Urine Metabolite Profiles after the Consumption of a Low- and a High-Digestible Protein Meal, and Comparison of Urine Normalization Techniques.

In the context of dietary transition toward plant proteins, it is necessary to ensure protein security in populations. It would thus be of interest to identify biomarkers of altered protein digestibility in populations. We examined the association between urinary metabolites and the acute intake of low- or highly digestible protein in healthy volunteers. The urine samples were collected before and 9 h after the ingestion of a meal containing either no protein, zein (low-digestible) or whey protein isolate (highly digestible). The liquid chromatography-high resolution mass spectrometry metabolomics approach was used for the profiling of the urinary metabolites. For the standardization of metabolomics data sets, osmolality-based, standard normal variates (SNV) and probabilistic quotient normalization (PQN) techniques were used. The ANOVA-based factorial method, AComDim_ICA, was used for chemometrics analysis. The osmolality adjustment has a beneficial effect and the subsequent mathematical normalization improves the chemometric analysis further. Some changes in the urinary metabolomes were observed 9 h after the meal in the three groups. However, there was no difference in the urine metabolome between groups. No biomarker of protein digestibility can be identified after the ingestion of a single meal, even when marked differences in the digestion efficiency of protein have been observed.

True Ileal Amino Acid Digestibility and Protein Quality of 15N-Labeled Faba Bean in Healthy Humans.

BACKGROUND: The recommended transition toward more plant-based diets, particularly containing legumes, requires a wider knowledge of plant protein bioavailability. Faba beans are cultivated at different latitudes and are used increasingly in human nutrition. OBJECTIVES: We aimed to assess the nutritional quality of faba bean protein in healthy volunteers equipped with an intestinal tube to implement the ileal 15N balance method. METHODS: Nine volunteers completed the study (7 males, 2 females, aged 33 ± 10 y, BMI: 24.7 ± 2.6 kg/m2). They were equipped with a nasoileal tube. After fasting overnight, they ingested a test meal consisting of cooked mash of dehulled faba bean seeds (20 g protein per serving of approximately 250 g) intrinsically labeled with 15N. Samples of ileal contents, plasma, and urine were collected over an 8-h postprandial period. Undigested nitrogen (N) and amino acids (AAs) were determined using isotopic MS, and subsequently, ileal digestibility and digestible indispensable amino acid score (DIAAS) were calculated. The measurement of postprandial deamination allowed calculation of the net postprandial protein utilization (NPPU). RESULTS: The ileal N digestibility was 84.1% ± 7.7%. Postprandial deamination represented 19.2% ± 3.6% of ingested N, and the NPPU was 64.7% ± 9.7%. The ileal digestibility of individual AAs varied from 85.1% ± 13.7% for histidine to 94.2% ± 3.6% for glutamine + glutamate. The mean AA digestibility was ∼6 percentage points higher than the digestibility of N, reaching 89.8% ± 5.9%, whereas indispensable AA digestibility was 88.0% ± 7.3%. Histidine and tryptophan were the first limiting AAs [DIAAS = 0.77 (calculated by legume-specific N-to-protein conversion factor 5.4); 0.67 (by default factor 6.25)]. Sulfur AAs were limiting to a lesser extent [DIAA ratio = 0.94 (N × 5.4); 0.81 (N × 6.25)]. CONCLUSIONS: Protein ileal digestibility of cooked, dehulled faba beans in humans was moderate (<85%), but that of AAs was close to 90%. Overall protein quality was restricted by the limited histidine and tryptophan content. This trial was registered at clinicaltrials.gov as NCT05047757.

Minimal processed infant formula vs. conventional shows comparable protein quality and increased postprandial plasma amino acid kinetics in rats.

