True ileal amino acid digestibility and digestible

indispensable amino acid scores (DIAASs) of

plant-based protein foods
Yohan Reynaud, Caroline Buffière, Benoît Cohade, Mélissa Vauris, Kevin
Liebermann, Noureddine Hafnaoui, Michel Lopez, Isabelle Souchon, Didier
Dupont, Didier Remond

To cite this version:

Yohan Reynaud, Caroline Buffière, Benoît Cohade, Mélissa Vauris, Kevin Liebermann, et al.. True
ileal amino acid digestibility and digestible indispensable amino acid scores (DIAASs) of plant-based
protein foods. Food Chemistry, 2021, 338, pp.1-7. �10.1016/j.foodchem.2020.128020�. �hal-02946774�

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 True ileal amino acid digestibility and digestible indispensable amino acid

scores (DIAASs) of plant-based protein foods

Yohan Reynauda,b, Caroline Buffièrec, Benoît Cohadec, Mélissa Vaurisc, Kevin

Liebermannc, Noureddine Hafnaouic, Michel Lopeza, Isabelle Souchond, Didier Dupontb,

Didier Rémondc*
a
IMPROVE SAS, F-80480, Dury, France

b
STLO, INRAE, INSTITUT AGRO, F-35042, Rennes, France

c
Université Clermont Auvergne, INRAE, UNH, Unité de Nutrition Humaine, F-63000

Clermont-Ferrand, France

d
Université Paris-Saclay, INRAE, AgroParisTech, SayFood, F-78850 Thiverval-Grignon,

France

Corresponding author: Didier Rémond, Unité de Nutrition Humaine, INRAE de Clermont-

Ferrand/Theix, 63122 St Genès-Champanelle, France. Tel: 33 473 624 074. Fax: 33 473 624
755. e-mail: [email protected]

Authors’ e-mail addresses: [email protected], [email protected],

[email protected], [email protected], [email protected],
[email protected], [email protected], [email protected],
[email protected], [email protected]

Conflict of interest: The authors declare no conflicts of interest.

Funding: The project was funded by IMPROVE SAS and INRAE. A PhD grant was funded
by IMPROVE SAS through a CIFRE procedure (CIFRE n°2016/0719)
Role of the funding source: IMPROVE SAS provided foods and INRAE performed the
experimental work.

1
Running title: DIAAS of plant-based protein foods

Abstract

Plant-based protein foods are increasingly common, but data on their nutritional protein

quality are scarce. This study evaluated it for seitan (wheat-based food), tofu (soya-based food),

soya milk, and a pea emulsion. The true ileal digestibility (TID) of their amino acids was

determined in minipigs, to calculate the digestible indispensable amino acid score (DIAAS).

The TID of the proteins was high and not significantly different between the foods tested: 97%

for seitan, 95% for tofu, 92% for soya milk and 94% for pea emulsion. There were only minor

differences in individual amino acid TIDs. DIAAS ranking was thus essentially driven by the

amino acid composition of the food: soya-based food > pea emulsion > seitan. Nevertheless,

the lower TID of sulphur-containing amino acids in tofu than in soya milk induced a significant

decrease in DIAAS (from 117% to 97%), highlighting the importance of the matrix effect on

nutritional protein quality.

Keywords: plant protein, pea, soya, wheat, food matrix, digestibility, DIAAS

2
1 Introduction

Gross domestic product (GDP) per capita is positively correlated with dietary energy

intake. Since it does not influence the contribution of protein to dietary energy (i.e., protein

provides 5–15% of total energy) (Gerbens-Leenes, Nonhebel, and Krol 2010), demand for

protein may increase worldwide as global per-capita GDP climbs. Indeed, research has found

that, as standards of living improve, so does the percentage of energy provided by animal-based

products. However, in general, the production of animal protein has a greater environmental

impact than does the production of plant protein, as measured via the quantity of greenhouse

gases emitted per gram of protein (González, Frostell and Carlsson-Kanyama, 2011). Since

2004, the availability of commercial plant-protein-based foods has increased dramatically in

France; the source of protein in these foods is most often soya, wheat, and pea (GEPV, 2019).

