrogen species to the total nitrogen content (Prusisz et al., 2007).
Testing for N
The modern version of the proximate analysis uses mostly the Kjeldahl method of testing for N. Few other companies in history had such a dramatic effect on chemistry in general and food chemistry in particular as the Danish beer producer, Carlsberg. S.P.L. Sørensen was Director of the Carlsberg Laboratory’s Department of Chemistry from 1901 to 1938. In 1909, he developed the pH scale – a method for specifying the level of acidity or alkalinity of a solution on a scale from 0-14 and demonstrated the significance of pH for biochemical reactions, including those involved in brewing. With the invention of the pH scale, Carlsberg could ensure high quality of every beer. The applications of the pH scale have since been countless throughout all fields. (carlsberggroup.com)
It is remarkable that more than 20 years before the pH scale was invented, his predecessor from the same institution invented the definitive measurement for N in protein. Here is the story, told by Sáez-Plaza, et al. in their Overview of the Kjeldahl Method of Nitrogen Determination, Part I and Part II.
“The Danish chemist Johan Gustav Christoffer Thorsager Kjeldahl (1849–1900), Head of Chemistry Department of the Carlsberg Foundation Laboratory of the Danish Brewing Carlsberg Company, introduced a method known later under the eponym the Kjeldahl method that basically is still in use. It was first made public at a meeting of the Danish Chemical Society (Kemisk Forening) held on March 7, 1883 (Burns, 1984; Johannsen, 1900; McKenzie, 1994; Oesper, 1934; Ottensen, 1983, Veibel, 1949). Within the same year, the method was published in the German journal Zeitschrift f¨ur Analytische Chemie (Kjeldahl, 1883a), and written in French and Danish languages in communications from the Carlsberg Laboratory (Holter and Møller, 1976; Kjeldahl, 1883b, 1883c; Ottesen, 1983).” (Sáez-Plaza, et al, 2013)
“Because of the respect that the founder of the laboratory, the Danish brewer J. C. Jacobsen, had for Pasteur and his work for the French wine industry (Burns, 1984), extensive French summaries of the Carlsberg papers were also published. As an extended summary of the Kjeldahl paper appeared in Chemical News in August (Kjeldahl, 1883d), the method was quickly taken up (Sella, 2008). The Analyst first gave details of the method in 1885 “for the benefit of those who may have missed the original paper” (Burns, 1984; Editor of The Analyst, 1885, p. 127), although the method had been briefly mentioned by Blyth (1884), who gave Kjeldahl’s name incorrectly as Vijeldahl. A surprisingly short period went by between the publication of the Kjeldahl method and the appearance of publications affecting further improvements (Dyer, 1895; Hepburn, 1908; Kebler, 1891; Vickery, 1946a), both in Europe and the U.S., due to the tremendous impact that the Kjeldahl work had on others, especially in Germany (McKenzie, 1994).
Most of the earlier contributions were discussed by Fresenius in the Zeitschrift, often to a length of several pages (Vickery, 1946a). Throughout the history of analytical chemistry, none of the methods has been as widely adopted, in so short a time, as the “Kjeldahl Method” for the estimation of nitrogen, as stated by Kebler (1891) at the beginning of an annotation in which he compiled references on the estimation of nitrogen by the Kjeldahl method (some 60) and by all other methods (about 200).” (Sáez-Plaza, et al, 2013)
“The Kjeldahl method was originally designed for the brewing industry as an aid in following protein changes in grain during germination and fermentation (Bradstreet, 1940; Kjeldahl, 1883b); the lower the amount of protein in the mush, the higher the volume of beer produced. It was Berzelius who suggested the use of the word “protein” in 1838 in a letter to Mulder because it was derived from the Greek word meaning “to be in the first place” (Zelitch, 1985). The Kjeldahl protein content is strictly dependent on total organic nitrogen content (Wong et al., n.d.); i.e., protein structure will not interfere
with the accuracy of protein determination.”