During industrial processing, heat treatments applied to infant formulas may affect protein digestion. Recently, innovative processing routes have been developed to produce minimally heat-processed infant formula. Our objective was to compare the in vivo protein digestion kinetics and protein quality of a minimally processed (T−) and a heat-treated (T+++) infant formula. Sixty-eight male Wistar rats (21 d) were fed with either a diet containing 40 % T− (n 30) or T+++ (n 30), or a milk protein control diet (n 8) during 2 weeks. T− and T+++ rats were then sequentially euthanised 0, 1, 2, 3 or 6 h (n 6/time point) after ingestion of a meal containing their experimental diet. Control rats were euthanised 6 h after ingestion of a protein-free meal to determine nitrogen and amino acid endogenous losses. Nitrogen and amino acid true caecal digestibility was high for both T− and T+++ diets (> 90 %), but a tendency towards higher nitrogen digestibility was observed for the T− diet (96·6 ± 3·1 %) compared with the T+++ diet (91·9 ± 5·4 %, P = 0·0891). This slightly increased digestibility led to a greater increase in total amino acid concentration in plasma after ingestion of the T− diet (P = 0·0010). Comparable protein quality between the two infant formulas was found with a digestible indispensable amino acid score of 0·8. In conclusion, this study showed that minimal processing routes to produce native infant formula do not modify protein quality but tend to enhance its true nitrogen digestibility and increase postprandial plasma amino acid kinetics in rats.

Context and Perspectives for Establishing a Novel Database for Protein Quality of Human Foods, as Proposed by a Joint Food and Agriculture Organization of the United Nations/International Atomic Energy Agency Expert Technical Meeting in October 2022.

United Nations agencies have a long history of leading work on establishing global human nutrient requirements. Dietary protein contributes to metabolism and homeostasis and plays an essential role in human health for growth, maintenance, reproduction, and immune function (or immunity). Accurately defining the quantity and quality of protein provided by foods and diets required to meet human nutritional needs is essential to achieving global environmental and nutrition goals. There have been many scientific developments related to protein quality over the past decades, with the preferred method being the scoring approach that relates the capacity of protein sources to provide an adequate amount and proportion of nitrogen and indispensable amino acids (IAAs) in a bioavailable form (often referred to as digestibility). Questions surrounding the scoring approach and IAA metabolic availability have been discussed during past and recent expert consultations. Recently, an Food and Agriculture Organization of the United Nations/International Atomic Energy Agency technical meeting, held in Vienna, 10-13 October, 2022, reviewed and updated evidence and related methods on protein requirements and protein quality assessment and designed a framework for the development of a Protein Digestibility Database to aid dialog on the evaluation of protein quality and protein sufficiency in different populations. The database should be a living document and align with national food compositional databases.

Values for the Digestibility of Pea Protein Isolate or Casein Amino Acids Determined using the Dual Isotope Method Are Not Similar to Those Derived with the Standard Ileal Balance Method in Healthy Volunteers.

BACKGROUND: The measurement of ileal amino acid (AA) digestibility is invasive and inappropriate when applied to vulnerable populations. The dual isotope method has been developed over the past 5 y as an alternative method. OBJECTIVE: The aim of this work was to compare the indispensable amino acid (IAA) digestibility values of 2 different proteins obtained using the dual isotope and the standard ileal balance methods in the same subjects. METHODS: Fifteen healthy adults completed the study. Over 4 h, they ingested 9 successive portions of mashed potatoes containing the test protein (pea protein or casein) labeled intrinsically with 15N and 2H, and a 13C-free AA mixture as a reference for the dual isotope method. Plasma was sampled regularly over the 8-h postprandial period, whereas the ileal digesta was collected continuously via a naso-ileal tube. Isotopic enrichments (15N and 13C) were measured in the digesta for the direct determination of ileal IAA digestibility, whereas plasma enrichments (2H and 13C) were measured to determine IAA digestibility using the dual isotope method. RESULTS: The 4-h repeated meal procedure enabled the almost complete digestion of test proteins at 8 h and the attainment of a plasma isotopic plateau between 2.5 and 4 h. These conditions were necessary to perform the ileal balance and dual isotope methods simultaneously. For pea protein, the mean IAA digestibility was similar between the 2 methods, but significant differences (from 10% to 20%) were observed for individual IAA values. For casein, IAA digestibility was significantly lower with the dual isotope method for all the IAA analyzed. CONCLUSIONS: Under our experimental conditions, the degree of agreement between the dual isotope and ileal balance methods varied among AAs and depended on the protein source. Further research is needed to validate the dual isotope method. This study was registered at clinicaltrials.gov as NCT04072770.

