Protein Digestibility-corrected Amino Acid Score Research Articles

165 Characterizing protein quality with in vitro methodologies

Abstract Nutritional assessment of the quality of a protein source is a complex process with different requirements depending on the jurisdiction being discussed. The Protein Efficiency Ratio (PER) is a growth measurement required in Canada, whereas the United States uses the Protein Digestibility Corrected Amino Acid Score (PDCAAS), which requires the determination of the amino acid profile and digestibility of the protein source. Notably both PER and PDCAAS are in vivo methods, necessitating the use of animals for measurement of growth and protein digestibility. Understandably there is significant interest from both industry and consumers to reduce reliance on animal experimentation for content claims. It is notable that outside of North America there are multiple examples of in vitro protein quality assessment. In Europe protein content claims are based on whether the protein content contributes >12% of the total energy, and in Australia content claims begin at 5 g of protein per serving. As the use of in vitro techniques for protein quality assessment has not led to indices of protein malnutrition in Europe and Australia, should be possible to use in vitro techniques in methods such as PER and PDCAAS thereby removing the reliance on animal use for quality assessment in North America. This can be accomplished by using in vitro methods in the assessment of protein digestibility and relying on recent changes by Health Canada to allow for calculation of PER from PDCAAS values. This presentation will introduce how protein quality is calculated via PER/PDCAAS, and subsequently discuss modifications of these methods to incorporate in vitro techniques to generate values indicating their nutritional quality with positive correlations to in vivo data.

Evaluation of the nutritional quality of yeast protein in comparison to animal and plant proteins using growing rats and INFOGEST model

Yeast, identified as a microorganism, boasts a considerable protein content, positioning yeast protein as a highly promising alternative in the quest for sustainable protein sources. The primary aim of this study is to evaluate the protein quality of yeast protein and compare it with animal proteins (whey concentrate/isolate proteins) and plant proteins (soy, wheat, pea proteins). Notably, yeast protein exhibits the highest ratio of indispensable/dispensable amino acids (IAAs/DAAs, 0.91). However, in both in vivo and in vitro digestion experiments, yeast protein demonstrated lower true protein digestibility (TPD) and true ileal digestibility (TID) compared to other proteins. Despite this, the yeast protein's amino acid score (AAS, 1.37 for >3 years), protein digestibility-corrected amino acid score (PDCAAS, 100 % for >3 years), and digestibility-corrected amino acid score (DIAAS, 82.42 % for >3 years) of yeast protein surpassed those of plant proteins, yet remained lower than animal proteins primarily due to its lower digestibility.

Canavanine Content Quantification in Processed Bitter Vetch (Vicia ervilia) and Its Application as Flour in Breads: An Analysis of Nutritional and Sensory Attributes.

Bitter vetch (Vicia ervilia Willd.) is a traditional Mediterranean-West Asian legume, mainly used as livestock feed because of its toxic non-proteinogenic amino acid, canavanine. However, historical sources suggest its past human consumption. Currently, bitter vetch is a minor crop confined to marginal soils in semi-arid regions, presenting a potential alternative protein source amid projected climate changes. This study evaluated the nutritional and sensory attributes of bitter vetch seeds processed through various household methods. Germination and cooking significantly reduced the canavanine content by 28% and 60%, respectively. Incorporating bitter vetch flour (BVF) into wheat bread enhanced protein and fiber contents without substantially altering carbohydrate and lipid levels, and the baking process reduced the canavanine content by 40%. Bitter vetch flour enriched the bread with iron and calcium, contributing significantly to their daily nutritional intakes. Sensory evaluations indicated positive reception for bread with 12% BVF, achieving a balance between nutritional enhancement and consumer acceptance. This study identifies bitter vetch seeds as a valuable resource for improving bread formulations with corrected gluten contents and enhanced protein quality, as measured using protein-digestibility-corrected amino acid score (PDCAAS) values. With strategic processing and formulation adjustments, bitter vetch has the potential to re-emerge as a feasible high-protein grain crop, promoting sustainable farming.

