Early history of food protein research and analytical methodology
Scientists in the 18th century were curious about how vegetable foods are converted into animal substances, which led them to investigate commonalities between these substances and eventually to discover protein (Rosenfeld 2003). The characteristics of animal-sourced substances, such as glue, gelatin, and egg white have been described for thousands of years. Analogous physical properties for a plant-based glutinous substance (now known as gluten) and for certain animal substances were first reported by Beccari in 1746 (Osborne 1908). Animal substances that coagulated upon heating, such as egg white, were defined as early as 1777 by the term albuminous (McCollum 1957a). Fourcroy in 1789 recognized that similar albuminous substances in plants and animals were a distinct class of biological molecules (Fourcroy 1789; McCollum and others 1939a). Mulder is credited with coining the term protein in 1838 (McCollum and others 1939b), following a suggestion by Berzelius (Vickery 1950; Brouwer 1952; Rosenfeld 2003).
During the 18th century, developments in the analysis of albuminous proteins correspond with progress in analytical chemistry, when Lavoisier developed combustion methods to analyze the organic chemical composition of substances (Szabadváry 1966). In 1786 Berthollet, using a distillation procedure, first reported that nitrogen was a constant constituent of animal extracts (Berthollet 1786; McCollum and others 1939a; Szabadváry 1966). Based on Lavoisier's procedures for combustion analysis, Gay-Lussac and Thénard developed the 1st combustion method for nitrogen determination and determined the nitrogen (azote) content of egg white protein in 1810 (Davy 1815; Szabadváry 1966).
From the early to middle 19th century, 2 types of food analysis methods involving protein were developed by those interested in the composition of food and the role of nitrogenous compounds in nutrition. Mulder and his contemporaries agreed that nitrogenous protein substances in the diet played an important role in nutrition (McCollum and others 1939a; Brouwer 1952; McCollum 1957b). Led by Liebig and Boussingault in the 1830s, this thinking led to food analyses based solely on nitrogen content. Liebig in particular asserted that the nutritional value of food was based solely on its “plastic”“body-forming” protein content, which could be assessed by measuring the total nitrogen content of a food, ignoring other constituents and analytical methods (McCollum and others 1939a; McCollum 1957b, 1957c; McCosh 1984; Rosenfeld 2003). Liebig measured the nitrogen content of foods using combustion methods based on his improvements on methods discovered by Lavoisier, Gay-Lussac, and Thénard's, and Boussingault used improved combustion methods developed by Dumas (McCollum 1957b; Szabadváry 1966; Rosenfeld 2003).
A 2nd approach for the analysis of food composition was pioneered by Einhof, Vogel, Gorham, Hermbstaedt, and others beginning around 1800. This approach incorporated methods to separate and quantify constituents, such as proteins, fat, starch, and others from cereal grains (Osborne 1908; McCollum and others 1939a; McCollum 1957c). On the basis of solubility properties, individual types or fractions of proteins were separated from other plant constituents (Osborne 1908; McCollum and others 1939a; McCollum 1957c). This approach is consistent with current understanding of nutrition and food compositional analyses but did not gain acceptance until later due to the authority then given to Liebig's theories. McCollum, in his review of nutrition history, suggests that the dominance of Liebig's theory, even though it turned out to be incorrect, promoted the nutritional analysis of food based solely on nitrogen content (1st approach) and stifled further development of methods initiated by Einhof and others until the late 19th century (McCollum 1957c).
In the middle to late 19th century, a number of significant developments in total nitrogen content methods for food protein analysis included early versions of the methods still used today. The 1st quantitative combustion method using total nitrogen to measure proteins in foods, albeit unreliably, is credited to Dumas (Dumas 1831; Szabadváry 1966; Rosenfeld 2003). More dependable combustion procedures for nitrogen were improvements on the Dumas method and were reported by Shiff, and Varrentrapp and Will (the Soda-lime Process) from 1841 to 1868. These never enjoyed widespread popularity because a much simpler and more reliable wet chemistry method for total nitrogen determination was developed shortly thereafter by Kjeldahl (Szabadváry 1966).
Although it is not clear who first reported the use of a total nitrogen-to-protein conversion factor to quantify the total (crude) protein of food, the analytical methods developed by Henneberg and Stohmann in 1864 at the Weende Experimental Station in Germany used a factor of 6.25 (Henneberg 1865; Atwater and Woods 1896; McCollum and Simmonds 1929). This factor was based on the assumptions that protein consistently contained 16% nitrogen and that all nitrogen in food was from protein (McCollum and Simmonds 1929; Jones 1931/41; Koivistoinen 1996; Salo-Väänänen and Koivistoinen 1996). Early 19th-century protein nitrogen studies such as those conducted by Mulder and Gay-Lussac supported this factor, but the combustion methods used at that time were not necessarily reliable or accurate (Brouwer 1952; Szabadváry 1966).