As we have mentioned earlier in this article, a drawback of the Kjeldahl method is that it lacks the “analytical selectivity because it does not distinguish protein-based nitrogen from nonprotein nitrogen. Adulteration incidents (e.g., adulteration of protein-based foods with melanine and related nonprotein compounds) exploiting this analytical vulnerability have been recently detected (Breidbach et al., 2010; Levinson and Gilbride, 2011; Moore et al., 2010; Tyan et al., 2009) and are new examples of a problem that dates back to before the Kjeldahl method was introduced (M¨oller, 2010a).” (Sáez-Plaza, et al, 2013)
“The presence of non-protein nitrogen (NPN) compounds in foods (aminoacids, ammonia, urea, trimethylamine oxide) overestimates their true protein content (M¨oller, 2010a; van Camp and Huyghebaert, 1996; Yuan et al., 2010) as derived from the current nitrogen determination methods. Separation of NPN from true protein nitrogen may be carried out by adding a protein precipitating agent such as trichloroacetic acid or perchloric acid (Rowland, 1938a, 1938b). The process conditions applied during protein precipitation, however, affect the composition and the amount of NPN, so it is mandatory to specify the type and concentration of precipitating agents used in each case. Alternative techniques such as dialysis and gel filtration are probably more accurate in removing the NPN fraction (van Camp and Huyghebaert, 1996), but they remain unacceptable for routine analysis. Reviews of NPN determination methods in cow milk, and on aspects concerning the composition of NPN fraction, are given by Wolfschoon-Pombo and Klostermeyer (1981, 1982). The Kjeldahl method measures what is termed total protein (American Jersey Cattle Association, n.d.). The alternative use of true protein (total nitrogen minus the NPN) has been under debate for some years (Grappin, 1992; Harding, 1992; Rouch et al., 2007; Salo-V¨a¨ananen and Koivistoinen, 1996). A fundamental change in milk pricing in the U.S. was introduced January 1, 2000, with the implementation of producer payments in Federal Milk Marketing orders on the basis of the true protein content (American Jersey Cattle Association, n.d.; Stephenson et al., 2004; Zhao et al., 2010).” (Sáez-Plaza, et al, 2013)
“The protein content in a foodstuff is estimated by multiplying the nitrogen content by a nitrogen-to-protein conversion factor, usually set at 6.25 (Comprehensive Review of Scientific Literature . . . , 2006; Mariotti et al., 2008), which assumes the nitrogen content of proteins to be 16%. It is not clear who first reported such a factor for use (Moore et al., 2010). This general conversion factor is used for most foods because their non-protein content is negligible. However, pure proteins differ in terms of their nitrogen content because of differences in their amino acid composition, ranging from 13.4% to 19.3%; different multiplying factors are suitable for samples of different kinds. The factor 5.7 is applied for wheat and 6.38 for dairy products (O’Sullivan et al., 1999) and 6.394±0.004 for cheddar cheese, as shown recently (Rouch et al., 2008). The proximate system where protein is measured as total nitrogen multiplied by a specific factor clearly dominates food composition studies (Greenfield and Southgate, 2003). As a matter of fact, most cited values for protein in food composition databases derive from total nitrogen or total organic nitrogen values.” (Sáez-Plaza, et al, 2013)
“A large variety of food proteins, either from animals (milk, meat, eggs, blood, fish) or plants (seeds, cereals), is nowadays available in the food industry. The determination of protein in foods and food products has important nutritional, functional, and technological significance (Van Camp and Huyghebaert, 1996). The protein content determines the market value (Krotz et al., 2008; Wiles et al., 1998) of major agricultural commodities (cereal grains, legumes, flour, oilseeds, milk, and livestock feeds). In addition, the quantitative analysis of protein content is necessary for quality control, and also a prerequisite for accurate food labelling (Owusu-Apenten, 2002). Protein analysis is required for a very wide range of animal and human nutrition products. Consumer interest in soy protein products has increased rapidly in Western cultures in recent years. This trend is due in part to the high-quality protein of soy foods and soy protein ingredients and in part to their associated health benefits (Jung et al., 2003); 25 g of soy protein per day may improve cardiovascular health (U. S. Food and Drug Administration, 1999). Consequently, precise determinations of protein content of soy products are very important. Total nitrogen concentration in soils is one of the most frequently measured nutrients in soil-testing laboratories (Sharifi et al., 2009). Determination of nitrogen content plays a key role in assigning values to insulin reference materials (Anglow et al., 1999). Primarily devised for the determination of protein nitrogen, the Kjeldahl method has been extended to include the determination of various other forms of nitrogen, e.g., in soils, plant materials, biological tissues, and wastewater matrices (Chemat et al., 1998). (Sáez-Plaza, et al, 2013)
