Nutrient Requirements and Interactions

The Protein DigestibilityCorrected Amino Acid Score Method Overestimates Quality of Proteins Containing Antinutritional Factors and of Poorly Digestible Proteins Supplemented with Limiting Amino Acids in Rats1
Ghulam Sarwar
Health Canada, Nutrition Research Division, Bureau of Nutritional Sciences, Health Protection Branch, Banting Research Centre, Tunneys Pasture, Ottawa, Ontario, K1A 0L2, Canada
ABSTRACT The validity of the protein digestibility-corrected amino acid score (PDCAAS) method in predicting the quality of fourteen protein products was compared with the commonly used protein quality methods, protein efciency ratio (RER) and net protein ratio (NPR). A rat growth and balance study was conducted to determine protein digestibility and quality of the animal and vegetable protein products by the PER and NPR methods. Amino acid compositions of the products were also determined, and PDCAAS were calculated using a rat and a human pattern of amino acid requirements. Compared to the biological methods, the scoring method overestimated protein quality of mustard our [PDCAAS of 8492% vs. relative PER (RPER) or relative NPR (RNPR) of 0], raw black beans (PDCAAS of 4572% vs. RPER or RNPR of 0), alkaline-treated lactalbumin and soybean protein isolate (PDCAAS of 4467% vs. RPER or RNPR of 0) and heated skim milk (PDCAAS of 2931% vs. RPER and RNPR of 05%). The scoring method also overestimated the protein quality of zein (true protein digestibility of 63%) supplemented with Lys, Met, Thr and Trp (PDCAAS of 6371% vs. RPER and RNPR of 344%). These data demonstrate that the PDCAAS method is inappropriate for predicting protein quality of those protein sources which may contain naturally occurring growth-depressing factors or antinutritional factors formed during alkaline and/or heat processing. J. Nutr. 127: 758764, 1997 KEY WORDS: rats protein digestibility amino acid score protein efciency ratio antinutritional factors growth

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The protein digestibilitycorrected amino acid score (PDCAAS)2 method has been considered to be a simple and scientically sound approach for routine assessment of dietary protein quality for humans (FAO/WHO 1991). FAO/WHO (1991) recommended use of the FAO/WHO/UNU (1985) amino acid requirement pattern for children of preschool age for the evaluation of dietary protein quality for all age groups except infants. The PDCAAS is now a federally approved alternative method to the protein efciency ratio (PER) rat bioassay procedure still ofcial in Canada and U.S.A. Major questions have, however, been raised about the validity of the PDCAAS relative to its inability to credit the extra nutritional values of proteins having scores higher than that of the reference protein, its failure to fully account for the possible adverse effects of antinutritional factors, and its assumption about complete biological efciency of supplemental amino acids in improving quality of proteins, which may not be true in the case
1 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 USC section 1734 solely to indicate this fact. 2 Abbreviations used: FP, fecal protein; IDRC, International Development Research Centre; MFP, metabolic fecal protein; NPR, net protein ratio; PDCAAS, protein digestibility-corrected amino acid score; PER, protein efciency ratio; PI, protein intake; RNPR, relative net protein ratio; RPER, relative protein efciency ratio; SBM, soybean meal; SPI, soybean protein isolate.

of poorly digestible, low quality proteins (Food Chemical News 1991). These concerns about the PDCAAS, however, require proper documentation (Young 1995). Antinutritional factors may occur naturally or may be formed during heat processing. Some examples of naturally occurring antinutritional factors include glucosinolates in mustard and rapeseed protein products (Fenwick et al. 1982), trypsin inhibitors and hemagglutinins in legumes (Rackis and Gumbmann 1981), phytates in cereals and oilseeds (Sandberg 1991), and gossypol in cottonseed protein preparations (Martinez and Hopkins 1975), which could adversely affect nutrient utilization and may contribute to growth depression in animals. During processing of foods, protein sources are treated with heat, oxidizing agents (such as hydrogen peroxide), organic solvents, alkalis, and acids for a variety of reasons such as to sterilize/pasteurize, to improve avor, texture, and other functional properties, to deactivate antinutritional factors, and to prepare concentrated protein products (Cheftel 1979, Friedman et al. 1984, Schwass and Finley 1984). These processing treatments may cause the formation of Maillard compounds, oxidized forms of sulfur amino acids, D-amino acids, and crosslinked peptide chains (such as lysinoalanine and lanthionine), resulting in lower amino acid bioavailability and protein quality. The purpose of this study was to assess the validity of the

0022-3166/97 $3.00 1997 American Society for Nutritional Sciences. Manuscript received 9 August 1996. Initial review completed 25 September 1996. Revision accepted 28 January 1996. 758

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PDCAAS method in evaluating the quality of six protein sources containing antinutritional factors, and of one poorly digestible protein supplemented with limiting amino acids. A rat growth and balance study was conducted to determine protein digestibility and quality of fourteen test diets. Amino acid proles of test proteins were also determined to calculate scores by the PDCAAS method, using a rat and a human pattern of amino acid requirements. The PDCAAS values were compared to the protein quality indices based on rat growth, such as protein efciency ratio (PER), net protein ratio (NPR), relative PER (RPER) and relative NPR (RNPR).

