Protein digestibility-corrected amino acid scores for bean and bean-rice infant weaning food products

Vegetable proteins are an integral part of infant weaning diets in Latin America. Protein quality in plant-based products, however, is constrained by amino acid composition and intrinsically present antinutritional factors. The goal of this study was to improve bean protein quality by utilizing fermentation and germination processing. The objectives were to determine if protein quality, as measured by Food and Agricultural Organization (FAO) approved True Protein Digestibility (TPD) and Protein Digestibility-Corrected Amino Acid Scores (PDCAAS), of formulated bean-based weaning products could be improved upon fermentation and germination and if protein quality could be further improved when processed beans were combined with cooked rice. Results showed that the highest TPD and PDCAAS values were obtained for cooked germinated beans combined with rice. The TPD values for products ranged from 80 to 91%, and the PDCAAS values were 0.38-0.51. There was no significant increase (P < 0.05) of either TPD or PDCAAS values upon fermentation. Germination increased TPD of cooked bean products; this increase was not, however, accompanied by an increase in PDCAAS. When combined with rice, the PDCAAS values for all bean products improved significantly, thus supporting the concept of cereal-legume complementation. In conclusion, this study showed the range of PDCAAS in processed black bean and bean-rice infant weaning food products. The potential for incorporation of these products into the diets of weaning age Latin American children would, however, be confirmed only after validation with growth or metabolic balance studies in human infants.     

Evaluating the quality of protein from hemp seed (Cannabis sativa L.) products through the use of the protein digestibility-corrected amino acid score method

The macronutrient composition and the quality of protein of hemp seed and products derived from hemp seed grown in Western Canada were determined. Thirty samples of hemp products (minimum 500 g), including whole hemp seed, hemp seed meal from cold-press expelling, dehulled, or shelled, hemp seed and hemp seed hulls, were obtained from commercial sources. Proximate analysis, including crude protein (% CP), crude fat (% fat) and fiber, as well as full amino acid profiles, were determined for all samples. Protein digestibility-corrected amino acid score (PDCAAS) measurements, using a rat bioassay for protein digestibility and the FAO/WHO amino acid requirement of children (2-5 years of age) as reference, were conducted on subsets of hemp products. Mean (±SD) percentage CP and fat were 24.0(2.1) and 30.4(2.7) for whole hemp seed, 40.7(8.8) and 10.2(2.1) for hemp seed meal, and 35.9(3.6) and 46.7(5.0) for dehulled hemp seed. The percentage protein digestibility and PDCAAS values were 84.1-86.2 and 49-53% for whole hemp seed, 90.8-97.5 and 46-51% for hemp seed meal, and 83.5-92.1 and 63-66% for dehulled hemp seed. Lysine was the first limiting amino acid in all products. Removal of the hull fraction improved protein digestibility and the resultant PDCAAS value. The current results provide reference data in support of protein claims for hemp seed products and provide evidence that hemp proteins have a PDCAAS equal to or greater than certain grains, nuts, and some pulses.     

Near-infrared reflectance spectroscopy enables the fast and accurate prediction of the essential amino acid contents in soy, rapeseed meal, sunflower meal, peas, fishmeal, meat meal products, and poultry meal

Near-infrared reflectance spectroscopy (NIRS) calibrations were developed to enable the accurate and fast prediction of the total contents of methionine, cystine, lysine, threonine, tryptophan, and other essential amino acids, protein, and moisture in the most important protein-rich feed ingredients. More than 1000 samples of global origin collected over four years were analyzed on amino acids following the official methods of the United States and the European Union. Detailed data and graphics are given to characterize the obtained calibration equations. NIRS was validated with independent samples for soy and meat meal products and compared to the amino acid predictions using linear crude protein regressions. With a few exceptions, validation showed that 85-98% of the amino acid variance in the samples could be explained using NIRS. NIRS predictions compared to reference results agree excellently, with relative mean deviations below 5%. Especially for meat and poultry meals, NIRS can predict amino acids much better than crude protein regressions. By enabling the amino acid analysis of many samples to be completed in a short time, NIRS can improve the accuracy of feed formulation and thus the quality and production costs of mixed feeds.     