The use of 15N-labelled protein to account for the endogenous nitrogen contribution to in vitro protein digestibility measurement.

Protein digestibility, a key indicator of dietary protein quality for human nutrition, can be estimated using an in vitro digestion model, however its definition and determination remain variable across studies. The present study aimed to determine the contribution of the endogenous nitrogen (N) to the plant and animal protein digestibility values obtained in vitro. 15N-labelled gluten and caseins (4, 8 and 16 % of the model meal) were used to differentiate dietary and endogenous N and were digested using the INFOGEST in vitro digestion model with no oral phase. The dietary and endogenous N were measured before and during digestion after centrifugation and 10 kDa ultrafiltration. The proteolysis degree was measured by the OPA method. The endogenous and dietary N were determined by elemental analyser coupled with isotopic ratio mass spectrometry. Apparent and true digestibility were determined and values of 135, 92 and 71 % for apparent vs. 78, 69, 60 % for true digestibility were obtained for 4, 8 and 16 % dietary protein level, respectively, with a significant effect of protein level. Differences between apparent and true digestibility pointed out the important contribution of the endogenous nitrogen. Our results showed that 40 % of the N below 10 kDa, i.e., the digestible fraction, were from endogenous origin (i.e. from the pancreatin) and was even present before digestion. An average value of 27 % for pancreatin N autolysis was estimated independently of the protein levels or sources. The use of 15N-labelled protein to evaluate in vitro protein digestibility highlighted the important contribution of the endogenous N, in particular when low dietary protein solution (4 %) are digested. This gives new keys to overcome drawbacks of in vitro models for determining protein digestibility.

Differential effects of milk proteins on amino acid digestibility, post-prandial nitrogen utilization and intestinal peptide profiles in rats.

OBJECTIVE: The aim of this study was to analyze the protein digestibility and postprandial metabolism in rats of milk protein matrices obtained by different industrial processes. MATERIAL AND METHODS: The study was conducted on Wistar rats that consumed a meal containing different 15N-labeled milk proteins. Four milk matrices were tested: native micellar caseins (C1), caseins low in calcium (C2 low Ca2+), a matrix containing a ratio 63:37 of caseins and whey proteins (CW2) and whey proteins alone (W). Blood and urine were collected during the postprandial period and rats were euthanized 6 h after meal intake to collect digestive contents and organs. RESULTS: Orocaecal digestibility values of amino acids ranged between 96.0 ± 0.2% and 96.6 ± 0.4% for C1-, C2 low Ca2+- and W-matrices, while this value was significantly lower for CW2 matrix (92.4 ± 0.5%). More dietary nitrogen was sequestered in the splanchnic area (intestinal mucosa and liver) as well as in plasma proteins after ingestion of W matrix, especially compared to the C1- and C2 low Ca2+-matrices. Peptidomic analysis showed that more milk protein-derived peptides were identified in the caecum of rats after the ingestion of the matrices containing caseins compared to W matrix. CONCLUSION: We found that demineralization of micellar caseins did not modify its digestibility and postprandial metabolism. The low digestibility of the modified casein-to-whey ratio matrix may be ascribed to a lower accessibility of the protein to digestive enzymes due to changes in the protein structure, while the higher nitrogen splanchnic retention after ingestion of whey was probably due to the fast assimilation of its protein content. Finally, our results showed that industrial processes that modify the structure and/or composition of milk proteins influence protein digestion and utilization.