TABLE 1
Composition of experimental diets fed to rats
Diet1 Protein source Sucrose Cornstarch

g/kg Casein / L-Met Casein Lactalbumin Lactalbumin, treated Skim milk Skim milk, heated SPI2 SPI, treated SBM,3 raw SBM, heated Black beans, raw Black beans, heated Mustard our Zein (corn protein) Zein / AA Protein-free 88.9 88.9 93.9 99.7 235.8 335.8 93.6 97.7 190.9 190.9 372.1 352.9 167.9 89.9 89.9 0.0 234.4 234.4 232.7 230.8 185.5 185.5 232.7 231.5 200.4 200.4 140.0 146.4 208.4 237.1 238.2 267.7 467.7 467.7 464.4 460.5 369.7 369.7 464.7 461.8 399.7 399.7 278.9 291.7 414.7 464.0 464.4 535.3

MATERIALS AND METHODS

Proteins. Casein (Animal Nutrition Research Council Reference Protein), lactalbumin, zein (ICN, Cleveland, OH) and soybean protein isolate (SPI, Supro 620, Les Aliments UFL, Montreal, Quebec, Canada) were purchased commercially. Mustard our (dehulled and defatted yellow mustard) was prepared by the Department of Crop Science and Plant Ecology, University of Saskatchewan, Saskatoon, Saskatchewan. Raw soybeans, raw black beans and skim milk powder were purchased locally. Raw soybeans and raw black beans were nely ground (1 mm seive, Thomas Wiley Mill, Model ED5, Philadelphia, PA). The ground soybeans were defatted with hexane to obtain raw soybean meal (SBM). Heated SBM was prepared by autoclaving a portion of raw SBM at 103 kPa and 121C for 20 min (IDRC 1977). Heated skim milk powder was prepared by autoclaving a portion of the powder at 103 kPa and 121C for 1 h. Heated black bean sample was prepared by soaking and autoclaving raw black beans (IDRC 1977). The heated black beans were subsequently dried and ground. Treated SPI and lactalbumin were prepared by subjecting SPI or lactalbumin to alkaline treatment (0.1 mol NaOH/L) at room temperature for 1 h, followed by heat treatment at 75C for 3 h, neutralization with 10 mol HCl/L to pH 7.5, ultraltration to remove salts and spray drying of the ultraltered retentate (G. Sarwar, unpublished data). All the protein sources were equilibrated to room temperature and humidity before analysis and diet formulation. Total nitrogen was determined (in duplicate) by the microKjeldahl method using a Kjeltec Auto 1030 Analyzer (Tecator, Herndon, VA). Protein was calculated using a nitrogen-to-protein factor of 6.25. Protein sources were hydrolyzed (in duplicate) with 6 mol HCl/ L at 110C for 22 h for the determination of total amino acids except tryptophan and sulfur amino acids (AOAC 1990). Hydrolysis with NaOH (4.2 mol/L) was used for quantitative recovery of tryptophan (AOAC 1990). Hydrolysates for determination of methionine as methionine sulfone and cyste(i)ne as cysteic acid were prepared by performic acid oxidation of protein followed by hydrolysis with HCl (6 mol/L) (AOAC 1990). All amino acids, except tryptophan, in hydrolysates were determined by liquid chromatography of precolumn phenylisothiocyanate derivatives (Sarwar et al. 1988). Tryptophan in alkaline hydrolysates was determined by a simple liquid chromatography method requiring no derivatization (Sarwar et al. 1988). Amino acid analysis by liquid chromatography of precolumn phenylisothiocyanate derivatives has been successfully validated by conventional ionexchange methods (Beaver et al. 1987; Sarwar et al. 1988; White et al. 1986). Experimental design. A rat feeding study was conducted to determine protein digestibility and quality of test products based on rat growth. The effects of antinutritional factors (present naturally or formed during processing) in six protein sources (lactalbumin, skim milk powder, SPI, SBM, black beans and mustard our) on protein digestibility and quality were studied. Moreover, the inuence of amino acid supplementation (lysine, methionine, threonine / tryptophan) on protein digestibility and quality of one poorly digestible protein source (zein) was investigated. The casein / L-methionine (0.2% of diet) control and test products (fourteen) were fed as the sole source of protein in diets containing 8 g protein/100 g diet (Table 1). A protein-free diet (Table 1) was also included in the feeding study to enable calculation of NPR, and for providing an estimate of metabolic fecal protein (MFP) needed for calculating true protein digestibility. The protein diets were made isonitrogenous by the addition of L-glutamic acid (Table