Nutritional responses of rats to diets based on chickpea ( Cicer arietinum L.) seed meal or its protein fractions

The aim of this study was to isolate the protein fractions from chickpea, var. IAC-Marrocos, as well as to evaluate its in vivo nutritional protein quality. Among the proteins, albumins showed better nutritional value in the in vivo assays and amino acid contents, despite their higher trypsin inhibitor contents. Trypsin inhibitors were found to be heat labile in all samples, but the digestibility results for unheated and heated flour and albumins suggest that their contents are not very decisive. The PER values for casein (not supplemented) were very similar to those of heated flour and unheated or heated albumin and total globulins. The albumin and glutelin fractions showed the best results for PDCAAS, however, lower than those of casein. Despite the high digestibility of the globulin the very low essential amino acid content lowered its PDCAAS, and it had the lowest values.     

Determination of sulfur amino acids and tryptophan in foods and food and feed ingredients: collaborative study

Samples of 4 foods, 1 animal feed, isolated soy protein, and beta-lactoglobulin were analyzed by 9 laboratories to determine concentrations of cysteine as cysteic acid, methionine as methionine sulfone, and tryptophan. Sulfur amino acids were determined by AOAC method 43.A08-43.A13 for food and feed ingredients, in which samples are oxidized with performic acid before protein hydrolysis with 6N HCl. Tryptophan was determined after protein hydrolysis with 4.2N NaOH. In both methods, free amino acids were separated by ion-exchange or reverse-phase chromatography. Each laboratory was provided with detailed methods and with sealed vials containing solutions of standards. Samples were analyzed in duplicate, and variation between laboratories was determined. Coefficients of variation between laboratories for the 6 samples ranged from 5.50 to 11.8% for methionine as methionine sulfoxide, 8.59 to 17.3% for cysteine as cysteic acid, and 3.87 to 16.1% for tryptophan. Amino acid recoveries were determined by analysis of beta-lactoglobulin and were based on expected levels of each amino acid obtained from amino acid sequence data. The mean recovery of cysteine was 97% with a range of 88-119%. For methionine, mean recovery was 98% (range 89-115%) and for tryptophan, 85% (range 59-102%). Method 43.A08-43.A13 for food and feed ingredients has been adopted official first action for determination of cysteine and methionine in processed foods. The alkaline hydrolysis method has been adopted official first action for determination of tryptophan in foods and food and feed ingredients.     

Oxidation and hydrolysis determination of sulfur amino acids in food and feed ingredients: collaborative study

Samples of 6 food and feed ingredients and a purified protein, beta-lactoglobulin, were analyzed by 7 laboratories to determine the concentrations of cysteine as cysteic acid and methionine as methionine sulfone. Samples were oxidized by reaction with performic acid before hydrolysis with 6N HCl. The free amino acids were then separated and measured by ion-exchange chromatography on dedicated amino acid analyzers. Each laboratory was provided with a detailed method as well as sealed vials containing solutions of standards. For the determination of cysteine as cysteic acid, the coefficients of variation between laboratories for duplicate samples ranged from 7.13 to 10.8% for the 6 ingredients. For the determination of methionine as methionine sulfone, the coefficients of variation between laboratories for duplicate samples ranged from 1.18 to 12.8% for the 6 ingredients. Cysteine and methionine recoveries were determined by analysis of beta-lactoglobulin and were based on expected levels of each amino acid from amino acid sequence data. The mean recovery of cysteine was 95% with a range of 91-101%. The mean recovery of methionine was 101% with a range of 98-106%. This method has been adopted official first action.     

Development of a new methanethiol quantification method using ethanethiol as an internal standard

Development of a new method to quantify methanethiol in which ethanethiol was employed as an internal standard is reported. Recovery yields for methanethiol from an aqueous model system and a soy protein concentrate (SPC) aqueous slurry determined with this method ranged from 97 to 107% and from 103 to 121%, respectively. The methanethiol content of two commercial SPCs and two commercial soy protein isolate (SPI) samples, on a dry basis, ranged from 835 to 1190 times greater than the odor threshold for methanethiol. Relative standard deviations for quantifying methanethiol with the method from these samples were <5%, indicating its good reproducibility in quantifying methanethiol from soy protein products. Also investigated in the current study was the feasibility of using 5',5-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent) to determine the concentration of methanethiol in the aqueous solutions used to prepare the standard curve for quantifying methanethiol from soy protein products. The concentrations of methanethiol obtained from Ellman's reagent method were comparable with those from a weighing method and theoretical calculation.     