Indispensable Amino Acid Digestibility of Moroccan Fava Bean Using the Dual Isotope Method in Healthy Adults.

BACKGROUND: Assessment of protein quality is necessary to satisfy the nutritional needs of populations across the world. In addition to indispensable amino acid (IAAs) composition, protein digestibility is a major component of IAA bioavailability, playing a crucial role in human health and affecting the linear growth of children. OBJECTIVES: This study aimed to evaluate IAA digestibility of fava beans, a legume widely consumed in Morocco using the dual-tracer method. METHODS: 2H-intrinsically labeled Fava beans supplemented with 12 mg/kg BW of 13C spirulina were given to 5 healthy volunteers (3 men and 2 women), aged 25.8 ± 3.3 y, with a mean BMI of 20.0 kg/m2. The meal was spread in small portions and was given hourly throughout 7 h. Blood was sampled at baseline and hourly from 5 to 8 h after meal ingestion. IAA digestibility was evaluated by gas chromatography-combustion-isotope ratio mass spectrometry using the 2H/13C ratio in plasma IAA. Digestible indispensable amino acid ratios (DIAAR) were calculated using the scoring pattern for people older than 3 y. RESULTS: Fava beans had an adequate level of lysine but were limiting in several IAAs, especially methionine. Under our experimental conditions, the average IAA digestibility of fava bean was 61.1% ± 5.2%. Valine had the highest digestibility (68.9% ± 4.3%) and threonine had the lowest (43.7% ± 8.2%). In consequence, the lowest DIAAR was 67% for threonine and only 47% for sulfur amino acids (SAA). CONCLUSIONS: The present study is the first to determine the digestibility of fava bean amino acids in humans. The mean IAA digestibility was moderate, and consequently, we conclude that fava bean provides a limited amount of several IAAs, especially SAA, but adequately for lysine. Preparation and cooking methods of fava beans should be improved to increase digestibility. This study was registered at ClinicalTrials.gov as NCT04866927.

Digestive and metabolic bioavailability in healthy humans of 15N-labeled rapeseed and flaxseed protein incorporated in biscuits.

BACKGROUND: In the search to diversify protein sources for humans, oilseeds are good candidates due to the high protein content of their coproducts after oil extraction. Among them, rapeseed presents a well-balanced amino acid (AA) profile. Flaxseed is an emerging source but the nutritional value of its protein is not yet documented. OBJECTIVES: This study aimed to determine the nitrogen (N) and AA bioavailability of these protein sources. METHODS: Nineteen healthy volunteers were intubated with a naso-ileal tube. They ingested 156 g biscuits containing intrinsically labeled 15N rapeseed (n = 10) or flaxseed (n = 9) protein over a 4-h period. Ileal digesta, blood, and urine were sampled over 8 h after the first meal ingestion. N and 15N enrichment and AAs were measured to determine digestive and deamination losses. Ileal digestibility, the digestible indispensable AA score (DIAAS) and net postprandial protein utilization (NPPU) were calculated. RESULTS: Real ileal digestibility was 80.7 ± 6.5% for rapeseed protein and 92.2 ± 2.0% for flaxseed protein (P = 0.0002). Mean indispensable AA (IAA) digestibility reached 84.1 ± 6.9% and 93.3 ± 6.7% for rapeseed and flaxseed, respectively, lysine being the lowest digestible IAA for both sources. Despite moderate digestibility, the DIAAS was 1.1 for rapeseed but only 0.6 for flaxseed due to lysine insufficiency. Deamination losses accounted for 20.0 ± 6.5% of dietary N for flaxseed and 11.0 ± 2.8% for rapeseed (P = 0.002). The NPPU did not differ between the protein sources, with 71.3 ± 6.5% for flaxseed and 69.7 ± 7.6% for rapeseed. CONCLUSIONS: Despite good digestibility, flaxseed protein cooked in biscuits was penalized by both lysine insufficiency and poor lysine digestibility that decreased its DIAAS and increased deamination. By contrast, rapeseed was moderately digestible but presented no limiting IAA, resulting in an excellent DIAAS and low deamination. This study was registered at clinicaltrials.gov as NCT04024605.