Fourier‐transform mid‐infrared spectroscopy for in vitro protein digestibility measurement of pulse crops

AbstractThe standard method of estimating in vitro protein digestibility, the protein digestibility corrected amino acid score (PDCAAS) assay, does not support the expected workflow of a pulse breeding program. This is mainly due to its low‐throughput design and long processing time (∼16–24 h) per sample. Fourier‐transform mid‐infrared (FT‐MIR) spectroscopy has been developed as a high‐throughput phenotyping tool to estimate protein digestibility in pulses. The mid‐infrared region representing the amide I band (1756.81–1586.27 cm−1) was utilized to perform chemometric modeling with partial least squares regression (PLSR) to estimate in vitro protein digestibility in dry pea (Pisum sativum L.), lentil (Lens culinaris Medik.), and chickpea (Cicer arietinum L.) flours. The root mean square error of predictions of the developed PLSR models for dry pea, lentil, and chickpea were 0.00039, 0.00024, and 0.00017, respectively. Phenotyping with the FT‐MIR approach is more rapid than with the PDCAAS assay and estimates protein digestibility from the flour of a single seed in a shorter time (∼1–2 min). The FT‐MIR approach is robust as the spectroscopic data are consistent and chemically fingerprint this nutritional trait. Accordingly, FT‐MIR can resolve the phenotypic bottleneck of pulse breeding related to in vitro protein digestibility measurements by enabling a high‐throughput phenotyping workflow.

Use of Directed Lactic Fermentation to Obtain Plant-Based, Upcycled Beverage from Milk Thistle Endosperm

This study aimed to evaluate the possibility of using milk thistle endosperm (MTE) extract with a protein content of >2 g/100 mL to obtain a fermented product, an alternative to milk beverages. Directed lactic fermentation with Lacticaseibacillus rhamnosus was carried out. The course of the process was monitored. The changes in pH, the saturation of the medium with dissolved oxygen, and temperature were measured. The values of the main nutritional parameters, silymarin content, amino acid profile/content, and the PDCAAS value (Protein Digestibility-Corrected Amino Acid Score) were determined in the extract before and after fermentation. The lactic acid fermentation of the MTE extract took place in four phases, with the activity of L. rhamnosus being the most intense between 4 and 8 h into the process. As a result of fermentation, there were changes in the total amino acid content in the tested medium, suggesting the catabolism of aspartic acid, arginine, proline, and isoleucine via LAB took place. Particularly significant was the increase in the content of lysine and valine (4.95 and 4.68 g/100 g of total AA, respectively), which are the limiting amino acids in MTE. Although MTE contained approximately 1% silymarin, no presence of this flavonolignan complex was found in the extracts.

Low protein digestibility-corrected amino acid score and net nitrogen-to-protein conversion factor value of edible insects

The amount of chitin, a nitrogen-containing dietary fiber, in edible insects can mislead the exact nitrogen-to-protein conversion factor (NPF) and true protein content. We determined the amino acid score (AAS), protein digestibility-corrected AAS (PDCAAS), chitin content, and net NPF of five edible insects. Additionally, the effect of the amino acid composition of migratory locust on rat growth were investigated. The AAS of the insects were ranged from 63 to 94. The chitin contents were ranged from 1.6 g/100 g to 10.7 g/100 g. The PDCAAS, calculated by AAS and gut-intestinal digestibility, ranged from 44 to 81, which was lower than casein (97). The net NPF ranged from 4.93 to 5.76, which were lower than the conventional value. Dietary migratory locust, whose PDCAAS was the lowest, decreased growth and altered lipid metabolism. Therefore, a lower PDCAAS and overestimation of net NPF of insects can affect the true protein calculations and growth.

In vivo protein quality of pistachios (Pistacia vera L.)