Chlorogenic acid moderately decreases the quality of whey proteins in rats

During processing and storage, phenolic compounds (PCs) may react with food protein bound amino acids (AAs). Such reactions have been reported to change physicochemical and to decrease in vitro digestion properties of proteins. A rat growth and nitrogen (N) balance study was conducted to prove whether derivatization with chlorogenic acid (CA) affects the nutritional quality of beta-lactoglobulin (beta-LG). Test diets (10% protein level) contained nonderivatized beta-LG (LG, treated under omission of CA), low derivatization level beta-LG (LGL), high derivatization level beta-LG (LGH), or casein supplemented with l-methionine (0.3% of diet; C+met) as an internal standard. An additional group received untreated beta-LG supplemented with pure CA (1.03% of diet; LG+CA). The AA composition of test proteins, plasma AAs, and liver glutathione (GSH) concentrations were determined. Protein digestibility-corrected amino acid score (PDCAAS) was calculated using human or rat AA requirement patterns and rat fecal digestibility values. N excretion was significantly higher in feces and lower in urine of rats fed with LGH as compared to LG and LGL. Consequently, true N digestibility (TND) was significantly lower with LGH as compared to LG and LGL. The lower content of methionine, cysteine, lysine, and tryptophan in LGH corresponded to a reduced TND. Net protein utilization (NPU) was not different between treated beta-LG fed diet groups but was lower than in LG+CA and C+met fed groups. Only at a relatively high level of derivatization with CA, the otherwise good nutritional quality of beta-LG is affected so that TND is reduced, while NPU still remains unaffected. Derivatization of beta-LG with CA does not seem to lead to an additional deficiency in a specific indispensable AA in growing rats fed with 10% protein.     

Protein Quality and Physical Characteristics of Kisra (Fermented Sorghum Pancake‐like Flatbread) Made from Tannin and Non‐Tannin Sorghum Cultivars

Kisra is a naturally lactic acid bacteria‐ and yeast‐fermented sorghum thin pancake‐like flatbread produced in Sudan. Kisra has considerable potential as the basis for development of a gluten‐free sandwich wrap. To help direct cultivar selection for commercial production of these products, two white, tan plant non‐tannin Type I, one white Type II tannin, and one red Type III tannin sorghum cultivars were evaluated with respect to kisra protein quality and physical characteristics. Kisra from the non‐tannin sorghums were flexible and had an open‐textured structure with many regular gas cells, whereas those from the tannin sorghums were more brittle, denser in structure, and contained far fewer and smaller gas cells. Kisra from the tannin sorghums had the lowest reactive lysine content, in vitro protein digestibility, and Protein Digestibility Corrected Amino Score (PDCAAS), with values being lowest for the Type III sorghum. PDCAAS of kisra from the Type III sorghum was only 0.12, less than half of that from the Type I sorghums. As the tannins in tannin sorghums adversely affect kisra protein quality and physical characteristics, white tan plant, non‐tannin sorghum cultivars are most suitable for kisra production and for development of wrap‐type sorghum‐based baked goods.     

Comprehensive analytical method for the determination of hydrophilic metabolites by high-performance liquid chromatography and mass spectrometry

A method for the comprehensive analysis of hydrophilic metabolites, based on a combination of high-performance liquid chromatography and mass spectrometry, is described. We evaluated three types of stationary phases to achieve the separation of highly hydrophilic metabolites. Good chromatographic retention and separation of these metabolites were achieved on a pentafluorophenylpropyl-bonded silica column with gradient elution, using 0.1% aqueous formic acid and acetonitrile as the mobile phase. The optimized conditions allowed the comprehensive determination of the standard 49 kinds of amino acids, 6 kinds of amines, 45 kinds of organic acids, 18 kinds of nucleic bases, 5 kinds of nucleosides, and 14 kinds of nucleotides, and then the linearity, dynamic range, detection limit, and precision of the retention time and the peak area were validated. We applied this method for the targeted analysis of the components in soy sauce. The results from the quantitative determination of amino acids were compared to those obtained with an amino acid analyzer, and the accuracy was in the range between 85 and 119%. The accuracy of other detected components was confirmed to be 105-133% by the recovery rate after the addition of standard compounds. We also applied the method for the nontargeted metabolic profiling of the components in several kinds of soy sauces with the principal component analysis. They were classified by the manufacturing methods, and the components that corresponded to the differences were identified. This method could be useful for the targeted analysis of hydrophilic metabolites as well as their nontargeted metabolic profiling.     