Caecal digestibility as an approximation of ileal protein digestibility evaluation in rats.

The rat model can be used to assess ileal protein digestibility rapidly and in first intention, but no standardised method exists. Our objective was to compare methods to assess protein digestibility, depending on collection site (ileum/caecum) and use of a non-absorbable marker. A meal containing either casein, gluten or pea protein and chromium oxide as non-absorbable marker was given to male Wistar rats and the entire digestive content was collected 6 h later. Total chromium recovery was incomplete and variable, depending on protein source. We observed no significant difference in digestibility between the methods for any of the protein sources tested. Although none of the methods tested is optimal, our results suggest that caecal digestibility can be used as a proxy of ileal digestibility in rats without using a non-absorbable marker. This simple method makes it possible to evaluate protein digestibility of new alternative protein sources for human consumption.

Moderate adiposity levels counteract protein metabolism modifications associated with aging in rats.

PURPOSE: Physiological parameters such as adiposity and age are likely to influence protein digestion and utilization. The aim of this study was to evaluate the combined effects of age and adiposity on casein protein and amino acid true digestibility and its postprandial utilization in rats. METHODS: Four groups were included (n = 7/8): 2 months/normal adiposity, 2 months/high adiposity, 11 months/normal adiposity and 11 months/high adiposity. Rats were given a calibrated meal containing 15N-labeled casein (Ingredia, Arras, France) and were euthanized 6 h later. Digestive contents were collected to assess protein and amino acid digestibilities. 15N enrichments were measured in plasma and urine to determine total body deamination. Fractional protein synthesis rate (FSR) was determined in different organs using a flooding dose of 13C valine. RESULTS: Nitrogen and amino acid true digestibility of casein was around 95-96% depending on the group and was increased by 1% in high adiposity rats (P = 0.04). Higher adiposity levels counteracted the increase in total body deamination (P = 0.03) that was associated with older age. Significant effects of age (P = 0.006) and adiposity (P = 0.002) were observed in the muscle FSR, with age decreasing it and adiposity increasing it. CONCLUSION: This study revealed that a higher level of adiposity resulted in a slight increase in protein and individual amino acid true digestibility values and seemed to compensate for the metabolic postprandial protein alterations observed at older age.

Using the dual isotope method to assess cecal amino acid absorption of goat whey protein in rats, a pilot study.

Measurement of ileal amino acids (AA) bioavailability is recommended to evaluate protein quality. A dual isotope tracer method, based on plasma isotopic enrichment ratios, has been proposed to determine true digestibility in humans. In a pilot study, we aimed to evaluate whether this method could be implemented in rats to determine AA bioavailability based on isotopic enrichment ratios measured in cecal digesta or plasma samples. Goat milk proteins were intrinsically labeled with 15N and 2H. Wistar rats were fed a meal containing the doubly labeled goat whey proteins and a tracer dose of 13C-spirulina. Blood samples were collected 0, 1 h and 3 h after meal ingestion from the tail vein. The rats were euthanized 4 h (n = 6) or 6 h (n = 6) after meal to collect plasma and intestinal contents. True orocecal protein digestibility and AA bioavailability were assessed by means of 15N and 2H enrichment in cecum content and compared with absorption indexes determined at the plasma or cecum level using isotopic ratios. Plasma kinetics of isotopic enrichment could not be completed due to the limited quantity of plasma obtained with sequential blood collection. However, the absorption indexes determined from cecal 15N or 2H/13C ratios gave coherent values with true orocecal AA bioavailability. This dual isotope approach with measurements of isotopic ratios in digestive content could be an interesting strategy to determine true AA bioavailability in ileal digesta of rats.

Real ileal amino acid digestibility of pea protein compared to casein in healthy humans: a randomized trial.