“Though the Kjeldahl procedure is hazardous, lengthy, and labour-intensive, it has become the industry standard; it remains an accurate and reliable method and is used to standardize other methods (ISO, 2009a, 2011; Orlandini et al., 2009a; Orlandini et al., 2009b; Rayment et al., 2012). Semiautomated or fully automated nitrogen (protein) analysis systems based on the classical Kjeldahl procedure (Rhee, 2001; Wright and Wilkinson, 1993) are preferable in order to cut costs and to save time when a large number of samples need to be analyzed.” (Sáez-Plaza, et al, 2013)
The automation of chemical methods used routinely in research can lead to a considerable saving in time and labor and, thus, efficiency in carrying out a particular piece of work (Davidson et al., 1970; Feinberg, 1999). Automation makes it possible to avoid direct handling of dangerous reagents (Pansu and Gautheyrou, 2006), such as boiling sulfuric acid or concentrated soda. Ferrari (1960) succeeded in automating the Kjeldahl nitrogen procedure, describing the new concept of continuous nitrogen determination. The automated macro Kjel-Foss analyzer was introduced in 1973, with which one can routinely perform 20 analyses/hour (Oberreith and Neil, 1974). Various degrees of automation are available for the Kjeldahl method, including automated digestion and distillation followed by manual titration; fully automated digestion, distillation, and titration; and the use of block digesters and autosamplers for the unattended analysis of a maximum of 60 samples per batch. Semiautomated equipment is available with digestion and distillation determination units at macroscale and microscale from, for example, the manufacturers Bicasa, B¨uchi, Gerhardt, Skalar, Foss- Tecator, and Velp (Pansu and Gautheyrou, 2006; Pansu et al., 2001). Depending on the analysis procedure used, the scale of operation applied, and the degree of automation installed, the analysis time of the procedure could be further reduced, corresponding to frequencies of analysis up to 20 samples/hour.” (Sáez-Plaza, et al, 2013)
Jones Factors
An important feature of the Proximate Analysis is the conversion of measured nitrogen to protein by multiplying total N by a factor to estimate the total protein content. It is still used for its simplicity and relative accuracy. The commonly used factor is 6.25 derived from the fact that protein contains 16% nitrogen. 100/16 = 6.25. Who the first person is to use this factor is not known. N in food can be measured by the Kjeldahl Method (1883) in which protein-N is dissociated from its combination with other elements by digestion in concentrated sulfuric acid (H2SO4) followed by conversion to the hydroxide and subsequent distillation and titration.” (Dryden, 2008)
“Jones, Munsey and Walker (1942) measured the nitrogen content of a wide range of isolated proteins and proposed a series of specific factors for different categories of food. These factors have been widely adopted and were used in the FAO/WHO (1973) review of protein requirements. These are listed in the table below. Several authors have criticized the use of these traditional factors for individual foods (e.g. Tkachuk, 1969). Heidelbaugh et al. (1975) evaluated three different methods of calculation (use of the 6.25 factor, use of traditional factors and summation of amino acid data) and found variations of up to 40 percent. Sosulski and Imafidon (1990) produced a mean factor of 5.68 based on the study of the amino acid data and recommended the use of 5.70 as a factor for mixed foods.
In principle, it would be more appropriate to base estimates of protein on amino acid data (Southgate, 1974; Greenfield and Southgate, 1992; Salo-Väänänen and Koivistoinen, 1996) and these were incorporated in the consensus document from the Second International Food Data Base Conference held in Lahti, Finland, in 1995, on the definition of nutrients in food composition databases (Koivistoinen et al., 1996).