Evaluation of detoxification methods on toxic and antinutritional composition and nutritional quality of proteins in Jatropha curcas meal

The Jatropha curcas meal was detoxified by different methods, and the effect of detoxification was evaluated in this study. The method that hydrolysis of enzymes (cellulase plus pectinase) followed by washing with ethanol (65%) had a significant (p < 0.05) effect on the toxin, antinutritional components, and nutritional quality of proteins. After this treatment, the phorbolesters (PEs) were decreased by 100%. The antinutritional components (phytates, tannins, saponins, protease inhibitor, and lectin activities) were decreased to tolerable levels, which were lower than those in soybean meal. The crude protein in detoxified meal was 74.68%, and the total content of amino acids was 66.87 g/100 g of dry matter. The in vitro protein digestibility (IVPD) increased from 82.14 to 92.37%. The pepsin-insoluble nitrogen was only 4.57% of the total nitrogen, and about 90% of the protein was true protein. The protein-digestibility-corrected amino acid score (PDCAAS) of the meal was 0.75. The results showed that this treatment was a promising way to detoxify J. curcas meal, and the nutritional quality of detoxified meal can be simultaneously enriched and improved.     

Near-infrared reflectance spectroscopy for predicting amino acids content in intact processed animal proteins

Near-infrared calibrations were developed for the instantaneous prediction of amino acids composition of processed animal proteins (PAPs). Two sample presentation modes were compared (ground vs intact) for demonstrating the viability of the analysis in the intact form, avoiding the need for milling. Modified partial least-squares (MPLS) equations for the prediction of amino acids in PAPs were developed using the same set of samples (N = 92 PAPs) analyzed in ground and intact form and in three cups differing in the optical window size. The standard error for cross validation (SECV) and the coefficient of determination (1-VR) values yielded with the calibrations developed using the samples analyzed in the intact form showed similar or even better accuracy than those obtained with finely ground samples. The excellent predictive ability (1-VR > 0.90; CV < 3.0%) obtained for the prediction of amino acids in intact processed animal proteins opens an enormous expectative for the on-line implementation of NIRS technology in the processing and marketing of these important protein feed ingredients, alleviating the costs and time associated with the routine quality controls.     

Comparison of interlaboratory variation in amino acid analysis and rat growth assays for evaluating protein quality

Estimates of inter- and intralaboratory variation of protein efficiency ratio (PER), relative PER (RPER), net protein ratio (NPR), relative NPR (RNPR), and nitrogen utilization (NU) were compared with those of amino acid analysis in the same batches of 7 protein sources (ANRC casein, egg white solids, minced beef, soy assay protein, rapeseed protein concentrate, pea flour, and whole wheat flour). Interlaboratory variation (estimated as between-laboratories coefficients of variation, CV) of NPR and RNPR (up to 6.0%) was lower than that of PER (up to 20.2%) and RPER (up to 18.5%). The interlaboratory determination of NPR and RNPR was also more reproducible than that of most essential amino acids (CV up to 10.0%), especially tryptophan (CV up to 23.7%), cystine (CV up to 17.6%), and methionine (CV up to 16.1%). Intralaboratory variation (estimated as within-laboratories CV) of amino acid analysis (up to 4.7%), however, was comparable to that of protein quality indices in most protein sources (up to 6.0%). The significant (P less than 0.01) positive correlations (r = 0.68-0.74) between amino acid scores and protein quality indices based on rat growth were further improved when amino acid scores were corrected for digestibility of protein (r = 0.73-0.78) or individual amino acids (r = 0.79-0.82).     