Pistachios (Pistacia vera L.), similar to other nuts, fall under the protein foods category, according to the current U.S. Dietary Guidelines and Canada’s Food Guide. When substantiating claims for protein content of pistachios, protein quality data must be available to support these claims. In North America, the Protein Digestibility-Corrected Amino Acid Score (PDCAAS) is used for this purpose, however, Canada requires that this value be used to calculate a Protein Efficiency Ratio (PER). PER can be used for estimating protein quality. The current investigation aimed to offer current assessments of PER and PDCAAS, through the utilization of established in vivo rodent bioassays, for pistachio samples (raw, dry-roasted, and oil-roasted). Amino acid scores established lysine as the most limiting amino acid, with values of 0.933, 1.073, and 1.058 for raw, dry-roasted and oil-roasted pistachios, respectively. PDCAAS values ranged from 83 to 94%, being highest for dry-roasted pistachios. The measured PER values (corrected to casein = 2.50) were 2.53, 2.48, and 2.58 for the raw, dry-roasted, and oil-roasted pistachios, respectively, and were not significantly different from casein. These data support the position that pistachios can be labeled as “good sources of protein” in the U. S. and Canada.

Simultaneous biogas upgrading and single cell protein production using hydrogen oxidizing bacteria

CO2, a primary byproduct of anaerobic digestion, significantly reduces the calorific value of biogas, necessitating its enhancement through biogas upgrading to increase the CH4 content. The hydrogen oxidizing bacteria (HOB) Cupriavidus necator H16 demonstrates potential as a candidate for biological biogas upgrading due to its efficient CO2 capture and biosynthesis capabilities. Results indicated that in batch experiments, the concentration of CH4 could be elevated from 57.09 % to 98.46 % within 72 h by HOB, with a maximum CO2 assimilation efficiency of 27 mL/(L·h), meeting the requirements for biomethane. In bioreactor scale-up experiments, the CH4 concentration was increased to 94.22 % within 96 h. Beyond biogas upgrading, HOB also produces biomass usable as single cell protein (SCP), with its protein content varying between 43.75–70.83 % depending on the gas supply ratio. A total of 17 amino acids were identified, including eight essential amino acids. The Protein Digestibility Corrected Amino Acid Score (PDCAAS) indicated that the essential amino acid content in the HOB-based protein was well-balanced, closely approximating the quality of fishmeal and pork. A techno-economic analysis revealed that the net revenue from the anaerobic digestion process could be enhanced by 48.49 % using HOB-based biogas upgrading. In contrast, biogas upgrading processes based on hydrogenotrophic methanogens (HM) resulted in a 57 % reduction in net revenue. This study establishes a carbon flow pathway from organic solid waste to biomethane and utilizable protein sources, facilitating sustainable nutrient recovery. This approach not only enhances economic benefits but also reduces carbon emissions associated with the anaerobic digestion process.

Effect of fortifying sorghum and wheat with Longhorn grasshopper (Ruspolia differens) powder on nutritional composition and consumer acceptability of biscuits.

This study aimed at improving the nutrient composition and protein quality of biscuits made from sorghum and wheat through fortification with Longhorn Ruspolia differens powder (RDP) for use as a supplementary food targeting children vulnerable to protein-energy malnutrition (PEM). Ten biscuit formulations were prepared by replacing a part of wheat and sorghum-wheat flours with 5, 15, 20, and 40% RDP. To establish the nutrient content of biscuits, proximate and mineral compositions were determined. The amino acid composition, reactive lysine and invitro protein digestibility were determined for protein quality. Compositing wheat or wheat-sorghum biscuits with RDP increased the protein, fat, ash, and crude fiber by percentages as high as 118, 37, 133, and 573%, respectively. Mineral content increased with, iron, zinc, and potassium as high as 161, 219, and 169%, respectively. The lysine, reactive lysine and invitro protein digestibility of the fortified biscuits increased significantly, relative to the 100% cereal biscuits. Fortification with RDP significantly improved the amino acid content of the biscuits but had a marginal effect on improvement of the lysine score and did not meet the reference pattern for children aged 3-10 years. The Protein Digestibility Corrected Amino Acid Score (PDCAAS) of wheat-sorghum and wheat biscuits improved by 6% to 47% and 2% to 33%, respectively, compared to the control biscuits. The fortified biscuits were liked by the consumers. The RDP-fortified biscuits have the potential to alleviate PME in developing countries.

