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Protein efficiency ratio (PER, see Basic Protocol 1) is an animal growth assay that uses rats as test subjects. Two groups of rats are fed a diet consisting either of the test protein or the reference protein, which is usually casein. The rats are fed for a specified period of time with weight gain and food consumption measured. PER was formerly the standard assay for all protein quality analysis; however uncertainties regarding the reliability of this rat growth assay to accurately predict the quality of a protein source in human nutrition resulted in its replacement with other tests. Because PER is a biological assay, it can provide an indication of protein requirements for cell maintenance, and protein digestibility if other tests are conducted along with it. For ranking protein quality, PER results correlate well with biological value (BV) and net protein utilization (NPU) tests (see Alternate Protocol 4). A modification of the PER that takes maintenance into account by comparing the growth between a lactalbu-min control group and test protein groups is the relative nitrogen utilization (RNU) test. This method is an improvement over NPR, since NPR, which uses casein as a reference protein, tends to overestimate the protein quality for lysine-deficient proteins (Hackler, 1977).

An in vivo protein digestibility assay is often conducted in conjunction with a PER (see Alternate Protocol 3). For protein digestibility, the nitrogen ingested in the food is compared with that recovered in the feces (Dong et al., 1987). In vitro protein digestibility (see Basic Protocol 3 and Alternate Protocol 5) can be determined by incubating the test protein with a mixture of proteolytic enzymes and monitoring the degree of hydrolysis (Dimes et al., 1994).

The PER has been replaced by tests that determine a chemical score for a protein (see Basic Protocol 2). In this method, the biological value of a protein, including the limiting amino acid(s), can be predicted by comparing the amino acid profile of a test protein to that of a reference protein. The problem with chemical scoring methods is that they do not take into account how digestible a protein is for animals or people, nor do they predict whether amino acids are bioavailable or whether the test protein contains antinutritional or toxic factors, let alone whether the test protein is palatable as a sole or primary source of dietary protein. A related problem with the PDCAAS is that it

Biochemical Compositional Analyses of Proteins does not reflect the higher nutritional value of a protein that scores higher than the reference protein. Also, because it is an in vitro determination, PDCAAS cannot account for adverse antinutritional factors in a protein ingredient such as those present in plants, i.e., glucosino-lates (mustard and canola), gossypol (cottonseed), phytates (cereals and oilseeds), hemag-glutins and trypsin inhibitors (legumes). Neither can it account for antinutritional factors formed during heat processing or via chemical additions (e.g., acids, oxidizing agents). The method does not determine the true digestibility of poorly digestible or low-quality proteins with supplemental amino acids added; the bioavailability of individual amino acids may be as much as 44% lower than the overall digestibility of protein in the same food product (Sarwar, 1996). Because of this limitation, it is difficult to determine the actual limiting amino acid in a protein, and this affects the accuracy of the overall protein score.

Additional factors that may affect the reliability of the chemical scoring methods lie with the inherent difficulties of amino acid analysis. The analytical procedure for amino acid analysis can affect both the recovery and reliable quantitation of amino acids. Proteins must first be hydrolyzed to amino acids before analysis. Hydrolysis methods affect the amino acid recovery. Cystine, methionine, tryptophan, threonine, serine, and tyrosine can be destroyed during hydrolysis. Valine and isoleucine are released slowly and may not be completely recovered. There can also be significant differences in the amino acid profile for the same protein, depending upon the chromatographic methods chosen for amino acid analysis (Selig-son and Mackey, 1984).

The most common chemical scoring method is the protein digestibility-corrected amino acid score (PDCAAS; see Basic Protocol 2). This method has been adopted by the FDA for nutritional labeling of foods in the United States and by FAO/WHO for routine analysis of protein quality for humans (Sarwar, 1996). A nutritionally complete protein is used as the reference protein for scoring protein quality. There are different scoring patterns for infants (which use the amino acid composition of human milk), preschool-aged children (2 to 5 years), school-aged children (10 to 12 years), and adults (see Table B2.1.2). The PDCAAS corrects the chemical score for protein digestibility. Digestibility is determined through an in vitro method. These in vitro methods use a series of enzymes that simulate the mix of digestive proteolytic enzymes in the small intestine.