Characterization and quantification of proteins in lecithins

Several methods for extraction and quantification of proteins from lecithins were compared. Extraction with hexane-2-propanol-water followed by amino acid analysis is the most suitable method for isolation and quantification of proteins from lecithins. The detection limit of the method is 15 mg protein/kg lecithin, and the quantification limit is 50 mg protein/kg. The relative repeatability limits for samples containing 0-500 and 500-5000 mg protein/kg sample were 12.6 and 7.5%, respectively. The protein recovery ranged between 101 and 123%. The protein content has been determined in different kinds of lecithins. The results were as follows: standard soy lecithins (between 232 and 1338 mg/kg), deoiled soy lecithin (342 mg/kg), phosphatydylcholine-enriched soy lecithins (not detectable and 163 mg/kg), sunflower lecithins (892 and 414 mg/kg), and egg lecithin (50 mg/kg). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein patterns of the standard soy and sunflower lecithins are very similar to those of soy flour. The protein profile of the egg lecithin shows several bands with a broad range of molecular masses. The molecular masses of the main proteins of soy lecithins and soy flour have been determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and ranged from 10.5 to 52.2 kDa. Most of the major proteins from soy and sunflower lecithins identified by MALDI-MS and electrospray tandem MS belong to the 11S globulin fraction, which is one of the main fractions of soy and sunflower seeds. In addition, the seed maturation protein P34 from the 7S globulin fraction of soy proteins has also been identified in soy lecithins. This protein has been reported as the most allergenic protein in soybean.     

Low molecular weight compounds responsible for savory taste of Indonesian soy sauce

Indonesian soy sauce is made using only soybeans as the nitrogenous source. Moromi obtained from fermentation of yellow soybeans using Aspergillus sojae as the starter was investigated. The fraction with molecular weights of less than 500 Da obtained by stepwise ultrafiltration was then fractionated by several chromatographic procedures, including gel filtration chromatography and RP-HPLC. Several chemical analyses, CE profiles, and taste profiles were performed to obtain the most intense umami fraction. The main components eliciting or enhancing the umami taste present in the fraction were purified and identified by protein sequencing, ESI-MS, and (1)H NMR at 400 MHz. Besides free l-glutamic acid and aspartic acid, free aromatic amino acids such as l-phenylalanine and l-tyrosine may also play an important role in impressing savory or umami taste of Indonesian soy sauce at their subthreshold concentrations and in the presence of salt and free acidic amino acids. This is reported as a new phenomenon of the so-called bitter amino acids.     

Evaluation of protein digestibility-corrected amino acid score method for assessing protein quality of foods

The current concepts of protein quality evaluation were reviewed. A detailed examination of existing animal assays and more promising amino acid scoring methods has been carried out by an Ad Hoc Working Group on Protein Quality Measurement for the Codex Committee on Vegetable Proteins during the last 5 years. Several factors such as inadequacies of protein efficiency ratio (PER, the poorest test) and other animal assays, advancements made in standardizing methods for amino acid analysis and protein digestibility, availability of data on digestibility of protein and individual amino acids in a variety of foods, and reliability of human amino acid requirements and scoring patterns were evaluated. On the basis of this evaluation, amino acid score, corrected for true digestibility of protein, was recommended to be the most suitable routine method for predicting protein quality of foods for humans. Amino acid scores corrected for true digestibility of protein (as determined by rat balance method) were termed "protein digestibility-corrected amino acid scores." A detailed method for the determination of the protein digestibility-corrected amino acid score was proposed, and information about the range of scores to be expected in foods or food products was provided in the present investigation. The protein digestibility-corrected amino acid score method is a simple and scientifically sound approach for routine evaluation of protein quality of foods. Accuracy of the method would, however, be confirmed after validation with growth or metabolic balance studies in humans.     

Generic combustion method for determination of crude protein in feeds: collaborative study

Nine laboratories participated in a collaborative study on determination of crude protein in animal feeds to compare a generically described combustion method with the AOAC mercury catalyst Kjeldahl method (7.015). The combustion method was written in general terms of method principle, apparatus specifications, and performance requirements. The sample set comprised closely matched pairs of feed ingredients and mixed products ranging from 10 to 90% protein. Ten pairs ground to 0.5 mm were the focus of the study; 4 pairs were ground to 1.0 mm for comparison. Nicotinic acid and lysine monohydrochloride were included as standards. Collaborators were instructed to report their results for performance checks using materials supplied. Only one laboratory failed to meet the proposed limits. Seven laboratories used the LECO Model FP-228 analyzer and 2 used the LECO CHN 600 analyzer. For the 0.5 mm pairs, repeatability standard deviations (Sr) ranged from 0.09 to 0.58 for the Kjeldahl method and from 0.14 to 0.33 for the combustion method, with a pooled Sr value of 0.28 and relative standard deviation (RSDr) of 0.59%. Reproducibility standard deviations (Sg) ranged from 0.23 to 0.86 (Kjeldahl) and from 0.30 to 0.61 (combustion), with a pooled Sg value of 0.52 and RSDg of 1.10%. Grand means for the samples ground to 0.5 mm were 47.65% protein by the combustion method and 47.41% protein by the Kjeldahl method. For samples ground to 1.0 mm, corresponding values were 31.82 and 31.50% protein. The generic combustion method has been approved interim official first action.     