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1 Each diet also contained (g/kg): AIN-76 mineral mixture (AIN 1977), 35; AIN-76A vitamin mixture (AIN 1980), 10; choline bitartarate (Sigma Chemical, St. Louis, MO), 2; lard, 38.5, soybean oil, 61.5 and cellulose (Alfa-oc, Teklad Test Diets, Madison, WI), 50. The control diet also contained (g/kg): L-Met, 2.0; L-Glu, 10.0. All test protein diets except zein / AA also contained (g/kg): L-Glu, 12.0. The zein / AA diet also contained (g/kg): L-LysrHCl, 5.5; L-Met, 1.0; L-Thr, 1.0; L-Trp, 1.0. 2 Soybean protein isolate. 3 Soybean meal.

1). The actual analysis revealed that the casein / Met, casein, lactalbumin, skim milk, treated lactalbumin, heated skim milk, SPI, treated SPI, raw soybean meal, heated soybean meal, raw black beans, heated black beans, mustard, zein and zein / amino acids diets contained 9.00, 9.01, 8.80, 8.78, 8.90, 8.92, 8.94, 8.86, 8.88, 9.05, 8.90, 8.76, 9.00, 8.78 and 8.97 g protein/100 g diet (N 1 6.25), respectively. The determined protein values were used in calculating protein intake data, required for the determination of protein digestibility, PER and NPR. Animals. Weanling male Sprague-Dawley rats (Charles River Canada, S. Constant, Quebec) were used in the feeding study. A total of sixteen diets (control, fourteen test diets and a protein-free diet, Table 1) were tested. The experimental diets were fed to groups of eight rats in a completely randomized design. Rats were housed individually in stainless steel screen-bottom cages in a temperature; and humidity-controlled facility. To minimize contamination of feces with urine, and to catch spilled food, highly absorbent paper was placed under each cage. Animals were given free access to water and food for 2 wk, and records of weekly food consumption and body weights were kept. The Health Canadas guide for the care and use of laboratory animals was followed, and the protocol was approved by the animal care committee. Protein digestibility determinations. Total collection of feces from individual rats was carried out during the last four days of experiment, and records of daily food consumption were also kept during the collection period. Total feces from each rat were lyophilized, weighed and ground. The ground feces from eight rats fed the same diet were pooled and analyzed for total nitrogen. Eight individual protein digestibility values per diet were then calculated using the protein intake and fecal output data for each rat. The balance method used in this investigation has been shown to give protein digestibility values similar to those obtained using metabolic cages (Sarwar 1984). The use of screen-bottom cages, as done in the present investigation, has been a common practice in determining protein and amino acid digestibilities (Happich et al. 1984, Keith and Bell 1988). Protein digestibility determinations have also been compared in our laboratory by using nitrogen determination(s) on pooled vs. individual feces samples from rats fed the same diet. The differences in true protein

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TABLE 2
A comparison of essential amino acid requirements for rat growth, as recommended by NRC (1978) and NRC (1995)
Amino acid NRC (1978) NRC (1995)

RPER (PER of test diet/PER of control diet) 1 100, RNPR (NPR of test diet/NPR of control diet) 1 100, where PER weight gain of test rat/protein consumed by test rat, and NPR (weight gain of test rat / weight loss of nonprotein rat)/ (protein consumed by test rat). The PDCAAS was calculated as follows (Sarwar and McDonough 1990):

g/100 g protein Arg His Ile Leu Lys Met / Cys Phe / Tyr Thr Trp Val 5.00 2.50 4.17 6.25 5.83 5.00 6.67 4.17 1.25 5.00 2.87 1.87 4.13 7.13 6.13 6.53 6.80 4.13 1.33 4.93