BACKGROUND: It is necessary to propose plant alternatives to animal proteins that are of good nutritional quality. Pea is a good candidate owing to its high protein content and its well-balanced amino acid (AA) profile. OBJECTIVES: This study aimed to assess the real ileal AA and nitrogen digestibility (RIDAA and RIDN) of pea protein isolate as compared to milk casein in humans. It also aimed to evaluate their nutritional quality through calculation of the digestible indispensable amino acid score (DIAAS) and to determine the net postprandial protein utilization (NPPU). METHODS: Fifteen healthy volunteers were included in a randomized, single-blinded, 2-arm, parallel-design trial. They were equipped with a naso-ileal tube. They ingested the test meals, which consisted of 9 successive portions of mashed potatoes containing either pea protein or casein, intrinsically labeled with nitrogen 15. Ileal content, plasma, and urine samples were collected regularly over an 8-h postprandial period. RESULTS: The mean RIDAA values were 93.6% ± 2.9% for pea protein and 96.8% ± 1.0% for casein, with no difference between the sources (P = 0.22). Leucine, valine, lysine, and phenylalanine were significantly less digestible in pea than in casein. The RIDN values were 92.0% ± 2.7% and 94.0% ± 1.7% for pea protein and casein, respectively, and were not different (P = 0.11). The DIAAS was 1.00 for pea protein and 1.45 for casein. The NPPU was 71.6% ± 6.2% and 71.2% ± 4.9% for pea protein and casein, respectively (P = 0.88). CONCLUSIONS: Although some AAs are less digestible in pea protein than in casein, the real ileal digestibility and the NPPU were not different. The DIAAS of 1.00 obtained for pea protein demonstrated its ability to meet all AA requirements. This study shows the potential of pea isolate as a high-quality protein. This study was registered at clinicaltrials.gov as NCT04072770.

Evaluation of Protein Quality in Humans and Insights on Stable Isotope Approaches to Measure Digestibility - A Review.

The recent Food and Agricultural Organization/World Health Organization/United Nations University expert consultations on protein requirements and quality have emphasized the need for the new Digestible Indispensable Amino Acid Score (DIAAS), as a measure of protein quality. This requires human measurements of the true ileal digestibility of individual indispensable amino acids (IAAs) until the end of the small intestine. Digestibility is measured using standard oro-ileal balance methods, which can only be achieved by an invasive naso-ileal intubation in healthy participants or fistulation at the terminal ileum. Significant efforts have been made over the last 2 decades to develop noninvasive or minimally invasive methods to measure IAA digestibility in humans. The application of intrinsically labeled (with stable isotopes like 13C, 15N, and 2H) dietary proteins has helped in circumventing the invasive oro-ileal balance techniques and allowed the differentiation between endogenous and exogenous protein. The noninvasive indicator amino acid oxidation (IAAO) technique, which is routinely employed to measure IAA requirements, has been modified to estimate metabolic availability (a sum of digestibility and utilization) of IAA in foods, but provides an estimate for a single IAA at a time and is burdensome for participants. The recently developed minimally invasive dual isotope tracer method measures small intestinal digestibility of multiple amino acids at once and is suitable for use in vulnerable groups and disease conditions. However, it remains to be validated against standard oro-ileal balance techniques. This review critically evaluates and compares the currently available stable isotope-based protein quality evaluation methods with a focus on the digestibility and metabolic availability measurements in humans. In view of building a reliable DIAAS database of various protein sources and subsequently supporting protein content claims in food labeling, a re-evaluation and harmonization of the available methods are necessary.

The True Amino Acid Digestibility of 15N-Labelled Sunflower Biscuits Determined with Ileal Balance and Dual Isotope Methods in Healthy Humans.