A promising Artemisia capillaris Thunb. Leaf proteins with high nutrition, applicable function and excellent antioxidant activity

The nutritional and functional properties of leaf proteins is a decisive factor for their use in food. This work was aimed to extract defatted Artemisia capillaris Thunb. (ACD) leaf proteins (ACLP), and assess ACLP nutritional quality, functional properties and in vitro antioxidant activity, as well characterize the structure. ACLP had a balanced amino acid profile and high bioavailability (protein digestibility corrected amino acid score (PDCAAS) 99.29 %). Solubility, foaming capacity and emulsifying ability of ACLP correlated positively with pH. Water and oil holding capacity were increased with temperature. Gel electrophoresis shown the protein molecular size was mainly ∼25 kDa, and random coil was the mainly secondary structure while β-sheet was dominant regular conformation as indicated by circular dichroism (CD). ACLP performed in vitro antioxidant activity which was better after digestion. All data implied ACLP met the WHO/FAO protein quality expectations and had application potential in food.

Estimating In Vitro Protein Digestion and Protein Digestibility Corrected Amino Acid Score of Chicken Breasts Affected by White Striping and Wooden Breast Abnormalities.

An understanding regarding impacts of growth-related myopathies, i.e., white striping (WS) and wooden breast (WB), on the quality of dietary protein from cooked chicken breast is still limited. This study aimed at comparing protein content and in vitro protein digestion and estimating the in vitro protein digestibility corrected amino acid score (PDCAAS) of cooked chicken meat exhibiting different abnormality levels (i.e., normal, WS, and WS + WB). The results show that the WS + WB samples exhibited lower protein content, greater cooking loss, and greater lipid oxidation than those of normal samples (p < 0.05). No differences in protein carbonyls or the myofibril fragmentation index were found (p ≥ 0.05). Cooked samples were hydrolyzed in vitro using digestive enzyme mixtures that subsequently mimicked the enzymatic reactions in oral, gastric, and intestinal routes. The WS + WB samples exhibited greater values of free NH2 and degree of hydrolysis than the others at all digestion phases (p < 0.05), suggesting a greater proteolytic susceptibility. The in vitro PDCAAS of the WS + WB samples was greater than that of the other samples for pre-school children, school children, and adults (p < 0.05). Overall, the findings suggest that the cooked chicken breast with the WS + WB condition might provide greater protein digestibility and availability than WS and normal chicken breasts.

Protein Quality of African Locust Bean-A High-Value Gathered Tree Food Contributing Protein and Palatability to Plant-Based Diets.

The African locust bean tree Parkia biglobosa (Jacq.) R.Br. ex G. Don is a leguminous species native to the Sudanian parkland of western Africa. The seed obtained from pods collected from trees by rural women is fermented into a dense and aromatic paste known as soumbala, dawadawa, or iru-a protein-rich condiment underlying much of West African cuisine, its rich umami base lending a depth of flavor as a "meat substitute" in plant-based diets for which animal protein is a limiting component. Here, we assess the protein quality of P. biglobosa seed and its fermented product (soumbala) from three locations of southern Mali comprising three different eco-climatic zones, to determine whether variation in nutritional composition and protein quality could be correlated with the geographic variables of latitude and elevation. Proximate composition was determined, and amino acid profiles were compiled for 19 amino acids, with results compared by location and eco-climatic zone. A protein quality test was conducted in the aggregate and for each zone using the WHO/FAO Protein Digestibility-Corrected Amino Acid Score (PDCAAS) method. Principal component analysis (PCA) was used to assess patterns of amino acid compositional variation between the three origins. The results underline the nutritional significance of African locust bean as a source of dietary protein and of a depth of flavor providing enhanced palatability to plant-based diets. Although the PCA biplot for the amino acid profiles does indicate geographic clustering, the variation in nutritional composition and protein quality is insignificant for the raw seed, but highly significant for the fermented product (soumbala). The results indicate no correlation between phytochemical parameters and geographic variables of latitude and elevation, suggesting that management and processing may contribute more to nutritional quality than product provenance. Further studies should assess specific processing methods and the ambient microbiome as factors contributing to protein quality.