大豆蛋白粉的辐照灭菌研究

研究了60Coγ射线辐照大豆蛋白粉的杀菌效果与对大豆蛋白粉的主要营养成分、尿素酶(脲酶)活性以及感官品质的影响。结果表明,2.0kGy辐照对大豆蛋白粉中菌落总数、大肠菌群的杀菌率达70%,对霉菌的杀菌率达53%;4.0kGy辐照杀菌率达95%;经6.0kGy辐照处理后,杀菌率达到97%以上,辐照的大豆蛋白粉样品中微生物指标均达到食品卫生国家标准;8.0kGy辐照杀菌率达到100%。与对照相比,剂量为2.0~8.0kGy的辐照对大豆蛋白粉中蛋白质、粗纤维、总糖、氨基酸(除Leu)含量的影响不明显;而粗脂肪及卵磷脂的含量有极显著变化(P<0.01),尤其是卵磷脂的含量显著增加,异黄酮的含量则显著降低(P<0.05);辐照对大豆蛋白粉中尿素酶活性没有显著影响;6.0kGy以下剂量辐照对大豆蛋白粉色泽、气味、口感无明显改变。综合试验结果,确定了大豆蛋白粉辐照杀菌的适宜工艺剂量范围为3.0~5.0kGy。     

Technological and Nutritional Properties of Flours and Tortillas from Nixtamalized and Extruded Quality Protein Maize (Zea mays L.)

Nixtamalized and extruded flours from quality protein maize (QPM, V‐537C) and tortillas made from them were evaluated for some technological and nutritional properties and compared with the commercial brand MASECA. Both QPM flours showed higher (P < 0.05) protein content, total color difference, pH, available lysine, and lower (P < 0.05) total starch content, Hunter L value, water absorption index, gelatinization enthalpy, resistant starch, and retrograded resistant starch than nixtamalized MASECA flour. Tortillas from nixtamalized and extruded QPM flours had higher contents of essential amino acids than tortillas from MASECA flour, except for leucine. Tortillas from processed QPM flours also showed higher (P < 0.05) values of the nutritional indicators calculated protein efficiency ratio (C‐PER 1.80–1.85 vs. 1.04), apparent and true in vivo protein digestibility (78.4‐79.1 vs. 75.6% and 76.4–77.4 vs. 74.2%, respectively), PER (2.30–2.43 vs. 1.31), net protein retention (NPR; 2.88–2.89 vs. 2.11), and protein digestibility corrected amino acid score (PDCAAS; 54–55 vs. 29% based on preschool children and 100 vs. 85% based on adults) than MASECA flour. The use of QPM for flour and tortilla preparation may have a positive effect on the nutritional status of people from countries where these products are widely consumed.     

Effect of hydrolysis time on the determination of amino acids in samples of soybean products with ion-exchange chromatography or precolumn derivatization with phenyl isothiocyanate

Accurate determination of amino acid levels in soy products facilitates optimum diet formulation and amino acid supplementation. A study was carried out to investigate the effect of hydrolysis time and method of amino acid measurement on amino acid levels. Correction factors to standardize amino acid levels to 24 h of hydrolysis were also determined. Six different soybean products were evaluated. Hydrolysis was carried out for 10 different periods of time. Amino acids were analyzed by both ion-exchange chromatography and precolumn derivatization with phenyl isothiocyanate. Both hydrolysis time and measurement method affected (P < 0.05) amino acid levels. Standard hydrolysis conditions (hydrolysis in 6 M HCl at 110 degrees C for 24 h) rarely provide the maximal amino acid values. Therefore, sequential hydrolyses curves were very useful and should be used.