PDCAAS (%) True protein digestibility 1 amino acid score (or the lowest amino acid ratio). 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 protein) 1 100] for nine essential amino acids (His, Ile, Leu, Lys, Met / Cys, Phe / Tyr, Thr, Trp and Val) were calculated using a rat growth pattern of amino acid requirements (NRC 1995, Sarwar et al. 1985) and a human pattern of amino acid requirements (FAO/WHO/UNU 1985 suggested pattern of amino acid requirements for preschool children, 25 y) as the reference proteins. Two rat growthbased patterns of essential amino acid requirements (NRC, 1978 and 1995) are compared in Table 2. The values are expressed as g amino acid/100 g dietary protein since the requirements remain constant or decrease slightly when expressed a percentage of protein (NRC 1995). The values for all essential amino acids except arginine and methionine / cystine are similar in both the patterns. The arginine value was considerably lower in the 1995 report compared to the 1978 report (2.87 vs. 5.00 g/100 g protein), while the methionine / cystine value was higher in the 1995 report than in the 1978 report (6.53 vs. 5.00 g/100 g protein, respectively). Since the ASNS-recommended methionine- or cystine-supplemented casein-based control diet for laboratory rodents (providing 5.0 g of methionine / cystine/100 g protein) meets or exceeds rat growth requirements for essential amino acids (AIN 1977, Reeves et al. 1993), the NRC 1995 rat requirement value for methionine / cystine is too high. Moreover, the methionine-supplemented casein (containing 4.74 g of methionine / cystine/100 g protein) was reported to meet or exceed the requirements of rats for essential amino acids

digestibility values of casein and zein by the two sampling methods were less than 1% (G. Sarwar and R. Mongeau, unpublished data). Therefore, the digestibility data reported in this study obtained by using nitrogen analysis of pooled feces samples may be generally assumed to be as accurate as those obtained using nitrogen analysis of individual feces samples. True protein digestibility values were calculated using the following equation (Sarwar and Peace 1986): True protein digestibility PI 0 [FP 0 MFP]/PI 1 100, where PI protein intake, FP fecal protein and MFP metabolic fecal protein. The amount of protein in the feces of rats fed the protein-free diet was used as the estimate for MFP. Relative protein efciency ratio (RPER) and relative NPR (RNPR) values (2-wk) were calculated using the following equations (Sarwar and Peace 1994):

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TABLE 3
Growth of rats and protein efciency ratios (PER), net protein ratios (NPR) and true protein digestibility values of experimental diets1
Diet Weight gain2 Food consumed PER NPR True protein digestibility

g/2 wk

Casein / Met Casein Lactalbumin Lactalbumin, treated Skim milk Skim milk, heated SPI3 SPI, treated SBM,4 raw SBM, heated Black beans, raw Black beans, heated Mustard our Zein Zein / AA Pooled SEM

80a 59c 68b 016g 58c 010g 39d 014g 15e 44d 028h 11e 030h 0 6f,g 1f 6

184a 170b,c 180a,b 129d 174a,b 153c 162c 99f 130e 160c 63g 145d 51h 63g 66g 8

4.83a 3.85c 4.29b 0 3.72c 0 2.69e 0 1.30f 3.04d 0 0.87g 0 0 0.17h 0.08

5.68a 4.76c 5.19b 0 4.65c 0 3.66e 0 2.51f 4.00d 0 1.97g 0 1.45h 2.53f 0.11

100a 99a 99a 73g 94b,c 77f 96b 68h 80e 83d 71g 83d 92c 63i 73g 1

1 Values are means (n 8, initial weight of rats 45 { 4 g). Within a column, values with different superscripts are signicantly different (P 0.05). PER or NPR values of less than zero (i.e. negative values) were considered zero, and were not included in the statistical analysis because of lack of variability. These included PER and NPR of alkaline-treated lactalbumin, PER of heated skim milk, PER and NPR of alkaline-treated SPI, PER and NPR of raw black beans, PER and NPR of mustard our, and PER of zein. 2 Average weight loss of rats fed the protein-free diet was 14 g. 3 Soybean protein isolate. 4 Soybean meal.

