AOAC Stakeholder Panel on Dietary Supplements
Our fitness for Purpose from March: “Method must identify and quantify specific proteins in presence of other proteins and potential adulterants. Quantitative method must provide accurate and precise concentrations of specific intact proteins in ingredients and finished goods.”
As of this presentation, our Protein Working Group Work to Date includes:
- 1 In Person Meeting
- 3 teleconferences (March 2016 – June 2016)
- 4 SMPRs Drafted
- Public comment period (August, 2016)
- SMPRs made ready for SPDS review and approval
Proteins are polypetides made of individual amino acids in a linear chain. They form the basis of life and perform functions in every system of the human body. Enzymes catalyze biochemical reactions. Hormones are used for cell signaling and communication. These proteins synthesize and repair DNA. They tranport materials across the cell and respond to stimuli. They also provide structural support.
It is estimated that 4 billion metric tons of food protein is produced globally. It is also estimated that $94M was lost by changing the nitrogen-to-protein factor for dairy products from 6.38 to 6.25 in Europe in 2006. Proteins make up $4.7B dollars in the Sports Nutrition industry, which represents 70% of the total revenue in that category.
Non-selective protein methods have fueled the potential to adulterate samples with non-proteins and give inaccurate results. Melamine, urea, and free amino acids cannot be differentiated using the Kjeldahl and the Dumas methods and have been the source of scandals. However, public health is still at risk and economics still push adulteration.
Existing Methods: Qualitative
Some proteins have FCC monographs. For example, Whey Protein is identified by testing for: Ash, Fat, Lactose, Loss on drying, Nitrogen (and apply conversion factor). There are also DNA Analysis and LC/MS/MS.
Existing Methods: Quantitative
The Kjeldahl is one of the most common quantitative method. In this method, wet digestion converts nitrogen to ammonium sulfate. It is then neutralized to convert to free ammonia. Then we distill ammonia into boric acid. We back titrate with alkali. After, we convert nitrogen concentration to protein using conversion ratio. “True Protein” can be determined by precipitating out protein, analyzing remaining nitrogen, subtracting from total nitrogen content.
The Dumas method is the other most common quantitative method. First, we combust samples at high temp with oxygen to form water, carbon dioxide, and nitrogen. Then, we remove water and carbon dioxide, and Nitrogen is measured using a thermal conductivity detector. We then convert nitrogen concentration to protein using conversion ratio.
In the Amino Acids method, we hydrolyze protein into amino acids and derivatize the amino acids. We determine protein by summing individual amino acids.
In the Dye-binding method, complex is formed with dye and protein using ionic or electrostatic forces.Dye concentration is determined using a spectrophotometer.
In the Copper-Binding method, copper ions react with proteins to form a complex. Absorbance is measured at 540 nm.
Other methods include UV absorption and infrared.
Challenges with Existing Methods
The Kjeldahl and Dumas method are not selective to protein. With true Protein Kjeldahl, non-protein, nitrogen-containing compounds may precipitate or form complex with a precipitated protein. With the Amino Acid method, there is sometimes Inaccurate quantitation due to variable recovery of amino acids. In the Copper and Dye-Binding methods: other constituents besides proteins form complexes.
There is also a lack of standards. Protein biosynthesis is expensive, time-consuming, and not as robust as other methods. Proteins samples can vary widely and usually include multiple proteins.
SMPR Key Points
The definition of Protein is same as IUPAC: “Naturally occurring and synthetic polypeptides having molecular weights greater than about 10000 daltons (the limit is not precise).” Right now, four SMPRs are being proposed: Quantitative (i.e. Determination), Qualitative (i.e. Identification), Plant, and Meat-Derived Proteins.
SMPR Plant-derived sources of proteins include: Algae, Canola (Rapeseed), Flax, Hemp, Pea, Potato, Pumpkin, Quinoa, Rice, Soy, Wheat.
SMPR Meat-derived sources of proteins include: Casein, Egg, Whey, Milk.
SMPR Non-Protein Ingredients Including Adulterants include: Melamine, Urea, Free amino acids, Creatine, Caffeine, Taurine, Surfactants, Peptides (less than 10,000 daltons).
Concerns and Solutions
There was a concern voiced that though the given protein definition is recommended by IUPAC, it will have little practical use for the food industry. For instance, in the newly revised FDA food and supplement nutrition fact labeling, FDA recognizes all peptides as proteins (a merely amino acid source). Some also felt that 10,000 daltons sounds over emphasized.
The proposed change is to say “Polymeric chains of amino acid residues connected with peptide bonds.” At the end of this presentation, there was a motion to accept the Standard Method Performance Requirements for: determination and identification of Meat and Plant derived proteins as presented.