In vitro protein digestibility assays provide an estimate of how a protein is digested, absorbed, and utilized. Differences in protein digestibility arise from the varying susceptibility of protein to enzymic hydrolysis in the digestive system. These differences can arise from processing treatments, such as thermal abuse, which reduce protein digestibility. These deleterious changes are due to alteration in the

Analyses of Protein Quality

Table B2.1.2 Suggested Amino Acid Profiles for Reference Proteins"

Amino acid (mg/g crude protein)

Age category

Infant

Pre-school 2-5 year

School age 10-12 year

Adult

Infant

Pre-school 2-5 year

School age 10-12 year

Adult

Histidine

26(18-36)

19b

19b

16

Isoleucine

46(41-53)

28

28

13

Leucine

93(83-107)

66

44

19

Lusine

66(53-76)

58

44

16

Methionine + cystine

42(29-60)

25

22

17

Phenylalanine + tyrosine

72(68-118)

63

22

19

Threonine

43(40-45)

34

28

9

Tryptophan

17(16-17)

11

9a

5

Valine

55(44-77)

35

25

13

Total

Including histidine

460(408-588)

339

241

127

Minus histidine

434(390-552)

320

222

111

"Adapted from: Comparison of suggested patterns of amino acid requirements with the composition of high quality animal proteins (Joint FAO/WHO Expert Consultation, 1991).

'Values interpolated from smoothed curves of requirements versus age.

"Adapted from: Comparison of suggested patterns of amino acid requirements with the composition of high quality animal proteins (Joint FAO/WHO Expert Consultation, 1991).

'Values interpolated from smoothed curves of requirements versus age.

primary and secondary structures of a protein through formation of chemical bonds that are not susceptible to digestive enzymes. How a protein has been treated is also important because processing or storage under abusive conditions can alter the three-dimensional structure of the protein, either improving or lessening its susceptibility to digestive enzymes. The presence of nonprotein dietary constituents can also affect how a protein is digested. Some of these components include phytate, dietary fiber, and various toxigenic agents. Amino acid digestibility can differ from the digestibility of the protein in a food. For certain foods such as legumes, the true digestibility of methionine, cystine, and tryptophan are 25% to 44% lower than the respective protein. However, differences between protein and amino acid digestibility for most proteins do not differ by >10%, making a correction of amino acid scoring using total protein digestibility sufficiently accurate.

In vitro digestibility assays are commonly conducted as part of a PDCAAS assay or in conjunction with the PER animal bioassay. Enzymes for the standard in vitro digestibility assay include porcine pancreatic trypsin, porcine intestinal peptidase, bovine pancreatic a-chymotrypsin, and often one or more bacterial proteases. The digestibility of the test protein is calculated relative to casein and is based upon the drop in pH resulting from protein hydrolysis. For this reason, these assays are called pH-shift procedures. One of the more popular in vitro digestibility assays is the three-enzyme procedure, which is the basis for the standard AOAC method 982.30. In vitro assays underestimate the digestibility of high-quality proteins such as dried egg and nonfat dry milk. The major limitation of the pH shift methods is that the pH is not constant during the course of the assay. Also, the buffering capacity of peptides, proteins, and other substances in the test protein may influence the pH to decline in a pH shift assay. Recently developed pH-stat methods are a more accurate means for measuring protein digestibility because the reaction pH can be controlled during the incubation period (Hsu et al., 1977; Satterlee et al., 1979; Dimes et al., 1994).

The advantages of an animal bioassay are that protein digestibility, amino acid bioavail-ability, and the presence of antinutritional factors can be ascertained. However, animal-based feeding studies can be difficult. First, these assays are expensive to conduct and time-consuming. Secondly, these assays require animals to be fed artificial diets that do not reflect what the animal would consume on a regular basis. For certain species, there are additional difficulties. In aquaculture, for example, collection of feces is difficult since these are dispersed in the water column and dissolve; urine collection is not possible without resorting to a system that restrains and stresses the fish, affecting its feeding behavior. Also, extrapolation of the results from animal feeding experiment, such as the PER bioassay to humans has been criticized because of the difference in amino acid requirements and growth rates between rats and people (Seligson and Mackey, 1984). Factors that affect the results of protein quality assays using animals include (Hackler, 1977): age and gender of animal, animal weight, protein quality and quantity, food intake, other dietary components in the feed (i.e., minerals, fat, carbohydrate, and moisture), animal husbandry practices, and environmental conditions (i.e., temperature, humidity, cage size, and light-ing/photoperiod).