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in terms of RPER and RNPR (Sarwar 1984, Sarwar et al. 1984). Pick and Meade (1971) reported that the sulfur amino acid requirement of growing rats was 4.26 g/100 g dietary protein. Peace et al. (1986) and Sarwar et al. (1985) estimated the methionine / cystine requirement of growing rats to be 4.1 and 4.0 g/100 g dietary protein, respectively, using RNPR as the response criterion. The NRC-specied requirement may be higher to make allowance for the active role of methionine as a methylating agent in addition to the minimum needed for protein synthesis. Since the vitamin mixture used in the studies of Peace et al. (1986) and Sarwar et al. (1985) provided an adequate level of dietary choline, the requirement for methionine as a methyl donor may have been reduced. Based on the ndings of Peace et al. (1986) and Sarwar et al. (1985), a value of 4.0 g/100 g dietary protein for growth requirements of rats for sulfur amino acids was used in calculating amino acid scores. For all other essential amino acids, the values reported by NRC (1995) were used in the calculation of the scores. Statistics. The PER or NPR values less than zero (i.e. negative values) were considered zero and were not included in the statistical analysis due to lack of variation. These included PER and NPR of alkaline-treated lactalbumin, PER of heated skim milk, PER and NPR of alkaline-treated SPI, PER and NPR of raw black beans, PER and NPR of mustard our, and PER of zein. PER and/or NPR data for other diets, and weight gain, food consumed, and true protein digestibility data for all diets were analyzed by one-way ANOVA and Tukeys highly signicant difference test (Steele and Torrie 1980) using the Statistical Systems for Personal Computers (SAS Institute, Cary, NC). Differences were considered signicant when P 0.05.

TABLE 4
Lysine, methionine / cystine, threonine and tryptophan in protein sources, expressed as a proportion of the respective amino acids for rat growth requirements1
Protein source Lys Met / Cys Thr Trp

% Casein / Met Casein Lactalbumin Lactalbumin, treated Skim milk Skim milk, heated SPI3 SPI, treated SBM,4 raw SBM, heated Black beans, raw Black beans, heated Mustard our Zein Zein / AA5 138 138 171 136 129 38 99 81 99 97 111 111 94 2 94 142 86 138 84 79 72 65 45 72 70 63 61 91 55 86 112 112 138 76 108 106 96 62 96 97 121 116 100 69 99 1002 100 159 159 91 90 85 95 85 85 84 79 94 13 94

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RESULTS True protein digestibility and protein quality indices of experimental diets based on rat growth (PER and NPR) are shown in Table 3. Casein (both unsupplemented and supplemented with methionine), untreated lactalbumin, untreated SPI, mustard our and untreated skim milk powder had high true protein digestibility values (94100%); zein (both unsupplemented and supplemented with limiting amino acids) had low true protein digestibility values (6373%); and heat processed SBM and black beans had intermediate true protein digestibility values (8384%). The alkaline treatment had signicant negative effects on the protein digestibility of lactalbumin (99 vs. 73%) and SPI (96 vs. 68%). Moderate heat processing treatment had a signicant benecial effect on the protein digestibility of black beans (71 vs. 83%) and SBM (80 vs. 83%), while overheating had a signicant deleterious effect on the protein digestibility of skim milk powder (94 vs. 77%). The rat growth and food intake data needed to determine PER and NPR of the experimental diets are also shown in Table 3. The PER and NPR values of mustard our were zero. The alkaline treatment had a drastic negative effect on the PER and NPR values of lactalbumin (4.29 vs. 0%; 5.18 vs. 0%) and SPI (2.69 vs. 0%; 3.66 vs. 0%). As noted in the case of protein digestibility, moderate heat processing improved the PER and NPR values of black beans (0 vs. 0.87%; 0 vs. 1.97%) and SBM (1.30 vs. 3.04%; 2.51 vs. 4.00%), while overheating caused a drastic reduction in the PER and NPR values of skim milk powder (3.72 vs. 0%; 4.65 vs. 0%). The PER and NPR values of the zein diet were 0% and 1.45%, respectively. Supplementation of the zein diet with limiting amino acids (lysine, methionine, threonine / tryptophan) caused signicant improvement in the NPR value (2.53%). The calculations of amino acid ratios (the lowest amino acid ratio being the amino acid score) for the four most limiting amino acids (lysine, methionine / cystine, threonine and tryptophan) using the rat growth pattern for amino acid requirements are shown in Table 4. Only casein / methionine and untreated lactalbumin met or exceeded all the essential amino acids requirements for rat growth (Table 4, data for all essential

1 As reported by NRC (1995) for all essential amino acids except methionine / cystine. For methionine / cystine requirement, the value reported by Sarwar et al. (1985) was used. 2 Underlining denotes the lowest amino acid ratio (or amino acid score). 3 Soybean protein isolate. 4 Soybean meal. 5 Lysine / methionine / threonine / tryptophan.

