Determination of protein rating

1. Application

This method shall be used to determine the protein rating of foods under Sections B.01.062, B.01.063, B.01.064, B.01.065, B.14.073, B.14.085, B.14.086, B.14.087, B.14.088, B.14.089, B.14.090, B.21.027, B.22.027, B.22.029 and B.22.032 of the Food and Drug Regulations.

2. Principle

A bioassay for the evaluation of the nutritional quality of the proteins, this method involves measurement of the protein efficiency ratio (grams gain per gram protein consumed) under standardized conditions. Male rats 20-23 days of age are fed ad libitum an otherwise adequate reference diet containing 10% protein supplied by a standard sample of casein. Foods to be assayed are added to the diet as the sole source of protein at the expense of casein and corn starch to maintain a protein level of 10%. Protein efficiency ratios (PER's) are calculated after 4 weeks and are adjusted to a constant value of 2.5 for casein. The protein rating is the product of the adjusted PER of the test food multiplied by grams protein in a reasonably daily intake of the test food. Foods containing much lipid (meats, nuts, whole milk powder, etc.) may require a lipid extraction. See Appendix A.

3. Procedure

The test shall be carried out in accordance with the following instructions:

1. Animals

1. use weanling male rats of single strain 20-23 days of age, 10 for each diet.

2. Diets

1. use a basal diet of the following percentage composition on an air-dry basis:

   Percentage composition per item of the basal diet

   Item	Percentage
   Corn starch	80%
   Corn oil or Cottonseed oil	10%
   Non-Nutritive cellulose	5% Footnote 1
   Salts, U.S.P. XIV	4% Footnote 2Footnote 1
   Vitamin mixture	1% Footnote 3

2. incorporate the protein food under test into the diet at the expense of corn starch to give 10% protein (N multiplied by 6.25);
3. the protein content of the final diet should be within the range 9.7 to 10.3% as determined by analyses ^{Footnote 4};
4. supply the water and diet ad libitum.

3. Assay Period

1. use a 4 week period.

4. Cages

1. use individual cages which have screen bottoms and are provided with feeders which will reduce food spillage to a minimum.

5. Randomization

1. use a randomized block design in which blocks represent variations in initial weight;
2. randomize the rats in each block for diet and cage;
3. a procedure for randomization is outlined in Appendix B.

6. PER Determination

1. in addition to the test group, maintain a reference standard group of rats on a diet consisting of the basal diet with casein at the level of 10% protein (N multiplied by 6.25) as determined by analyses;
2. for uniformity, this casein should be a product of uniform high quality and good stability;
3. the Test Diet High Nitrogen Casein approved by the Animal Nutrition Research Council and prepared by the Sheffield Chemical Company, Norwich, N.Y., has been found to be satisfactory;
4. all test animals must be maintained at a room temperature of 72 ± 1°F and a relative humidity of 45 ± 5%;
5. for each animal, maintain a weekly record of body weight and food consumption.

4. Calculations

1. At the end of 4 weeks calculate the PER for each food and for the Reference Standard casein using the following equation:

   

   Text description

   The equation is: PER is equal to weight gain in grams divided by weight of protein consumed in grams.

2. Assuming that the casein has an average PER of 2.5 when determined under these conditions, adjust the PER of the test food as follows:

   

   Text description

   The equation is: Adjusted PER equals PER of test food multiplied by 2.5 divided by the determined PER of standard reference casein.

3. Calculate the protein rating (PR) of the test food as follows ^{Footnote 5}:

   

   Text description

   The equation is: Adjusted PER multiplied by grams protein in a reasonable daily intake of the food.

The method described above, being comprised of six pages, Appendix A being comprised of two pages, A1-A2 inclusive, Appendix B being comprised of two pages, B1-B2 inclusive, and identified as FO-1, Determination of protein rating and dated October 15, 1981 is hereby designated the "Official Method" for the purposes of Sections B.01.062, B.01.063, B.01.064, B.01.065, B.14.073, B.14.085, B.14.086, B.14.087, B.14.088, B.14.089, B.14.090, B.21.027, B.22.027, B.22.029 and B.22.032 of the Food and Drug Regulations.

Appendix A

1. Application

This procedure shall be used for the defatting of samples prior to the determination of protein rating.

2. Apparatus

1. Soxhlet apparatus, 3000 mL size. Johns Scientific Co., Catalogue No. 27614-208 or equivalent.

3. Reagents

1. Diethyl ether, reagent grade.

4. Procedure

1. freeze dry appropriate amount of sample or place sample in a shallow pan inan air oven at 50°C for four hr;
2. place approximately (ca) 500 g of sample in the soxhlet apparatus ^{Footnote 1} and continuously extract using diethyl ether for ca 1hr ^{Footnote 2};
3. air dry fat-extracted sample in a shallow pan in a fume hood;
4. grind defatted sample to suitable particle size for mixing in diets for rat feeding studies.

5. Notes

1. If appropriate size thimbles are not available sample may be wrapped in cheese cloth.
2. Ether is extremely inflammable. Use great caution and defat sample in a fume hood.
3. A final concentration of 10-15% lipid in the test diet is acceptable.

Appendix B

The Randomized Block Design

In biological assays, variations due to individual animal differences are frequently encountered. While it is not always possible to completely eliminate all errors due to these differences, the use of animals of the same age, sex and strain, and the use of the Randomized Block Design minimize the errors due to the effects of individual differences.

The randomized block technique groups the animals into homogeneous groups referred to as blocks on the basis of some characteristic of the animal. Each block contains one of each diet being tested or compared. The number of blocks required in the Protein Rating procedure is 10.