BACKGROUND: Sunflower is a promising protein source but data on amino acid (AA) digestibility are lacking in humans. Classically, the determination of AA digestibility requires ileal digesta sampling. The dual isotope method is minimally invasive but has not been compared to the conventional approach. OBJECTIVES: This study aimed to determine the true ileal digestibility of sunflower AAs in healthy volunteers who ate biscuits containing 15nitrogen (N) protein isolate, in comparison with the dual isotope method. METHODS: Twelve healthy volunteers (men and women; 40.4 ± 10.5 years old; BMI, 23.7 ± 2.9 kg/m2) were equipped with a naso-ileal tube. For 4 hours, they consumed 9 repeated meals comprising 15N-sunflower protein biscuits together with 13carbon (C)-AAs, carried either in chocolate (SUN + Ch; n = 7) or apple puree (SUN + P; n = 5). Ileal digesta and blood were sampled throughout 8 hours after ingestion of the first meal. The 15N and 13C AA enrichments were measured in digesta to determine ileal digestibility directly and in plasma to determine lysine and threonine digestibility using the dual isotope method. Differences between methods and between vector groups were analyzed using paired and unpaired t-tests, respectively. RESULTS: The ileal digestibility of sunflower indispensable AAs (IAA) was 89% ± 5.3%, with threonine and lysine having the lowest digestibility. In the SUN + Ch meal, IAA digestibility was 3% below that of SUN + P (P < 0.05). The mean free 13C-AA ileal digestibility was 98.1% ± 0.9%. No matter which matrix was used to carry 13C-AAs, plasma 15N and 13C-AA kinetics displayed a 1-hour offset. Digestibility obtained with the dual isotope method (70.4% ± 6.0% for threonine and 75.9% ± 22.3% for lysine) was below the target values. CONCLUSIONS: The ileal digestibility of IAAs from a sunflower isolate incorporated in a biscuit was close to 90% in healthy adults. Under our experimental conditions, the dual isotope method provided lower values than the usual method. Further protocol developments are needed to validate the equivalence between both methods. This trial was registered at clinicaltrials.gov as NCT04024605.

Multi-criteria assessment of pea protein quality in rats: a comparison between casein, gluten and pea protein alone or supplemented with methionine.

The objective of this study was to assess the nutritional quality of pea protein isolate in rats and to evaluate the impact of methionine (Met) supplementation. Several protein diets were studied: pea protein, casein, gluten, pea protein-gluten combination and pea protein supplemented with Met. Study 1: Young male Wistar rats (n 8/group) were fed the test diets ad libitum for 28 d. The protein efficiency ratio (PER) was measured. Study 2: Adult male Wistar rats (n 9/group) were fed the test diets for 10 d. A protein-free diet group was used to determine endogenous losses of N. The rats were placed in metabolism cages for 3 d to assess N balance, true faecal N digestibility and to calculate the Protein Digestible-Corrected Amino Acid Score (PDCAAS). They were then given a calibrated meal and euthanised 6 h later for collection of digestive contents. The true caecal amino acid (AA) digestibility was determined, and the Digestible Indispensable Amino Acid Score (DIAAS) was calculated. Met supplementation increased the PER of pea protein (2·52 v. 1·14, P < 0·001) up to the PER of casein (2·55). Mean true caecal AA digestibility was 94 % for pea protein. The DIAAS was 0·88 for pea protein and 1·10 with Met supplementation, 1·29 for casein and 0·25 for gluten. Pea protein was highly digestible in rats under our experimental conditions, and Met supplementation enabled generation of a mixture that had a protein quality that was not different from that of casein.

Protein and amino acid digestibility of 15N Spirulina in rats.