The interpretation of results from protein-quality assays for foods should be cautiously interpreted. For example, the palatability of ingredients is a function of compounds inherent in the ingredient or a result of formation of off-flavors resulting from processing (e.g., thermal abuse creating browning reaction products). Since protein-quality assessments are conducted with the test ingredient being the sole source of dietary protein, often fed to animals for a significant period of time, low palatability can confound a study and lead to unreliable predictions of protein quality. Low palatability has been observed in aquaculture diets containing defatted soybeans, thermally abused fish meal, and co-dried ensiled mixed protein ingredients.

Critical Parameters

Diet formula

1. PER control group diet formula. The protein component in the diet for the control group should be pure (reference) casein (ANRC reference casein, Teklab Diets). Casein is assigned a PER of 2.5. Results for the test protein(s) are normalized against this value in an attempt to reduce interlaboratory variation. The diet must be isocaloric (see recipe for PER Test Group Diet, below).

2. PER test group diet formula. Diet for test group with the protein fraction for the diet being the protein of interest (i.e., test protein): Test diets are 10% protein. The protein ingredient

Biochemical Compositional Analyses of Proteins

Analyses of Protein Quality must contain 1.6% nitrogen if it is to be incorporated into the test diet at the proper level. Diets must be <10% moisture. The fat content of high-fat foods should be reduced to <10% by ether extraction.

Diets are isocaloric. The composition of the test and control (reference protein) diet (calculated on a dry weight basis) is 10% protein (1.6% nitrogen), 1 % AIN vitamin mix 76, 3.5% AIN mineral mixture 76 (Nutritional Bio-chemicals), 0.2% choline bitartrate, 5% cellulose (only if test food is <5% total dietary fiber), corn oil to 10% total fat, and corn starch to total 100%. To account for differences in the protein content of the test diet, the level of corn starch can be adjusted (Joint FAO/WHO Expert Consultation, 1991). The chemical composition (proximate analysis) of the test protein must be measured before test diets are formulated. The proximate analysis of the test and control diets are to be measured after the diets are formulated, but before they are fed, to ensure that the protein content is the same for all diets, and that the diets are isocaloric.

3. PER zero-protein diet formula. For determination of net protein ratio, a test diet that contains no protein is run as one of the test diets. This zero protein diet is used to derive a correction to account for the amount of protein required for cell maintenance.

Accurate diet preparation and analysis are critical for any feeding study. The diet must be chemically uniform from the beginning to the end of the study, and the composition of each diet treatment must be accurately conducted. Experimentally determined differences between diet treatments will be reliable only if the composition of the diets can be relied upon—do the different treatments have the same protein and calorie content? Representative sampling of diets for testing is a prerequisite, as well as experience conducting an accurate proximate analysis. Palatability of the diets is also an issue in some studies, as well as the ease the animal has in consuming the diet. Proper acclimation of the animals prior to the study, and the overall health and well-being of the animals during the course of the study, are important to study outcome. Lethargy and failure to eat are not uncommon, and reasons for this must be determined, as this affects the reliability of the test. Conducting the experiment with the least amount of stress to the animals is very important. Also, at the beginning of the study, it is important to ensure that animals are uniformly distributed between diet treatment groups based upon their initial weight at the beginning of the study. Taking accurate weights of the animals at regular intervals, as well as of the feed provided and consumed are necessary for reliable results.

Chemical scoring methods

There has been resistance to a wider adoption of chemical scoring methods for predicting protein quality because of the number of separate assays required. A standard amino acid analysis, plus separate assays for tryptophan and methionine/cysteine, are required. All the critical factors associated with protein hydrolysis, sample preparation for amino acid analysis, proper use of and selection of internal and external standards, selection of analytical method including derivatization and chroma-tographic method, sensitivity, and possible interference from sample constituents, as well as data acquisition and analysis, will all play roles in determining reliability of using a chemical scoring method for estimating protein quality. Correlation of these results with those for in vivo assays is not guaranteed. Analytical results for lysine may not accurately reflect chemically or biologically available lysine (Friedman, 1996), and so additional testing to predict bioavailability is needed. Protein digestibility is often conducted in conjunction with PDCAAS using an in vitro assay. Again, the critical issue with this method is whether it will accurately reflect how well a protein and its constituent amino acids can be used by an organism for growth and maintenance.

