Draft – Determination of the protein rating using the Protein Digestibility Corrected Amino Acid Score (PDCAAS)

This document describes the methodology to determine, for the purposes of the Food and Drug Regulations, the protein rating (PR) of certain foods using the Protein Digestibility Corrected Amino Acid Score (PDCAAS).

Date published: November 4, 2023

Table of contents

• 1. PDCAAS Methodology
  • 1.1 Amino Acid Analysis
  • 1.2 Amino Acid Scoring Pattern
  • 1.3 Protein Digestibility
  • 1.4 Determination of PDCAAS
  • 1.5 Examples of Calculating the PDCAAS of a mixture
• 2. Calculating the Protein Rating using PDCAAS

1. PDCAAS Methodology

The PDCAAS of a food shall be determined using the methodology below, which is based on the Report of the Joint FAO/WHO Expert Consultation on Protein Quality Evaluation ^{Footnote 1}, with minor adjustments.

Key steps in determining the PDCAAS include:

1. Analyzing the amino acid content in the test food. This requires hydrolysis of the protein to release the amino acids, followed by separation and quantification of the amino acids;
2. Comparing the amino acids in the test protein against an amino acid scoring pattern; and
3. Correction for the digestibility of the protein.

1.1 Amino Acid Analysis

See Section 5 of the Report of the Joint FAO/WHO Expert Consultation on Protein Quality Evaluation ^{Footnote 2} for more information.

Procedure for determination of amino acids

1. Three hydrolysates, in duplicate, are required:
   1. acid hydrolysis of unoxidized protein for determination of all required amino acids (i.e. all amino acids which are part of the amino acid scoring pattern described in section 1.2) except tryptophan, methionine and cystine;
   2. acid hydrolysis of oxidized protein for determination of methionine and cystine ^{Footnote 3}; and
   3. alkaline hydrolysis of unoxidized protein for determination of tryptophan.
2. Acid hydrolysis of unoxidized and oxidized protein should be conducted according to the detailed protocols given by Pellett and Young, ^{Footnote 4} Mason et al., ^{Footnote 5} AOAC (1984), ^{Footnote 6} AOAC (1985), ^{Footnote 7} or Finley. ^{Footnote 8} For acid hydrolysis of unoxidized protein, oxygen should be excluded. Similar procedures, with respect to sample weight, acid volume, temperature and time of hydrolysis should be followed.
3. Oxidation of protein should be carried out using performic acid prepared and used as described by Moore, ^{Footnote 9} Pellett and Young, ^{Footnote 10} Mason et al. ^{Footnote 11} or AOAC (1985). ^{Footnote 12} The ratio of performic acid to protein in these procedures is in the range 0.08 - 1.3 ml/mg crude protein.
4. Alkaline hydrolysis should be carried out by one of the procedures described by Slump and Schreuder, ^{Footnote 13} Pellett and Young, ^{Footnote 14} AOAC (1984), ^{Footnote 15} Nielsen and Hurrell, ^{Footnote 16} Finley, ^{Footnote 17} or Bech-Andersen. ^{Footnote 18} The use of 5-methyl-tryptophan or alpha-methyltryptophan as an internal standard carried through the entire procedure is recommended.
5. Amino acids in the hydrolysates should be determined by classical ion-exchange chromatography (IEC), by high performance liquid chromatography (HPLC) using cation exchange resins and post column derivatization or by prederivatization followed by reverse phase HPLC. In the latter case, derivatization and separation procedures which have been shown to give results with foods equivalent to classical IEC should be used. The phenylisothiocyanate (PITC) method has been found satisfactory in this respect.
6. Tryptophan and the internal standards 5-methyl-tryptophan or alpha-methyl-tryptophan are best separated by reverse phase HPLC and quantified by UV absorption or fluorescence without derivatization.
7. Results should be expressed as mg amino acid/g nitrogen (N) or be converted to mg amino acid/g protein by use of the factor 6.25. Results for threonine, serine, valine, and isoleucine should be corrected for hydrolytic losses by factors based on time-hydrolysis studies conducted once in each laboratory. Recovery of methionine and of cystine as methionine sulphone and cysteic acid, respectively, should be determined and correction factors applied as necessary. A correction factor for loss of tryptophan should be applied if the internal standard method is not used.
8. Where possible, the recovery of nitrogen from amino acids and ammonia should be calculated as a check on the quality of the analysis. For most food products the recovery of nitrogen should be greater than 90 %. For foods known to contain significant amounts of non-amino acid nitrogen, such as yeast rich in nucleic acids, recovery of nitrogen may be in the range 80 to 85 %.

Use of published amino acid data

The amino acid composition of a food shall be determined using the methods described above; however when reliable amino acid composition data already exists for the food it may be used for calculating the amino acid score. The data and the source of the data should be retained by the stakeholder and may be requested by Health Canada and the Canadian Food Inspection Agency (CFIA).

1.2 Amino Acid Scoring Pattern

See Section 6 of the Report of the Joint FAO/WHO Expert Consultation on Protein Quality Evaluation ^{Footnote 19} for more information.

The amino acid scoring pattern proposed in 1985 by the FAO/WHO/UNU ^{Footnote 20} for children of preschool age should be used to evaluate dietary protein quality of foods for all age groups, except for infants. Further information on the use of amino acid scoring patterns to calculate the PDCAAS is found in Section 1.4.

Adapted from Table 7 'Comparison of suggested patterns of amino acid requirements with the composition of high-quality animal proteins' by FAO of UN, 1991^{Footnote 21}

1.3 Protein Digestibility

See Section 7 of the Report of the Joint FAO/WHO Expert Consultation on Protein Quality Evaluation ^{Footnote 22} for more information.