1. Procedure

1. Rank the weanling male rats in order of weight from the lightest to the heaviest. Any unusually light or heavy rats should be replaced.
2. Make a systematic selection from the ranked weights starting with the lightest (or heaviest). Each selection group is to be equal in number to the number of diets including control.
3. By a random procedure, assign one treatment or diet to each rat in the group. A new randomization should be made for each group. Under no circumstance should the assignment of treatment (diet) to ranking of animals be the same from group to group.
4. Assign at random the n animals in a group to groups of n cages in a bank of cages. Each group is referred to as a block.
5. If there is any indication of cage location effect where the determination is to be carried out, the blocks should be randomly assigned to cage section.

A determination involving 5 different diets would require 60 rats (ten multiplied by 5 plus 1).

Graphically, the layout for a PER. Randomized Block Determination for this number of diets would be as follows:

Text description

The figure is a grid with 6 columns and 6 rows.

The first row is labelled Block 1.

Inside the box in the first column of row 1 is the number 46 in the top left corner representing the ranked weight, T2 in the centre representing diet 2, and C1 in the bottom right corner representing cage number 1.

Inside the box in the second column of row 1 is the number 44 in the top left corner representing the ranked weight, T1 in the centre representing diet 1, and C2 in the bottom right corner representing cage number 2.

Inside the box in the third column of row 1 is the number 47 in the top left corner representing the ranked weight, T5 in the centre representing diet 5, and C3 in the bottom right corner representing cage number 3.

Inside the box in the fourth column of row 1 is the number 48 in the top left corner representing the ranked weight, TC in the centre representing diet C, and C4 in the bottom right corner representing cage number 4.

Inside the box in the fifth column of row 1 is the number 43 in the top left corner representing the ranked weight, T4 in the centre representing diet 4, and C5 in the bottom right corner representing cage number 5.

Inside the box in the sixth column of row 1 is the number 45 in the top left corner representing the ranked weight, T3 in the centre representing diet 3, and C6 in the bottom right corner representing cage number 6.

The second row is labelled Block 2.

Inside the box in the first column of row 2 is the number 32 in the top left corner representing the ranked weight, T5 in the centre representing diet 5, and C7 in the bottom right corner representing cage number 7.

Inside the box in the second column of row 2 is the number 34 in the top left corner representing the ranked weight, TC in the centre representing diet C, and C8 in the bottom right corner representing cage number 8.

Inside the box in the third column of row 2 is the number 31 in the top left corner representing the ranked weight, T1 in the centre representing diet 1, and C9 in the bottom right corner representing cage number 9.

Inside the box in the fourth column of row 2 is the number 36 in the top left corner representing the ranked weight, T2 in the centre representing diet 2, and C10 in the bottom right corner representing cage number 10.

There are no boxes in the fifth and sixth columns of row 2.

The third row is labelled Block 3.

Inside the box in the first column of row 3 is C13 in the bottom right corner representing cage number 13.

There are no boxes in the second, third, fourth, fifth and sixth columns of row 3.

The fourth row is not labelled.

There are no boxes in the first, second, third, and fourth columns of row 4.

Inside the box in the fifth column of row 4 is the number 12 in the top left corner representing the ranked weight, TC in the centre representing diet C, and C47 in the bottom right corner representing cage number 47.

Inside the box in the sixth column of row 4 is the number 7 in the top left corner representing the ranked weight, T5 in the centre representing diet 5, and C48 in the bottom right corner representing cage number 48.

The fifth row is labelled Block 9.

Inside the box in the first column of row 5 is the number 37 in the top left corner representing the ranked weight, TC in the centre representing diet C, and C49 in the bottom right corner representing cage number 49.

Inside the box in the second column of row 5 is the number 39 in the top left corner representing the ranked weight, T5 in the centre representing diet 5, and C50 in the bottom right corner representing cage number 50.

Inside the box in the third column of row 5 is the number 42 in the top left corner representing the ranked weight, T3 in the centre representing diet 3, and C51 in the bottom right corner representing cage number 51.

Inside the box in the fourth column of row 5 is the number 38 in the top left corner representing the ranked weight, T4 in the centre representing diet 4, and C52 in the bottom right corner representing cage number 52.

Inside the box in the fifth column of row 5 is the number 40 in the top left corner representing the ranked weight, T2 in the centre representing diet 2, and C53 in the bottom right corner representing cage number 53.

Inside the box in the sixth column of row 5 is the number 41 in the top left corner representing the ranked weight, T1 in the centre representing diet 1, and C54 in the bottom right corner representing cage number 54.

The sixth row is labelled Block 10.

Inside the box in the first column of row 6 is the number 27 in the top left corner representing the ranked weight, T4 in the centre representing diet 4, and C55 in the bottom right corner representing cage number 55.

Inside the box in the second column of row 6 is the number 29 in the top left corner representing the ranked weight, T3 in the centre representing diet 3, and C56 in the bottom right corner representing cage number 56.

Inside the box in the third column of row 6 is the number 25 in the top left corner representing the ranked weight, T5 in the centre representing diet 5, and C57 in the bottom right corner representing cage number 57.

Inside the box in the fourth column of row 6 is the number 28 in the top left corner representing the ranked weight, TC in the centre representing diet C, and C58 in the bottom right corner representing cage number 58.

Inside the box in the fifth column of row 6 is the number 30 in the top left corner representing the ranked weight, T1 in the centre representing diet 1, and C59 in the bottom right corner representing cage number 59.

Inside the box in the sixth column of row 6 is the number 26 in the top left corner representing the ranked weight, TC in the centre representing diet C, and C60 in the bottom right corner representing cage number 60.

Where Tx refers to diet number X and Cx refers to cage number X

Number in the upper left corner refers to ranked weight.

Other randomization would give other caging arrangements. Further details on

Randomized Block Design and the proper procedure for analysis of the data may be found in most statistical textbooks.