Digestibility methods

High protein digestibility does not necessarily mean high protein quality. Protein quality measures the balance between the amino acids in the protein necessary for growth and cell maintenance.

In vivo tests require animal feeding studies, and all of the factors outlined under PER apply. In addition, for digestibility calculations, accurate measurement of feed consumed, complete recovery of feces (devoid of feed), possibly urine, and calculation of body composition are required. These are not the most pleasant experimental protocols to conduct, but have to be done carefully and correctly if meaningful results are to be obtained.

In vitro protein digestibility requires preparation of a treated sodium caseinate standard. Because of the empirical nature of these determinations, it is critical that results for the so dium caseinate are reliable. Preparation of this standard must be conducted exactly as described, and the standard must be tested to ensure that digestibility is 100±2% after it has been prepared. Nitrogen content of the protein standard and the test proteins must be accurately determined using an appropriate Kjeldahl method, since the digestibility assays are calculated on a constant-nitrogen basis. Protein samples must be held so that they do not take up or lose moisture between the time of the N determination and the digestibility assay. For certain protein ingredients, it may be necessary to correct the total N content for nonprotein nitrogen. Preparation of fresh enzyme solutions for each series of assays is critical. It is best that these solutions not be used for more than a couple of hours. Accurate preparation is important, and as the specific activity of each preparation may be somewhat different, the activity should be confirmed before the enzyme is used. Whether a pH shift or a pH stat assay is employed, accurate calibration of pH at the assay temperature is critical. Also, it will be important to select a reaction vessel that can be kept at a constant temperature and be stirred and into which a pH electrode can be inserted for measurement.

Troubleshooting

Protein efficiency ratio and in vivo digestibility methods

Maintaining animals in a comfortable environment, as stress free as possible, is important, including maintaining a constant temperature and uniform photoperiod. If animals appear sick or lethargic, or fail to eat, a veterinarian should be contacted and study terminated. Because some proteins are not highly palatable, conducting a test run, feeding a small number of rats a control diet and some others the experimental diet, is recommended to see how well the animals eat the diet. Sometimes replacing a small amount of the corn starch in the diet (~10%) with dextrose will improve palatability.

Animals should be watered and feed should be replaced at the same time of day, if possible, during the course of the study. Food consumption should be checked daily, and more often if necessary, depending upon feed consumption rate. Animal food dishes should not be left empty.

If animals spill food, the spilled food must be recovered as completely as possible and weighed. If this is a recurrent problem, switch to feed dishes of a different configuration, or alter the bulk density of the food (e.g., switch to a pelleted food, although this is not always possible). Line cages with paper to make collection of spilled feed easier. If paper becomes wet from spilled water or urine, dry the paper at room temperature before recovering and weighing the spilled feed. Spilled feed should be recovered and weighed daily.

The container used to weigh the rats should be large enough so that they cannot jump out. A small wire cage or basket is recommended.

Recovering feces for protein digestibility measurements can be tedious and unpleasant. It is necessary to recover as much material as possible from cage walls and floor, and from the cage liner, with as little contamination with food or urine as possible. Feces from each rat should be collected separately and stored in a sealed container, preferably in the freezer until analyzed. These should be air dried first before storage if possible. If it becomes difficult to recover feces from the cage liner because they stick, switch to a glossier paper to line the cage. In extreme situations, where there is a large amount of urine or water contamination of the feces, measuring nonprotein nitrogen may be necessary to correct for urine contamination (Varner et al., 1953).

Conducting body composition analysis on rat carcasses presents challenges that are usually not faced when taking representative samples, because of hair, bone fragments, etc. Taking additional samples for analysis is recommended, until the standard error for these measurements falls into line with lab averages with more conventional samples.

