protein Quality

INTRODUCTION

Protein quality measurements are used to predict the nutritional quality of food proteins for growth and maintenance(1-3). Biological(in vivo) assays measure growth or N balance as indicators of protein utilization and metabolism. These tests are designed to reflect the essential amino acid content, bioavailability of the amino acids present in the protein, and protein digestibility of the food or food ingredient being tested. Amino acid composition data is commonly compared to reference proteins, then corrected for digestibility(in vivo or in vitro) to obtain a protein quality estimate. Chemical methods to determine the availability of amino acids can also provide the basis for protein quality measurements when coupled with other test.

TABLE 16-1. Comparison of Suggested Patterns of Amino Acid Requirements for Humans with that of the Rat and with the Composition of Casein

SUGGESTED PATTERN OF REQUIREMENT

Amino Acid (mg/g crude protein)	Infant Mean(Range)2	Preschool Child (2-5 years)3	School Child(10-12 Years)	Adult	Laboratory Rat4	Reported Composition Casein5
Histidine	26(18-36)	(19)6	(19)	16	25	32
Isoleucine	46(41-53)	28	28	13	42	54
Leucine	93(83-107)	66	44	19	62	95
Lysine	66(53-76)	58	44	16	58	85
Methionine + Cystine	42(29-60)	25	22	17	507	35
Phenylalanine + tyrosine	72(68-118)	63	22	19	66	111
Threonine	43(40-45)	34	28	9	42	42
Tryptophan	17(16-17)	11	(9)	5	12.5	14
Valine	55(44-77)	35	25	13	50	63
Total
Including histidine	460(408-588)	339	241	127	407.5	531
Minus histidine	434(390-552)	320	222	111	382.5	499

the bioavailability of the amino acids contained within the food.

Regulatory Actions Related to Protein Quality Tests

The selection and approval of methods to assess protein quality are important from a regulatory perspective with regard to both public health needs and economic impact (11). Human clinical studies that measure growth and/or other metabolic indications(eg., N balance) provide the most accurate assessment of protein quality. However, use of such techniques is inappropriate and impractical to routinely measure protein quality, for reasons primarily of time and cost and sometimes of ethics. Therefore, animals are often used in protein quality evaluation tests.

One of the first widely used methods for assessing protein quality was the protein efficiency ratio(PER) test, developed in 1919. This method , which measures the ability of a protein to support growth in young, rapidly growing rats, has been used to predict protein quality for humans. Until recently, the PER procedure with casein as the reference protein was the only method specified by the FDA for nutritional labeling purposes. However, it has been recognized for some time that the PER is an inadequate method for assessing the nutritional quality of food proteins in human nutrition(11-13). It overestimates the value of some animal proteins for human nutrition and underestimates the value for some vegetable proteins. The PER method is also criticized for maintenance. This eventually led to a recommendation by the Joint FAO/WHO Expert Consultation on Protein Quality Evaluation that a protein digestibility-corrected amino acid score(PDCAAS) method be adopted internationally as the official method for routine evaluation of protein quality for humans.

METHODS

Growth and Nitrogen Balance Techniques

Of the growth methods, the Per test has been most widely used. An improvement upon the PER method is the net protein ratio(NPR), method. For N balance, the biological value (BV) method are utilized, with NPU being a modification and improvement of the BV method.

Protein Efficiency Ratio

The PER is a biological assay approved by the Association of Official Analytical Chemists International (AOAC Method 960.48) (15) to estimate the protein quality of different foods or food ingredients.

The PER method is based upon the weight gain of a group of male weanling rats fed a test protein, compared to those fed a control diet that contains casein as the sole source of protein. The better the nutritional quality of the test protein, the more rapidly the animals will grow. The quality of the test protein is reported relative to the casein control.

Since PER is an in vivo test, protein digestibility and amino acid bioavailability are encompassed to some extent within the assay. However , it is difficult from a PER assay to determine the individual contribution of each these factors toward the overall protein quality of any given food ingredient.

PROCEDURE

Two sets of male weanling rats from the same colony(21-28 days old) are fed 10 percent protein diets. There should be at least 10 animals per assay group. One set of animals is fed a reference casein(control) diet, and the other set, a diet containing the test protein. The test protein must contain a minimum of 1.80 percent nitrogen if it is to be incorporated into the test diet at the proper level. Diets are isocaloric and contain added carbohydrate in the form of corn starch, crude lipid as cottonseed oil, crude fiber as cellulose, and a balance of minerals and vitamins. To account for differences in the protein content of test materials, corn starch content of the diet is adjusted. Animals are housed in individual cages and re provided with water ad libitum.

The weight of each animal is recorded at the beginning of the assay, and body weight and food intake are measured at regular intervals during the course of the 28 day feeding trial. PER is calculated as the weight gain per g protein (%N X 6.25) fed. The quality of the test protein is reported as the ratio of the test portion PER (X 100) to the PER for reference casein. Casein is assigned a PER of 2.5, and results of the test protein are normalized to this value in an attempt to reduce the interlaboratory variation that has been observed in collaborative experiments. PER is calculated using the average weight gain and protein intake per each diet group(at 28 d)

PER= wt gain/protein intake

Relative PER(RPER) gives a value for a test protein relative to a certain run as part of the same experiment:

RPER= PER of test protein/PER of caseinX 25

Applications PER can discriminate between proteins based upon their nutritional quality even though the test has a tendency to overestimate the protein quality of certain diet and underestimate the value of certain vegetable protein sources, as mentioned above. This underestimation problem is due to the relatively higher need of the rapidly growing weanling rat for certain dietary essential amino acids compared to humans. From a public health standpoint, under estimating PER is not necessarily detrimental. However, there is a tendency of the PER assay to overestimate the nutritional requirements for histidine, isoleucine, threonine, valine, and sulfur-containing amino acids(methionine and cystine(. Casein is a less than ideal reference protein and is deficient by 15-30 percent in meeting the sulphur amino acid requirements of the rat.

The major flaw with the PER method is that it is a growth assay and, as such,does not adequately account for the protein used for maintenance. A protein that does not support growth has a PER of zero, even though it may be suitable for meeting the protein requirements of adults. Problems such as this one have led to recommendations to replace the PER with other methods.

Net Protein Ratio

The net protein ratio(NPR) is a method to correct the PER value for maintenance. This procedure is often run in conjunction with a PER. One group of animals is fed the test protein and a control group is fed a basal diet that contains no protein. The average weight loss of each group of animals is recorded at 10 and 14 d. The NPR value calculated accounts for the protein requirement for maintenance and represents the weight gain for animals on the test diet plus the average weight loss of animals on the zero protein diet per g protein consumed.

Biological Value and Net Protein Utilization

Unlike PER and NPR, which are growth techniques, biological value (BV) and net protein utilization (NPU) are determined from N balance (i.e, N intake – N losses) assays. The BV for a test protein is the proportion of absorbed nitrogen that is retained for maintenance and/or growth, corrected for metabolic and endogenous losses of nitrogen(BV=N retained/ N absorbed). The NPU is the proportion of nitrogen intake that is retained and is equivalent to BV X true digestibility. The NPU can be determined by comparing the carcass nitrogen content of a group of animals fed the test protein diet to that of animals fed a zero protein diet.

NPU = N retained/N intake= BV X true digestibilility.