amino acids not shown). Casein, skim milk powder, untreated SPI, alkaline-treated SPI, raw SBM, heated SBM, raw black beans, heated black beans, mustard our and amino acid-supplemented zein were all rst-limiting in sulfur amino acids with amino acid scores of 86, 79, 65, 45, 72, 70, 63, 61, 91, and 86%, respectively. Alkaline-treated lactalbumin was rstlimiting in threonine (amino acid score of 76%), while heated skim milk and zein were rst-limiting in lysine with amino acid scores of 38 and 2%, respectively. The calculations of amino acid ratios (the lowest amino acid ratio being the amino acid score) for four most limiting amino acids (lysine, methionine / cystine, threonine and tryptophan) using the human pattern for amino acid requirements are shown in Table 5. Casein / methionine, casein, untreated lactalbumin, skim milk powder, untreated SPI, raw SBM, heated SBM, raw black beans, heated black beans and mustard our met or exceeded all the essential amino acids requirements for humans using the FAO/WHO (1991) amino acid reference pattern (Table 5, data for all essential amino acids not shown). The overheated skim milk powder was rst-limiting in lysine (score of 40%), while the alkaline-treated SPI and lactalbumin were limiting in methionine / cystine (score of 72%) and threonine (score of 92%), respectively. Zein was rst-limiting in lysine (score of 2%), and the supplemented zein was marginally limiting in lysine (score of 99%). The protein digestibility-corrected scores, RPER and RNPR are shown in Table 6. Due to the higher amino acid requirements for rat growth compared to that of human (especially sulfur amino acids), the PDCAAS of most products based on rat requirements were considerably lower compared to those based on human requirements. The differences between the PDCAAS based on rat requirements and RNPR or RPER of casein, lactalbumin, skim milk powder, SPI, heated SBM were small (about 10%) (Table 6).

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TABLE 5
Lysine, methionine / cystine, threonine and tryptophan in protein sources, expressed as a proportion of the respective amino acids in human reference amino acid pattern (FAO-WHO 1991)
Protein source Lys Met / Cys Thr Trp

% Casein / Met Casein Lactalbumin Lactalbumin, treated Skim milk Skim milk, heated SPI2 SPI, treated SBM,3 raw SBM, heated Black beans, raw Black beans, heated Mustard our Zein Zein / AA4 146 146 181 144 136 40 105 85 105 102 117 117 146 2 99 227 138 221 134 127 115 105 72 115 112 101 102 146 88 138 136 136 168 92 132 129 117 75 116 118 148 140 122 84 121 1211 121 193 193 110 109 123 115 103 103 102 101 114 15 114

1 Underlining denotes the lowest amino acid ratio (or amino acid score). 2 Soybean protein isolate. 3 Soybean meal. 4 Lysine / methionine / threonine / tryptophan.

But, the PDCAAS based on rat requirements for alkalinetreated lactalbumin, heated skim milk powder, alkaline-treated SPI, raw SBM, raw black beans, heated black beans, mustard our and supplemented zein were up to 84% higher compared to the RPER or RNPR values for these products. The PDCAAS based on human requirements for casein, untreated lactalbumin, untreated skim milk, untreated SPI and mustard our were high (Table 6). The PDCAAS for the alkaline-treated lactalbumin and treated SPI were 67 and 49% of the values for untreated lactalbumin and SPI, respectively. The PDCAAS for overheated skim milk powder was 31% of that for the untreated powder. The difference between the PDCAAS of raw and heated SBM was small (80 vs. 83%), but the PDCAAS of heated black beans was higher than that of raw black beans (84 vs. 72%). The PDCAAS of amino acidsupplemented zein was 70% higher than that of unsupplemented zein.

Reeves et al. 1993), the RNPR method includes the use of methionine-supplemented ANRC casein as the reference protein. PER and NPR are commonly determined at the 10 g protein/100 g diet level. However, several researchers have used 8 g protein/100 g diet (Henry 1965, McLaughlan et al. 1980) or 9 g protein/100 g diet (Lowry and Baker 1989, Samonds and Hegsted 1977) in the determination of PER and/ or NPR values of protein products. Diets formulated to contain 8 g protein/100 g were used in this study because high quality proteins such as egg and methionine-supplemented casein show peak PER and NPR values at about that concentration (Henry 1965, Sarwar and McLaughlan 1981). As noted earlier, the experimental protein diets tested in this study contained (on analysis) 8.78-9.05 g protein/100 g. It is highly unlikely that protein quality of these diets would be much different if they were tested at the 10 g/100 g level of protein. The RNPR values of all the test protein sources were higher than RPER values (Table 6) because unlike the PER method, the NPR method credits protein used for both growth and maintenance. The protein required to prevent weight loss of rats fed the protein-free diet is assumed to be equivalent to the protein needed for maintenance. A comparison of the PDCAAS based on rat requirements and RPER or RNPR indicated that the scoring method clearly overestimated protein quality of those protein sources which contained antinutritional factors such as mustard our (PDCAAS of 84% vs. RPER or RNPR of 0), raw black beans (PDCAAS of 45% vs. RPER or RNPR of 0), alkaline-treated lactalbumin (PDCAAS of 55% vs. RPER or RNPR of 0), alkaline-treated SPI (PDCAAS of 44% vs. RPER or RNPR of 0), and overheated skim milk powder (PDCAAS of 29% vs. RPER/RNPR of 05%) (Table 6). The overestimation of protein quality of these protein sources by the PDCAAS method was even more marked when the scores were calculated based on human requirements. Some of these protein sources contain naturally occurring growth-depressing factors such as glucosinolates and isothiocy-