Options for substantiating protein content claims for conventional foods.

In Canada and the United States, front-of-package protein content claims require data to support the quality of the protein. In general, protein quality reflects the product of the amino acid composition of the food protein relative to human amino acid requirements and a measure of digestibility. The currently accepted method in both jurisdictions is the protein digestibility-corrected amino acid score (PDCAAS) that requires the measurement of true fecal protein (nitrogen) digestibility. The latter must be measured in vivo using a rat model. This requirement for animal testing is inconsistent with international efforts to reduce the usage of animals in testing for regulatory purposes. The current commentary positions four options to remove the need to use animal testing for determining protein quality, when considering protein content claim substantiation. These options include (i) a focus on protein quantity alone; (ii) the use of the amino acid score alone, with no correction for digestibility; (iii) the use of a fixed digestibility coefficient to estimate protein quality; and (iv) the use of in vitro methods to measure protein and/or amino acid digestibility. The relative merits and deficiencies of the options are positioned with the goal of encouraging dialogue within the regulatory agencies to move towards alternative approaches for substantiating protein content claims on foods, including those derived from plant-based sources.

Effect of full fat and defatted insect meals in breadmaking quality

Fat content and type of lipids in edible insect meals could affect bread quality when used to partially substitute wheat in bread formulations. This study evaluated the effect of Acheta domesticus (AD) and Tenebrio molitor (TM) meals in breadmaking. Full fat (FF), partially-(PD), and complete-defatted (DF) TM and AD meals were used to replace 10% (w/w) of wheat flour in bread formulations, resulting in 6 composite flours (FFAD/PDAD/DFAD/FFTM/PDTM/DFTM) and a 100% wheat control. Dough thermomechanical properties, breadmaking parameters, proximal composition, texture profile analysis, and Protein Digestibility-Corrected Amino Acid scores (PDCAAS) were assayed. Defatting insect meals increased water absorption of composite flours by 7 (TM) and 3% (AD) and TM fat increased the mixolab dough stability by 2.4 min. FFTM showed the highest bread volume (843 cm3) and lowest density (0.19 g/cm3) among composite loaves. The protein content of insect-supplemented bread increased up to 34% compared to the control. FFTM bread had the lowest hardness values through storage at room temperature. Defatted AD and TM had higher PDCAAS compared to the control. These findings indicated that TM fat contributes to breadmaking quality and DFAD increased the PDCAAS value, making it a nutritious alternative to partially substitute wheat in bread formulations.

The animal protein hydrolysate attenuates sarcopenia via the muscle-gut axis in aged mice

Age-related muscle loss and dysfunction, sarcopenia, is a common condition that results in poor quality of life in the elderly. Protein supplementation is a potential strategy for preventing sarcopenia and increasing muscle synthesis, but the effectiveness of protein type and level in improving sarcopenia is not well understood. In this study, we compared animal protein hydrolysate (APH), which has a high protein digestibility-corrected amino acid score (PDCAAS) and low molecular weight, with casein as a control group to investigate the effects and mechanisms of sarcopenia improvement, with a particular focus on the gut-muscle axis. APH supplementation improved age-related declines in muscle mass, grip strength, hind leg thickness, muscle protein level, muscle fiber size, and myokine levels, compared to the control group. In particular, levels of plasma cortisol, muscle lipids, and muscle collagen were markedly reduced by APH supplements in the aged mice. Furthermore, APH efficiently recovered the concentration of total SCFAs including acetic, propionic, and isovaleric acids decreased in aged mice. Finally, APH induced changes in gut microbiota and increased production of SCFAs, which were positively correlated with muscle protein level and negatively correlated with pro-inflammatory cytokines. In conclusion, APH can help to inhibit age-related sarcopenia by increasing muscle synthesis, inhibiting muscle breakdown, and potentially modulating the gut-muscle axis.

