Amino Acid, Protein Analysis & Labeling

Originally Published: May 25, 2017
Last Updated: February 6, 2021

May, 25, 2017 — Protein serves structural, functional, physiological and metabolic roles in the body. The quality of dietary protein is largely determined by its amount of indispensable (essential) amino acids. Of the 20 amino acids present in protein, nine are indispensable and must be supplied by the diet.  The topic of amino acid and protein analysis and labeling was addressed by Sneh Bhandari, Ph.D., Merieux NutriSciences, at the 2017 Protein Trends & Technologies Seminar. His presentation titled “Protein and Amino Acid Analysis in Relation to Nutrition Labeling and Protein Quality,” was given during the Technical Program: Formulating with Proteins.

Whey proteins are good sources of branched-chain amino acids, which are important for muscle synthesis. Cereals and grains are usually deficient in lysine; however, a mixture of pulses and grains can create a complete protein. Collagen lacks tryptophan and so is not a good-quality protein. “Additional nutrients present in the protein food can make it more or less valuable in the diet. Animal foods often contain saturated fat and cholesterol, whereas plant foods are rich in dietary fiber and phytonutrients,” said Bhandari.

In the U.S., Protein Efficiency Ratio (PER) is the required method of calculating protein quality in foods for infants one year of age and younger, and PDCAAS is used for other foods. Canada recognizes PER as the preferred method but allows PDCAAs when PER is not available.

Bhandari explained that the Kjeldahl method of protein analysis is based on total nitrogen content and has been widely used and often referred to as crude protein. Originally, all proteins were estimated to contain 16% nitrogen, and protein was calculated by multiplying the nitrogen value by 6.25. However, in 1941, factors ranging from 5.3-6.38 were established for specific food matrices. The FAO allows the conversion factor of 6.25 to be used for all foods, but the FDA requires AOAC-approved methods using specific conversion factors to be used when calculating PDCAAS. Non-protein nitrogen, which often exists as soluble nitrogen, must be deducted from crude protein values to calculate true protein.

Another common protein analysis is the Combustion/Dumas method, which measures oxides resulting from nitrogen combustion. Other less common methods use reflectance or transmission NIR.


The DIAAS method corrects some PDCAAS issues but, for now, the FDA is not replacing PDCAAS with DIAAS. [Click image to see large PDF of chart.]

To analyze individual amino acids, the protein is hydrolyzed in 6M HCL for 18-24 hours at 110° C. Some amino acids are readily oxidized or damaged by acid hydrolysis. For example, tryptophan can be almost completely lost by acid hydrolysis, so an alkaline hydrolysis is performed instead. The sulphur-containing amino acids methionine and cystine degrade during acid hydrolysis and are protected by prior performic acid oxidation. Individual amino acids are commonly separated by ion-exchange chromatography, followed by post-column derivatization. Tryptophan can be analyzed by ion-exchange or reverse-phase HPLC methods.

In 1993, the FDA adopted PDCAAS as the official method to determine protein quality, Bhandari noted. The first step is to analyze crude protein and essential amino acid concentrations. Next, the amino acids’ values must be scored against a reference pattern. The value for the limiting or lowest essential amino acid is the Amino Acid Score (AAS). The second step is to determine Protein Digestibility (PD). Human values are ideal, but rat values are often used. Finally, AAS is multiplied by PD, and values over 1.0 are rounded down to 1.0 (that is, when AAS is multiplied by PD and, if the resulting PDCAAS value is 1.05 or 1.8, it is rounded down to only 1.0).

There are several issues with the PDCAAS method, including the fact that fecal digestibility values are less accurate than ileal digestibility values. The Digestible Indispensable Amino Acid Score (DIAAS) method was recommended by FAO/WHO in 2013 to address these issues. The FDA agrees that DIAAS is a better method, but it declined to replace PDCAAS as the method for calculating nutrition labels in the 2016 final rule, because there was insufficient data to implement DIAAS. There are additional guidelines for reporting protein values on the Nutrition Facts panel.

Consumers continue to demand healthy quality protein. Accurate analysis of protein content will enable food manufacturers to make appropriate protein claims, Bhandari concluded.

Click on the link to see the PowerPoint for this presentation on amino acid and protein analysis and labeling. “Protein and Amino Acid Analysis in Relation to Nutrition Labeling and Protein Quality,” Sneh Bhandari, Ph.D., Merieux NutriSciences,


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