If these recommendations are to be adopted, the amino acid data should include values for free amino acids in addition to those for protein amino acids because they are nutritionally equivalent. The calculations require very sound amino acid values (measured on the food) as discussed below, and involve certain assumptions concerning the proportions of aspartic and glutamic acids present as the amides and correction for the water gained during hydrolysis. Clearly, this approach would not be very cost-effective when compared with the current approach.
At the present time, it is probably reasonable to retain the current calculation method, recognizing that this gives conventional values for protein and that the values are not for true protein in the biochemical sense. However, it is important to recognize also that this method is not suitable for some foods that are rich in non-amino non-protein nitrogen, for example, cartilaginous fish, many shellfish and crustaceans and, most notably, human breast milk, which contains a substantial concentration of urea.
| Factors for the conversion of nitrogen values to protein (per g N)* | |
| Foodstuff | Factor |
| Animal products | |
| Meat and fish | 6.25 |
| Gelatin | 5.55 |
| Milk and milk products | 6.38 |
| Casein | 6.4 |
| Human milk | 6.37 |
| Eggs | |
| whole | 6.25 |
| albumin | 6.32 |
| vitellin | 6.12 |
| Plant products | |
| Wheat | |
| whole | 5.83 |
| bran | 6.31 |
| embryo | 5.8 |
| endosperm | 5.7 |
| Rice and rice flour | 5.95 |
| Rye and rye flour | 5.83 |
| Barley and barley flour | 5.83 |
| Oats | 5.83 |
| Millet | 6.31 |
| Maize | 6.25 |
| Beans | 6.25 |
| Soya | 5.71 |
| Nuts | |
| almond | 5.18 |
| Brazil | 5.46 |
| groundnut | 5.46 |
| others | 5.3 |
| * (Where a specific factor is not listed, 6.25 should be used until a more appropriate factor has been determined.) | |
| Source: FAO/WHO, 1973. |
A number of direct methods for protein analysis have been developed for specific foods based on reactions involving specific functional groups of the amino acids present; these are thus not applicable to the measurement of proteins in general. Such methods include formol titration (Taylor, 1957) and the biuret reaction (Noll, Simmonds and Bushuk, 1974). A widely used group of colorimetric methods is based on reaction with Folin’s reagent, one of the most widely used biochemically in the dairy industry (Lowry et al., 1951; Huang et al., 1976). These methods are most commonly calibrated with bovine serum albumin, which is available at high purity.” (Greenfield and Southgate, 2003)
Nutrient makeup of proximate principles
The Proximate Analysis is not used for deriving nutritional values, but it is still important, before leaving this subject, that we should specifically relate it to several nutrients. “We shall thus also delimit the extent to which the Weende scheme can be expected usefully to describe specific nutrients and groups of nutrients found in the animal body and in its food.” (Loyed, et al. 1960) I quote this from Loyed, et al. who was published in the 1960s. Despite the age of the work, I find it remarkably complete as an introduction to the subject.
“Carbohydrates The total number of edible materials that are carbohydrate by definition is large. There are, for example, a dozen or more that are found in everyday foods, either as one of six or seven “sweet” sugars, or in combinations of them comprising numerous more complex molecules such as the starches, hemicelluloses, or celluloses. The monosaccharide sugars are classified according to the number of carbon atoms in their molecules. Thus there are 5-carbon or pentose sugars, and 6-carbon or hexose sugars. All pentose sugars have the same empirical formula, C5H10O5; the empirical formula for all hexoses is C6H12O6. The complex carbohydrates are merely polymers of the simple sugar units, as (C5H8O4)n or (C6H10O5)n. Cellulose, for example, has been estimated to consist of 1000-2000 hexose units polymerized into the long fibrous chains characteristic of the cellulose structure. The distribution of the carbohydrates between the Weende nitrogen-free extract and crude fibre fractions is shown in the table below.” (Loyed, et al. 1960)
“It will be seen that the carbohydrate portion of our foods and feeds consists either of single 1-carbon or 6-carbon units or of larger molecules formed by combinations of such structures. Before the larger molecules can be useful in nourishing the body they all must be degraded by enzymes in the digestive tract to their simple 5- or 6-carbon units; or, in the case of cellulose and perhaps of some of the hemicelluloses, to either the 2-, 3-, or 4-carbon molecules of acetic, propionic, or butyric acids, respectively. (These three acids are products of digestion by microflora inhabiting the digestive system of animals, such as herbivores.)” (Loyed, et al. 1960)
“The nutritional significance of the fact that carbohydrates are all assemblies