Nutritional characterization of quality protein maize (QPM) (Zea mays L.) protein concentrates

Maize is considered a staple food for the population; however, its protein contribution to the development of individuals is limited by its low quality. Quality protein maize varieties (QPM) have been developed to remedy this. The protein quality presents in protein concentrates obtained from two QPM varieties was evaluated: Sac Beh (SB) and Chichen Itza (ChI), which were compared with a traditional variety (Blanco Uxmal, BU) as a control. All were evaluated with non-nixtamalization (NN) and nixtamalization (N). Amino acid profile, in vitro digestibility, calculated protein digestibility-corrected amino acid score (PDCAAS), calculated protein efficiency ratio (c-PER), and biological value (BV) were determined. The extraction process of the QPM concentrates and nixtamalization had important effects on the essential amino acids Lys and Trp. In vitro digestibility showed significant differences (p < 0.05). The predominant limiting amino acid was tryptophan. The QPMs were closer to the requirements of the FAO/WHO standard, since they obtained chemical score values higher than 100% for His and Leu. PDCAAS values decreased in the amino acid tryptophan of SB and BU nixtamalized maize compared with the NN. PER7 and PER10 values were statistically higher (p < 0.05) in the QPMs compared to the BU control. The estimated biological value showed an imbalance in the concentration of essential amino acids. Lys and Trp in the QPM varieties were not sufficient evidence of increased biological value the proteins, due to the effect of the extraction processes and the alkaline heat treatment to which the maize grains were subjected.

The in vivo and in vitro protein quality of three hemp protein sources.

In this work, the protein quality of defatted hemp hearts and protein-enriched hemp fractions was determined. Protein quality was assessed using a rodent bioassay to evaluate growth and protein digestibility, while amino acid composition was determined via HPLC. A method for determining in vitro protein digestibility was compared to in vivo methodology and used to generate an in vitro protein quality score. The true protein digestibility of hemp protein 2, a hemp protein concentrate, was significantly lower than that of either defatted hemp hearts or hemp protein 1, a hemp protein concentrate (p < .05). While there was no relationship between the in vivo and in vitro measurements of protein digestibility (R 2 = .293, p = .459), there was a significant correlation between the protein digestibility-corrected amino acid score (PDCAAS) determined in vivo and in vitro PDCAAS (R 2 = .989, p = .005). The protein efficiency ratio of hemp protein 1 was significantly lower than that of either defatted hemp hearts or hemp protein 2 (p < .05). These data highlight the nutritional capacity of hemp protein sources while also demonstrating the relationship between in vivo and in vitro methods for determining protein quality.

Impact of extraction conditions and seed variety on the characteristics of pennycress (Thlaspi arvense) protein: a structure and function approach