Measuring Kjeldahl nitrogen is not trivial, particularly on samples with relatively low amounts of nitrogen. Running test proteins with known amounts of nitrogen will help to ensure that determinations are accurate. Titration values should be checked with known standards daily, particularly in the case of samples for which discerning the color change is difficult. Switching catalysts can sometimes help to improve results.

Chemical scoring methods

Amino acid analysis is fraught with literally dozens of pitfalls. For novel protein ingredients, those that have undergone chemical modification, or ones that have been subjected to high heat, conventional methods for protein hydrolysis may or may not be effective. It may be necessary to evaluate different hydrolysis methods to determine which provides the best recovery.

Biochemical Compositional Analyses of Proteins

Chromatographic separation of amino acids in the test protein is required for chemical scoring methods. The selectivity and sensitivity of chromatographic methods vary and comparing results of more than one method may be required, particularly when proteins with unusual amino acid profiles are being tested.

For routine regulatory and food labeling purposes, an accurate determination of amino acid content coupled with an assessment of protein provides a sufficient basis for estimating protein quality (Young and Pellet, 1991). However, this does not mean that values are accurate. Unlike animal bioassays, a chemical scoring method cannot factor in the value of a protein as part of a mixed diet. Also, it does not take into account mechanisms animals have for salvaging and reutilizing endogenous amino acids.

In vitro protein digestibility pH shift and pH stat assays are, in principle, fairly straightforward to conduct. However, it is critical the pH meter be calibrated at 37°C with 37°C buffer. Checking the sensitivity of the meter at the temperature is important.

Also, the effect of stirring on pH fluctuation should be checked for pH stat determinations.

Protein materials that are not highly soluble at pH 8 in water are difficult to test using this method. Hydrating a protein overnight is sometimes helpful.

Check the activity of enzymes separately if digestibility values seem to be low. Ensure that the correct enzyme, grade, and concentration have been used. If there is any question about enzyme quality, replace it.

Anticipated Results PER

PER values for test proteins will be lower than casein for conventional test materials. This is not the case for protein ingredients that have been specifically designed to have enhanced protein quality. Test results for other high-quality protein sources, such as meat, fish, and egg, may yield higher PER results than a casein control, and the relative rating between very high protein sources is related in part to the palatability of the respective diet. Sometimes the range of weights (weight loss) for animals fed zero-protein diets can be highly variable within the treatment group, reducing the precision of the assay.

Analyses of Protein Quality

Chemical score

Values for a test protein will be lower than for the reference protein. The reliability of results for chemical scoring methods depends upon the accuracy of the amino acid determinations that form the basis for these assays. Chemical scoring and animal bioassays tend to provide similar relative rankings of protein quality; however, the actual values may be different. Chemical scoring methods should not replace a bioassay for testing the quality of a food protein for which there is very little nutritional information.

Digestibility assays

The relative ranking of protein ingredients by in vivo and in vitro digestibility tests will generally track each other; however, the values on a percentage basis may be quite different. The digestibility of a protein will vary depending upon how it has been processed. Proteins that are highly soluble in water, or those that have been treated to improve solubility, will tend to have greater digestibilities. The digestibility values for proteins that have been partially hydrolyzed may not be reliable. For proteins that have been heated, or which have been derivatized (e.g., by formation of browning reaction products or exposure to alkali or acid), in vitro digestibility results may not provide a good indication of the bioavailability.

Time Considerations PER

The preparation time for a PER is extensive and involves several days for acquisition of rats and their acclimation before a feeding trial begins. Preparation of a consistent and uniform diet is not trivial. Adequate diet for each treatment for the course of the study should be prepared shortly in advance of the study. Testing of the diet involves chemical analysis for protein (i.e., Kjeldahl N takes several hours) and accurate determination of other diet constituents (e.g., ash, crude lipid, and dietary fiber) so that isocaloric diets can be formulated between treatment groups. These determinations take >1 day to complete. It is recommended that multiple samples from each diet be obtained for proximate analysis of diet before it is fed to ensure that each diet has the proper nitrogen content (and this is same between diet treatments) and that all diets are isocaloric.

This is a growth assay and takes 28 days to complete. Fecal N and body composition analyses each take an additional several days of sample preparation and analysis to complete.