of 5- or 6-carbon units is that they have potentially about the same energy value, roughly between 3.75 and 4.25 kilocalories per gram of dry substance. Except for small amounts of ribose, carbohydrates can be considered useful primarily as sources of energy. These facts make it clear that even though the carbohydrate portion of a food or a diet is estimated “by difference” in the Ween de scheme of analysis, little if any, useful information is lost by this group treatment.” (Loyed, et al. 1960)
“The digestible or the metabolizable energy the body ultimately obtains from the nitrogen-free extract, from the crude fibre, or from the total carbohydrate (i.e., the nitrogen-free extract plus the crude fibre) of a food, is a somewhat different matter since the completeness of the digestion of these two groups of carbohydrates is often appreciably different. This matter will be considered later when the question of digestibility is dealt with. For the moment it will suffice to note that celluloses and hemicelluloses yield less useful energy to nonherbivores than do the carbohydrates of the nitrogen-free extract category.” (Loyed, et al. 1960)
Crude protein is also a group name; it refers collectively to the sum of up to 20 nutrients, the amino acids, each of which has one or more specific roles in metabolism. In addition, each of these protein components, if present in excess of that needed for its specific function, may, following absorption, be split into a nitrogen-containing entity NH3 and a daemonized residue, the latter becoming a nonspecific source of energy.” (Loyed, et al. 1960)
“Most of the amino acid residues that can be metabolized for energy contain 3-carbon atoms. In any case, only that fraction of an amino acid that is equivalent to some intermediate in the metabolism of sugars (or of fats) is so used. However, the potential energy in proteins, as measured by their complete combustion in a bomb calorimeter, is considerably greater than that in carbohydrates. This is true because, with protein, oxygen is required not only – to oxidize the carbon, but, unlike carbohydrates, is required also to oxidize some of the hydrogen atoms; and the heat of water formation is much higher than that of carbon dioxide. Thus, typical pure proteins yield 5.25-5.75 kilocalories of gross energy per gram.” (Loyed, et al. 1960) “Nevertheless, the amount of nutritionally useful energy of protein is not greatly different from that of carbohydrate. This is so because the amino group that is split off in the deaminization of each “discarded” amino acid forms urea, which is eliminated in the urine. Urea contains combustible carbon and hydrogen, and this part of the potential energy from protein is lost the body. In humans it amounts to about 1.25 kilocalories per gram of protein so that the maximum usable energy from typical protein does not exceed 5.50 – 1.25 or 4.25 kilocalories per gram; this is usually reduced further by the incompleteness of digestion to about 4 kilocalories per gram. This can be stated in another way: whereas carbohydrate yields to the body, on average, about 95% of its potential energy, only some 70% of the potential energy of protein can be used to meet energy needs. Protein is obviously not normally the preferred source of energy in nutrition.” (Loyed, et al. 1960)
“Incidentally, crude protein, by itself, describes only the energy of this nitrogenous fraction of foods. Without other information, the figure for the amount of crude protein in a food gives no reliable clue to the makeup of its nutrient units, the amino acids. Of these acids, we shall learn more later. It is sufficient here to tabulate them as some of the nutrients that we must deal with in nutrition (see table below).” (Loyed, et al. 1960)
Lipids, fats, and ether extract “In a beginning course in nutrition there is a tendency to use almost interchangeably the terms lipid, fat, and ether extract. In particular, when we record the total of the ether extractives, We often designate it merely as fat. This rather loose usage of these terms does not often lead us astray, for reasons that will become obvious as one delves deeper into the subject. But it may be well at the outset to define these terms in a more specific way, in order to have a clearer conception of what substances are included in that fraction of the Weende analysis called ether extract.” (Loyed, et al. 1960)
“Lipids Ure naturally occurring substances soluble in organic solvents, such as diethyl ether. A classification of these substances is given in the table below.” (Loyed, et al. 1960)