AbstractAs the consumer demand for plant proteins continues to grow, the food industry is seeking novel and sustainable protein sources to incorporate in various food products. Pennycress (Thlaspi arvense), a sustainable cover crop, produces oilseeds high in protein, warranting investigation. Accordingly, protein extraction from pennycress was evaluated under various extraction conditions, using alkaline extraction and salt solubilization coupled with ultrafiltration. Given the superior color and functionality of the salt extracted pennycress protein isolate (PcPI), its production was scaled‐up about two hundred folds in a pilot plant. Furthermore, a new pennycress accession bred to have zero erucic acid (0EA) was evaluated to determine the impact of seed variety on protein characteristics. Structural and functional characterization was performed on PcPI and compared to native (nSPI) and commercial (cSPI) soy protein isolates. Salt extracted PcPI had comparable gel strength to cSPI, three times higher solubility under acidic conditions, and ~1.5 times better emulsification capacity. PcPI extracted from 0EA was mildly different in structure and functionality from that extracted from wildtype pennycress, with the slight variation attributed to genetic variance. Finally, the protein digestibility‐corrected amino acid score (PDCAAS) of the salt extracted PcPI, calculated in vivo (0.72) and in vitro (0.87), was superior or comparable to other plant protein sources. This research provided, for the first time, a comprehensive evaluation of different protein extraction protocols to produce a functional PcPI that can compete with soy protein for various food applications, such as acidic beverages, meat and dairy products, and emulsified systems.

Plant Proteins: Methods of Quality Assessment and the Human Health Benefits of Pulses.

As countries increase their standard of living and individual income levels rise, there is a concomitant increase in the demand for animal-based protein. However, there are alternative sources. One of the alternatives available is that of increased direct human consumption of plant proteins. The quality of a dietary protein is an important consideration when discussing the merits of one protein source over another. The three most commonly used methods to express protein quality are the protein efficiency ratio (PER), a weight gain measurement; protein digestibility-corrected amino acid score (PDCAAS); and the digestible indispensable amino acid score (DIAAS). The possibility that alterations in the quality and quantity of protein in the diet could generate specific health outcomes is one being actively researched. Plant-based proteins may have additional beneficial properties for human health when compared to animal protein sources, including reductions in risk factors for cardiovascular disease and contributions to increased satiety. In this paper, the methods for the determination of protein quality and the potential beneficial qualities of plant proteins to human health will be described.

Determination of Indispensable Amino Acid Digestibility of the Red Kidney Bean in Humans Using a Dual Stable Isotope Tracer Method

BackgroundProtein quality of the red kidney bean (RKB), a common source of dietary protein, has been assessed using the protein digestibility-corrected amino acid score (PDCAAS) determined in animal models using mainly oro-fecal digestibility. More recently, the FAO recommended to use digestible indispensable amino acid score (DIAAS) instead of PDCAAS but highlighted insufficient data on true ileal indispensable amino acid (IAA) digestibility of proteins because amino acids are absorbed in the ileum. ObjectivesUsing a recently developed dual stable isotope tracer method, we aimed to measure each IAA digestibility as representation of true ileal digestibility of the RKB, Phaseolus vulgaris, in humans consuming a typical Jamaican meal. MethodsRKB-IAAs were intrinsically labeled by adding 2H2O to the plants. Uniformly labeled-[13C]-spirulina (standard protein) was added to a meal prepared with the labeled RKB and fed to 10 healthy adults (5 males, 5 females) in a nonrandomized trial as primed/intermittent doses to achieve a steady state IAA enrichment in plasma. Enrichment of 2H- and 13C-labeled IAA in plasma and the bean was measured by mass spectrometry. Each IAA digestibility (except tryptophan and histidine) was calculated as the ratio of plasma 2H-IAA to meal 2H-IAA divided by the ratio of plasma 13C-IAA to meal 13C-IAA adjusted for loss of 2H-atom during transamination and digestibility of spirulina. ResultsAdequate IAA labeling (>1000 ppm 2H excess) and plasma plateau isotopic enrichment were achieved. Mean RKB-IAA digestibility (%) was 79.4 ± 0.5, ranging from 69.0 ± 1.2 (threonine) to 85.7 ± 1.7 (lysine). ConclusionThe dual stable isotope tracer digestibility data are similar to published oro-fecal digestibility supporting substantial nitrogen exchange in the colon. The individual IAA digestibility is useful to derive DIAAS to replace PDCAAS. Using published RKB-IAA composition, extrapolated DIAAS was 0.63 based on the lowest score for methionine. Clinical trial registrationhttps://register.clinicaltrials.gov; ID: NCT-04118517.