PURPOSE: Spirulina is often used as dietary supplement for its protein content and quality. However, in vivo data on protein digestibility are lacking. This study aims to determine nitrogen and amino acid digestibility in rats. A secondary objective was to test the effect of sonication prior to ingestion to break cell walls. METHODS: Wistar rats were fed a single test meal containing 15N Spirulina that was either sonicated (n = 11) or not (control, n = 13). Rats were euthanized 6 h after the meal ingestion. Spirulina nitrogen digestibility was measured by assessment of 15N recovery in digestive contents. Amino acid digestibility was measured by quantification of the caecal amino acid content and their 15N enrichment. RESULTS: Real fecal nitrogen digestibility was 86.0 ± 0.7%, without any differences between groups. Mean 15N amino acid caecal digestibility was 82.8 ± 1.3%, and values ranged between 77.9 ± 1.9% for serine and 89.4 ± 1.0% for methionine. No effect of sonication was observed. The most limiting AA was histidine, with a chemical score of 0.98 and a PD-CAAS of 0.84. Lysine was also limiting in a lesser extent. CONCLUSION: The nitrogen and amino acid digestibility of Spirulina is relatively low, and showed no effect of prior sonication. Its amino acid composition is relatively well balanced but not enough to compensate for the poor digestibility.

Determinants of amino acid bioavailability from ingested protein in relation to gut health.

PURPOSE OF REVIEW: The current review provides an update on the recent research developments regarding amino acid bioavailability in conditions of both good health and gut disorders. RECENT FINDINGS: Determination of amino acid bioavailability is complex and invasive. Minimally invasive methods using stable isotopes have been developed for humans. Data were collected in different models - humans, pigs and rats with various procedures - leading to interstudy variability. They mainly focused on either plant protein or the effect of food processing on animal protein. Plant protein in their original food matrix (legumes, grains, nuts) are generally less digestible (about 80%) than animal protein (meat, egg, milk; about 93%). Food processing has a limited impact on animal protein but its effect might be higher on plant protein. Few studies have documented the effect of gut disorders on protein digestibility, except in gastric bypass where paradoxical effects were reported. Data are needed to identify the amplitude of protein malabsorption in diseases such as inflammatory bowel disease or environmental enteric dysfunction. SUMMARY: The past 5 years have seen a renewed interest in amino acid bioavailability in view of assessing protein quality to support current shifts in protein sourcing. Methodological developments have been performed and several studies have reported values in various models. The question of protein digestibility in gut disorders remains poorly addressed.

Very low ileal nitrogen and amino acid digestibility of zein compared to whey protein isolate in healthy volunteers.

BACKGROUND: Whey protein and zein are of nutritional interest due to their high leucine content, but little data are available on their amino acid (AA) ileal digestibility. OBJECTIVE: This study aimed to determine ileal digestibility of whey protein isolate (WPI) and zein in healthy volunteers by use of the naso-ileal intubation method, which allows continuous collection of postprandial ileal digesta. METHODS: Twenty-two healthy volunteers were intubated with a naso-ileal sampling device positioned at the terminal ileum level. They received a single meal of protein-free biscuits and a drink containing zein (n = 8), WPI (n = 7), or no protein (protein free, n = 7). Ileal effluents and plasma samples were collected over a 9-h postprandial period. Total nitrogen and AA contents were quantified in effluents. True ileal digestibility was calculated after correction for endogenous losses evaluated in the protein-free group. RESULTS: True ileal nitrogen digestibility of zein was markedly lower than WPI (60.2 ± 4.5% and 91.2 ± 2.6%, respectively, P = 0.0003). True ileal digestibility of AAs ranged from 87.4 ± 2.7% for threonine to 98.4 ± 1.0% for methionine in the WPI group, and from 59.3 ± 5.6% for methionine to 69.0 ± 5.8% for arginine in the zein group. The digestible indispensable AA (IAA) score was 1.03 (histidine) for WPI and close to 0 for zein, owing to its negligible lysine content. Plasma IAA concentration significantly increased after WPI intake (P = 0.0319), whereas no effect of zein on aminoacidemia was observed, including plasma leucine, despite its high leucine content. CONCLUSIONS: Our findings provide data on ileal digestibility of WPI and zein AAs in healthy humans and, in contrast to WPI, zein is poorly digestible. This study was registered at clinicaltrials.gov as NCT03279211.