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TABLE 6
Protein digestibility-corrected amino acid scores (PDCAAS), relative protein efciency ratio (RRER), and relative net protein ratio (RNPR) values for several protein products
Product PDCAAS (rat) PDCAAS (human) RPER RNPR

DISCUSSION The PDCAAS of test protein sources were compared with RNPR or RPER values, an estimate of biological utilization of proteins (Table 6). The 2-wk RNPR method is the most appropriate rat test for routine assessment of protein quality of foods for humans (Codex Alimentarius Commission 1984, Pellett and Young 1980), and signicant correlations (r 0.90, P 0.01) have been reported between 2-wk PER or NPR and 4-wk PER values (Harris and Burns 1988). The RNPR method, which is a modied net protein utilization (NPU) method (based on weight gain), gives results similar to NPU and biological value (BV) methods (Pellett and Young 1980). Henry (1965) reported excellent agreement between NPU and NPR values of 23 protein sources which were tested at the 8 g protein/100 g diet level. Since casein is known to be limiting in sulfur amino acids for rat growth (AIN 1977,

Casein / Met Casein Lactalbumin Lactalbumin, treated Skim milk Skim milk, heated SPI1 SPI, treated SBM,2 raw SBM, heated Black beans, raw Black beans, heated Mustard our Zein Zein / AA3

100 85 100 55 74 29 62 44 58 58 45 51 84 1 63

100 100 100 67 100 31 100 49 80 83 72 84 92 1 71

100 80 89 0 77 0 56 0 27 63 0 63 0 0 3

100 84 91 0 82 5 64 0 44 70 0 70 0 0 44

1 Soybean protein isolate. 2 Soybean meal. 3 Lysine / methionine / threonine / tryptophan.

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anates (breakdown products of glucosinolates) in mustard our (Fenwick et al. 1982) and trypsin inhibitors and hemagglutinins in raw black beans and raw SBM (Rackis and Gumbmann 1981). Some other protein products contained antinutritional factors formed during alkaline and heat treatments such as lysinoalanine and lanthionine (Friedman et al. 1984, Robbins et al. 1980) in the alkaline-treated lactalbumin (containing 4.42 g lysinoalanine/100 g protein; G. Sarwar, unpublished data) or SPI (containing 1.94 g lysinoalanine/100 g protein; G. Sarwar, unpublished data), and Maillard compounds in the overheated skim milk powder. Lysinoalanine is an unnatural amino acid derivative formed (during alkaline treatment of proteins) mainly by the addition of the e-amino group of lysine residue to the double bond of a dehydroalanine residue, that has been generated by the belimination reaction of cystine, phosphoserine or glycoserine residues (Friedman et al. 1984, Mega 1984). The formation of lysinoalanine adversely affects the nutritional value of proteins because bioavailable lysine, cystine and phosphoserine are lost. Moreover, sufciently large doses of lysinoalanine, especially free lysinoalanine released from the protein-bound form during the digestion process, can induce a reversible form of renal toxicity known as nephrocytomegaly in rats. It has been proposed that lysinoalanine, which chelates certain metal ions, may exert its toxic effect by metal-binding in renal tubule cells (Hayashi 1982). Since extracts from human kidney were less effective in metabolizing lysinoalanine than corresponding extracts from several animal species, human kidney cells may be more susceptible to lysinoalanine toxicity than those from rats or other animals tested (Kawamura and Hayashi 1987). The formation of lanthionine during alkaline and heat processing of foods also results in signicant loss of bioavailable cystine (Robbins et al. 1980). The Maillard reaction between proteins and reducing sugars is primarily responsible for the loss in nutritional value of food proteins during heat processing (Hurrell 1984), such as overheating of skim milk powder in this study. The overheating of skim milk and the alkaline treatment of lactalbumin and SPI may also have resulted in racemization of amino acid residues (Friedman et al. 1981). Protein-bound D-amino acids formed during processing may have adverse effects on biological value and safety of processed foods (Friedman et al. 1984). Since the routine amino acid methodology used in the determination of PDCAAS does not distinguish between D- and Lforms of amino acids, the scoring method does not take into account the presence and nutritional implications of D-amino acids in processed foods. Special analyses for D-amino acid compositions of processed foods are needed to assess the importance of this problem to human nutrition. The PDCAAS method also does not take into account bioavailability of individual amino acids, which may be up to 44% lower than the overall digestibility of protein in the same food product (Eggum et al. 1989, Sarwar and Peace 1986). Therefore, the PDCAAS method may give misleading results about the quality of proteins co-limiting in more than one essential amino acid (Sarwar and Peace 1994). In the present study, it was difcult to identify the true rst-limiting amino acid in SPI, alkaline-treated SPI, raw SBM, heated SBM, raw black beans, heated black beans and AA-supplemented zein using the PDCAAS method based on human requirements (Table 5). Depending upon the bioavailability of individual amino acids, these protein sources could be rst-limiting in methionine / cystine, lysine, threonine or tryptophan which would affect the true score. Moreover, biological experiments would be required to document benecial effects of supplementation with limiting amino acids. Emmert and Baker (1995)