The amino acid scores must be corrected for the true digestibility of crude protein. Human nitrogen balance studies are ideal for determining the true crude protein digestibility, however the rat balance method and the pig nitrogen balance method are suitable for predicting protein digestibility by humans. Therefore, when human nitrogen balance studies cannot be used, the standardized rat fecal-balance method described in the AOAC Official Method 991.29, ^{Footnote 23} or the pig nitrogen balance method as described by Forsum et al., ^{Footnote 24} is recommended to measure protein digestibility.

Use of Established Digestibility Values

Established digestibility values of well defined foods may be taken from a published database for use in the amino acid scoring procedure, assuming all safety and toxicological criteria have been met. However if digestibility values are not yet established for the food, or the food has undergone a processing technique which was not used on the food in the established digestibility value, then the true protein digestibility value must be determined for the food using the methods described above. Caution should be taken when using established values for plant-based foods containing high levels of anti-nutritional factors, as digestibility values will vary widely depending on processing techniques.

1.4 Determination of PDCAAS

See Section 8 of the Report of the Joint FAO/WHO Expert Consultation on Protein Quality Evaluation ^{Footnote 25} for more information.

Individual Foods

To calculate a PDCAAS, a test food must be analysed for proximate composition, and the amino acid composition and protein digestibility value of the test food must be obtained from a database or other reliable source, or be determined using methodology described in Sections 1.1 and 1.3.

1. Proximate composition: Levels of total nitrogen, fat, total dietary fibre, and moisture should be determined according to appropriate AOAC methods. ^{Footnote 26} Protein can then be calculated by using a nitrogen-to-protein conversion factor of 6.25. Foods high in moisture (such as meats) should be dried to less than 10% before analysis. Similarly, foods high in fat (such as meat, nuts, whole milk powder, etc.) should have fat content lowered to 10% or less by lipid extraction prior to analysis.
2. Amino acid profile: The test food's composition of the eleven amino acids which make up the amino acid scoring pattern (i.e. the nine essential amino acids, cysteine and tyrosine) needs to be determined using methods described in Section 1.1, or obtained from a database or other reliable source.
3. Amino acid score: Amino acid ratios (mg of an essential amino acid in 1.0 g of test protein/mg of the same amino acid in 1.0 g of reference pattern) for the 9 essential amino acids plus tyrosine and cystine should be calculated by using the amino acid scoring pattern shown in Section 1.2. The lowest amino acid ratio is termed the amino acid score.

   For example, a pinto bean sample contained 30.0, 42.5, 80.4, 69.0, 21.1, 90.5, 43.7, 8.8, and 50.1 mg/g protein of Histidine, Isoleucine, Leucine, Lysine, Methionine + Cystine, Phenylalanine + Tyrosine, Threonine, Tryptophan, and Valine, respectively. The respective amino acid ratios for the bean sample would be 1.58, 1.52, 1.22, 1.19, 0.84, 1.44, 1.28, 0.80, and 1.43. This would then result in an uncorrected amino acid score of 0.80 with tryptophan as the first limiting amino acid.

4. Protein digestibility: The true protein digestibility for the test food should be determined using methodology described in Section 1.3, or obtained from a database if a value is already established for the food and processing technique.
5. PDCAAS of a test food should then be calculated by multiplying the lowest amino acid ratio by true protein digestibility. For the purposes of calculating the protein rating the score should be expressed as a decimal, with 100% being reported as 1.0. In the case of the pinto bean sample having the lowest amino acid ratio of 0.80 and a true protein digestibility of 73% [as shown by McDonough et al. ^{Footnote 27}], the PDCAAS would be 0.80 x 0.73 = 0.58.

   PDCASS above 1.00 are truncated and should be reported as 1.00.

Food Mixtures

For food mixtures, the full procedure for individual foods described above may need to be followed. However, when data for the amino acid composition and protein digestibility of the individual components are well established and only the proportions differ, the PDCAAS can be calculated. The weighted average procedure described in Section 8 of the Report of the Joint FAO/WHO Expert Consultation on Protein Quality Evaluation, ^{Footnote 28} or the weighted average digestible amino acid content procedure described in Section 6 of the Report of a Joint WHO/FAO/UNU Expert Consultation on Protein and Amino Acid Requirements in Human Nutrition ^{Footnote 29} may be used. Examples of these calculations are provided in the following section.

1.5 Examples of Calculating the PDCAAS of a mixture

Worked example for a test food which is a mixture of wheat, chickpea and milk powder, using the weighted procedure described in the Report of the Joint FAO/WHO Expert Consultation on Protein Quality Evaluation. ^{Footnote 30} In this scenario, established values for both the amino acid composition and the protein digestibility of wheat, chickpea and milk powder are obtained from a database.

Worked example for a test food which is a mixture of wheat, chickpea and milk powder, using the weighted average digestible amino acid content procedure described in the Report of a Joint WHO/FAO/UNU Expert Consultation on Protein and Amino Acid Requirements in Human Nutrition. ^{Footnote 31} In this scenario, established values for both the amino acid composition and the protein digestibility of wheat, chickpea and milk powder are obtained from a database.

2. Calculating the Protein Rating using PDCAAS

The protein rating (PR) of the food is calculated by multiplying the PDCAAS of the food ^{Footnote 32} by the amount of protein (grams) in a reasonable daily intake (RDI) ^{Footnote 33} of the food.

PR = PDCAAS for food x grams protein in a RDI of the food

For example, if a food containing 10% protein (N x 6.25) has a PDCAAS of 1.0 and if a RDI of this food is 75 grams, then the PR would be calculated as follows:

PR = 1.0 x 0.1 x 75

PR = 7.5