McCollum reports that in the late 19th century analysts recognized that nonprotein nitrogen substances were present in food and that the use of total nitrogen measurements to quantify total protein were therefore inadequate (McCollum 1957c). To overcome this analytical issue, Wagner, Sestini, Kellner, Dehmel, and Schulze from 1878 to 1879 reported methods that putatively precipitated “true protein” and then quantified this fraction by total nitrogen measurements (Sestini 1878; Wagner 1878; Dehmel 1880; Kellner 1879; Schulze 1879; McCollum 1957c). These methods were based on the hypothesis that measuring this “true protein” fraction would provide a more accurate estimate of the nutritional value of nitrogenous substances in food (McCollum 1957c). This notion was contested by Weiske and others who in 1879, demonstrated in rabbit studies that a nonprotein, nitrogen-containing substance, asparagine, functioned as a nutritional substitute for protein (McCollum 1957c; Weiske and others 1879). This resulted in decreased interest in efforts to advance the chemical analysis of food by separating protein from nonprotein components (McCollum 1957c). Indeed, only one Official AOAC Method of Analysis using a similar method could be found from this period until the late 20th century. This Official Method is based on one reported by Van Slyke in 1893 to measure the casein content of milk by precipitating the casein with acetic acid and separating it from the supernatant before performing total nitrogen analysis (Van Slyke 1893; Wiley 1893; AOAC 1927).
In 1883 Kjeldahl developed a wet chemistry method for nitrogen analysis. It was faster, simpler, and more accurate than the previously established combustion procedures (Kjeldahl 1883; Dyer 1895; Szabadváry 1966). Kjeldahl's nitrogen determination method became pivotal in food and agricultural chemistry and became the authoritative reference method for total (crude) protein quantification in foods (Lynch and Barbano 1999). At least one authority suggests that the Kjeldahl method has been the subject of more studies than any other in analytical chemistry (Chen and others 1988).
Extensive work in the early 20th century at USDA's Protein and Nutrition Research Division improved assessment methods for protein quality and quantity (Jones 1931/41). These studies included isolation of individual proteins and amino acid analysis for different food ingredients, but such approaches were not then considered practical for quantitative protein analysis (Jones 1931/41). A major contribution of this group was improved measurement accuracy based on the development of nitrogen-to-protein conversion factors for specific food categories and food ingredients (Jones 1931/41).
In summary, the nutritional theories of the 19th century and analytical technological capabilities of the 20th century had a significant role in the evolution of food protein measurement. These conditions inhibited the development of analytical strategies to separate protein from other food matrix substances before analysis and promoted determinations based on total nitrogen to calculate total protein content. This assertion is supported by the failure of 3 attempts from the scientific community to develop and advance the former type of methods. The 1st was led by Einhof in the early 19th century to analyze the protein composition of grains, but this was stifled by Liebig's dominating nutrition theory. The 2nd effort was led by Wagner and others in 1879 to develop methods to precipitate and quantify the “true protein” contents of foods, but these approaches did not advance because of widespread acceptance of Weiske's nutritional theory. Finally, early 20th-century USDA scientists concluded that analytical technology of that time was not capable of efficiently separating and analyzing individual proteins. Instead, they promoted protein measurement by specific nitrogen-to-protein conversion factors. Unfortunately, protein-specific measurement methods were not accepted. Because of advances made in protein nutrition and analytical sciences since the 19th century, coupled with the aforementioned incidents of adulteration, a 4th attempt is now warranted.
Protein measurement for food valuation
For purposes of trade, the first occurrences of basing the market value of protein-based food ingredients on total protein content measurement is unknown, but the need for reliable methods dates to the late 19th century. In 1883 Kjeldahl justified his new method based on total nitrogen determination by noting the need for reliable tools to evaluate the protein contents of incoming barley ingredients used for brewing (Kjeldahl 1883). This challenge—developing reliable analytical methods that facilitated trade by providing reproducible and accurate analytical results for both buyers and sellers—was one of the principal reasons for the formation of the Association of Official Agricultural Chemists (AOAC, now known as AOAC International) in the late 19th century (Helrich 1984).