illustrated the benet of using amino acid analysis and bioavailability data in correctly predicting the protein quality of several processed soybean protein products. In chick assays, SBM was found to be limiting in methionine / cystine, while soybean protein concentrate and SPI were limiting in methionine / cystine as well as threonine (Emmert and Baker 1995). Between the two samples of SPI (functional and edible), the functional SPI was superior to the edible SPI in terms of protein quality. The amino acid scoring method also overestimated the protein quality of AA-supplemented zein (PDCAAS of 71% vs. RPER and RNPR values of 344%) (Table 6). The PDCAAS assumes complete biological efciency of supplemental amino acids in improving protein quality. This was, however, not true in the case of AA supplementation of zein, a protein of low digestibility and poor quality. A marked difference between the PDCAAS and RPER or RNPR of amino acid supplemented zein would suggest incomplete biological efciency of the supplemental amino acids. The poor biological response to amino acid supplemented zein may have been due to the poor bioavailability of essential amino acid(s) other than those supplemented. Since scores 100 are considered to be 100, the PDCAAS does not credit the extra nutritional value of a protein having a score higher than that of the reference protein such as egg, sh, milk and most meat protein products. There is a need to revise the calculation of PDCAAS to permit values 100 for high quality proteins, especially if they are intended to be used as supplements to other low quality proteins. However, this revision may not be required in calculating the PDCAAS for combined proteins, where the supplemental effect of excess amino acids is included in the calculation. In the amino acid scoring method, methionine and cyst(e)ine concentrations are combined. Due to lack of knowledge of the proportion of the total sulfur amino acid requirement which can be met by cystine, expression of protein values based on the sum total of methionine and cyst(e)ine has limitations (FAO-WHO 1991). Moreover, dietary methionine may be less effective in meeting the cellular requirements for cysteine as is a preformed dietary supply of cyst(e)ine (Baker and Han 1993, Young 1995). This may be especially applicable to protein sources with low cysteine:methionine ratios (0.2) such as casein, where some methionine would be needed for cysteine synthesis which is only about 80% efcient on a weight basis (Baker and Han 1993). Therefore, there may be a need for the inclusion of a desirable cysteine:methionine ratio in the scoring pattern. There is a large variation in cysteine:methionine ratios of dietary protein sources. For example, the cysteine:methionine ratios (molar basis) in beef, egg white, soy protein isolate, rapeseed protein concentrate, wheat our and pea our have been reported to be 0.5, 0.9, 1.2, 1.5, 1.6 and 1.8, respectively (Sarwar et al. 1983). Another protein source with one of the highest cysteine:methionine ratios (1.9) is mature human milk (Ra iha 1985). The cysteine:methionine ratios (2.32 to 2.38) were especially high in preterm and term transitional human milks (Sarwar et al. 1996). Since the introduction of the PDCAAS method by FAO/ WHO (1991), several questions about the validity of the method have been raised (Food Chemical News 1991, Sarwar and Peace 1994). Some of these concerns were also documented by this investigation. Therefore, there is a need to address these issues, and to suggest proper revisions to the scoring method. Meanwhile, the PDCAAS remains the preferred method for routine prediction of protein quality of properly processed (containing minimal amounts of residual antinutritional factors) and highly digestible (where the overall

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