Chemical scoring methods

A fairly complicated sample preparation is involved. Comparison of hydrolysis and chromatographic procedures may be necessary when testing unconventional protein sources. Knowing the sample nitrogen content is required. There is also the need to hydrolyze protein (an overnight procedure). Separate hydrolysis procedures are required for tryptophan and for the sulfur-containing amino acids, which take several hours (or overnight) to complete. Chromatographic analysis is relatively rapid (minutes to hours); however, becoming proficient at amino acid chromotography can take months or years.

Digestibility tests

In vitro assays themselves take a few hours to complete. The protein nitrogen content of a sample must be known. Sample hydration can take one to several hours. Preparation of enzyme solutions takes <1 hr, however, determination of the activity of the different enzymes within the preparation can take days. In vivo digestibility assays involve a feeding study, and the investigator faces similar issues as described for PER.

Literature Cited

Adler-Nissen, J. 1984. Control of proteolytic reactions and the level of bittnerness in protein hydrolysis processes. J. Chem. Technol. Biotech-nol. 34B:215.

Adler-Nissen, J. 1986. Enzymatic Hydrolysis of Food Proteins. Elsevier Applied Science Publishers. Barking, United Kingdom.

Dimes, L.E., Haard, N.F., Dong, F.M., Rasco, B.A., Forster, I.P., Fairgrieve, W.T., Arndt, R., Hardy, R.W., Barrows, F.T., and Higgs, D.A. 1994. Estimation of protein digestibility II. In vitro assay of protein in salmonid feeds. Comp. Biochem. Physiol. 180A:363-370.

Dong, F.M., Rasco, B.A., and Gazzaz, S.S. 1987. A protein quality assessment of wheat and corn distillers' dried grains with solubles. Cereal Chem. 64:327-332.

Dong, F.M., Rasco, B.A., Gazzaz, S.S., San Buenaventura, M.L., and Holcomb, L.M. 1990. Body composition and serum and liver lipids in rats fed distillers' dried grains. J. Sci. FoodAgric. 51:299-308.

Friedman, M. 1996. Nutritional value of proteins from different food sources. A review. J. Agric. Food Chem. 44:6-29.

Hackler, L.R. 1977. Methods of measuring protein quality: A review of bioassay procedures. Cereal Chem. 54:984-995.

Hsu, H.J.W., Satterlee, D.L., and Miller, G.A. 1977. A multi-enzyme technique for estimating protein digestibility. J. Food Sci. 42:1269-1271.

Joint FAO/WHO Expert Consultation. 1991. Protein quality evaluation. Bethesda, Md. 1989. Food and Agriculture Organization/World Health Organization. FAO Headquarters. Rome, Italy.

Pedersen, B. and Eggum, B.O. 1983. Prediction of protein digestibility by an in vitro enzymatic pH-stat procedure. Z. Tierphysio. Tierernachr. Futtermittelkd. 49:265-277.

Sarwar, G. 1996. The protein digestibility-corrected amino acid score method overestimates quality of proteins containing antinutritional factors and of poorly digestible proteins supplemented with limiting amino acids in rats. J. Nutr. 127:758764.

Satterlee, L.D., Marshall, H.F., and Tennyson, J. M. 1979. Measuring protein quality. J. Am. Oil Chem. Soc. 56:103-109.

Seligson, F.H., and Mackey, L.N. 1984. Variable predictions of protein quality by chemical score due to amino acid analysis and reference pattern. J. Nutr. 114:682-691.

Varner, J.E., Bulen, W.A., Vanecko, S., and Burrell, R.C. 1953. Determination of ammonium, amide, nitrite and nitrate nitrogen in plant extracts. Anal. Chem. 25:11528-1529.

Williams, A.P. 1982. Determination of amimo acids and peptides. In: HPLC in Food Analysis (R. Macrae, ed.), pp. 285-311. Academic Press, Boca Raton, Fla.

Young, V.R. and Pellett, P.L. 1991. Protein evaluation, amino acid scoring and the Food and Drug Administration's proposed food labeling regulations. J. Nutr. 121:145-150.

Contributed by Barbara Rasco Washington State University Pullman, Washington

Biochemical Compositional Analyses of Proteins

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