Calorie value of ether extract “This classification does not include all of the substances that may be found in the ether extract of foods or of tissue of the animal body. In general, the presence of substances other than triglycerides in ether extract dilutes its useful energy. They are mentioned here chiefly to show what a mixture the ether extract of foods may be. In foods of animal origin, such as meat fats, lard, or butter, it may be composed almost entirely of triglycerides. But in foods of plant origin, as much as half the total ether extract may be composed of sterols, waxes, and various other lipids. Since the nonglyceride lipids yield little utilizable energy to animals, the caloric value of ether extract is characteristic of specific foods: a single energy value, such as 9 kilocalories metabolizable energy per gram, while perhaps satisfactory for the fats of animal origin, the refined vegetable oils, or the shortenings prepared from them, is usually too high for the ether extract of foods of plant origin.” (Loyed, et al. 1960)
“The usefulness of the ether extract of the Weende scheme as a source of energy is dependent almost entirely on its total content of triglycerides. Ether extract values by themselves give no indication of the particular fatty acids in the fraction, nor of the amount of nonglyceride lipid. These values, therefore, are only an indication of the energy of a feed, which in turn is subject to considerable variation from one type of feed to another because of the possible variation in the composition of the lipid fraction.” (Loyed, et al. 1960)
Ash-the inorganic nutrients “The Weende analysis includes an inorganic fraction-the total of the noncombustible substances of the material. The quantity of ash in a feed or in some biological product does not of itself give information about any specific nutrient, and frequently the figure is used only to calculate the amount of carbohydrate by difference. The combination of mineral elements found in foods of plant origin is so variable that the ash figure of our analysis is useless as an index of the quantity of any particular element, or even of the total of the nutritionally essential ones. In the case of certain animal products, such as bone, milk, or cheese, whose composition is relatively constant, the approximate quantities of calcium and phosphorus can be predicted from the total ash figure. Thus, so far as useful information about the inorganic nutrients of foods is concerned, the ash figure is merely a starting point for specific analysis for one or another of some 21 to 26 mineral elements required by the body, and for a few about which information may be needed because of their toxic nature.” (Loyed, et al. 1960)
Classification of the nutrients
“To summarize this what we discussed here, the table below identifies the principal nutrients by name and indicates the fractions of the Weende analysis into which they fall.”
“The table makes it clear that the Weende analysis does not describe nutrients individually; when this is necessary, some other scheme of description must be used. But, in spite of limitations, the Weende analysis is the basis for the everyday chemical description of foods, body tissues, and excreta that are of concern in such calculations as the estimation of digestibility and utilization of foods and the establishment of feeding standards for all animal species.” (Loyed, et al. 1960)
Conclusion
The young man who took over from me as production manager at Woody’s Consumer Brands has a saying that managing a production department of a meat factory is a team sport. On the one hand, one needs the experienced manager’s approach of Albrecht Daniel von Thaer. On the other hand, I know from first-hand experience the financially devastating effect if a management team gets a call on a scientific matter related to meat science wrong. Managing a large food manufacturing concern is the job of an experienced team, not a lone ranger or a single night in shining armour and discipline in science and discipline in management has equally valid places.
A week ago an old friend visited from the United States and as we discussed these matters he commented that no matter what detours we take, we seem to always get back to clear management principles laid down by people like Peter Drucker. The study of the development of the proximate analysis and the examples of marrying good management and science as exemplified in the life of Carlsberg come to us through our analysis of the history of what led us to our current day determination of meat content in formulations. It is a subject so rich. We gain from it on every level.
(c) eben van tonder
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References:
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Sáez-Plaza, P., Michałowski, T., Navas, M. J., Asuero, A. C., & Wybraniec, S.. 2013. An Overview of the Kjeldahl Method of Nitrogen Determination. Part I Early History, Chemistry of the Procedure, and Titrimetric Finish, Critical Reviews in Analytical Chemistry, 43:4, 178-223, DOI: 10.1080/10408347.2012.751786 Link to this article: http://dx.doi.org/10.1080/10408347.2012.751786
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ISSN: 1040-8347 print / 1547-6510 online DOI: 10.1080/10408347.2012.751787
ANALYTICAL METHODS FOR PROTEINS IN FOODS
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