However, there are two major concerns regarding protein quality in these products: 1) the

products may contain antinutritional factors, such as trypsin inhibitors or tannins that limit

protein digestion (Gilani, Xiao and Cockell, 2012), and 2) the products may lack certain

indispensable amino acids (IAA). Assessing the nutritional quality of the protein in plant-based

products remains a significant challenge, complicating efforts to design innovative foods that

will respond to the growing demand for protein.

The Food and Agriculture Organisation (FAO) has defined an index for assessing

nutritional protein quality that integrates the notion of amino acid bioavailability, namely the

digestible indispensable amino acid score (DIAAS) (FAO, 2013). The DIAAS compares the

digestibility of individual amino acids at the end of the small intestine (i.e., true ileal

digestibility, which is a better proxy for bioavailability than whole-tract digestibility) to a

standard of reference. Protein digestibility has largely been studied using protein isolates: the

true ileal digestibility of soya, wheat, and pea proteins has been reported to be 91.5%

(Gaudichon et al., 2002; Bos et al., 2003), 93.4% (De Vrese et al., 2000), and 89.4–91.5%

3
(Gausserès et al., 1996, 1997; Mariotti et al., 2001), respectively. However, little is known

about the digestibility of these proteins when they are in foods that are traditionally consumed

by humans, such as tofu and seitan. However, it is now well known that the food matrix and its

transformation during processing affects the digestibility and use of the nutrients (Thorning et

al., 2017). Heat treatments, for instance, may inactivate antitrypsin factors in foods, thus

increasing protein digestibility, as illustrated by autoclaved and non-autoclaved soybean flour

(Li, Sauer and Caine, 1998). But these treatments may also impact protein structure (German,

Damodaran and Kinsella, 1982) and food structure (Le Feunteun et al., 2014), which influence

such factors as enzyme diffusion, substrate accessibility and, subsequently, protein digestibility.

It is thus crucial to estimate the digestibility of proteins in foods as they are actually consumed.

We hypothesized that protein digestibility in commonly consumed plant-based food could be

different that observed in protein isolates, because of the manufacturing processes involved

(heat treatment, coagulation), as previously observed for animal products (Bax et al.,

2012)(Barbé et al., 2013).

The aim of this study was to determine the nutritional quality of protein in four plant-based

foods (seitan, tofu, soya milk, and a pea emulsion) that differed in their structure (gel vs. liquid)

and protein source (wheat, soya and pea). Seitan and tofu were chosen because of the growing

interest of French consumers towards these plant-based protein foods. Protein digestibility of

the tested foods was evaluated in minipigs, and the DIAAS was calculated.

2 Materials and methods

2.1 Animal handling and surgery

All the procedures were carried out in accordance with European guidelines (Directive

2010/63/EU) and approved by the Auvergne Animal Experimentation Ethics Committee

(CEMEAA) and the French government (APAFIS#11001-2017082312525562v2). For the

experiment, we used six adult Yucatan miniature pigs (mean mass at 8 months = 20.2 ± 1.5 kg).

4
Three weeks before the experiment began, the pigs were surgically fitted with two permanent

catheters, which were placed in the hepatic portal vein and the upper hepatic vein. They were

also fitted with a T-cannula (made of silicone rubber; ID: 12 mm, OD: 17 mm) placed 10 cm

upstream from the ileocaecal valve.

To differentiate endogenous and dietary proteins, the pigs were given a continuous

perfusion of 13C-leucine solution through the upper hepatic vein catheter (Hess et al., 2000) for

five days before the experiment. They received a loading dose of 1 mg.kg-1 and then a

continuous dose of 1 mg.kg-1.j-1 at a rate of 1.5 ml.h-1. Prior to initiating the perfusion, a blood

sample was taken to determine the basal level of 13C-leucine enrichment.

The pigs were housed in individual pens in a ventilated room kept at a constant temperature

(21°C). Between the experimental trials, the pigs were given 500 g/d of a concentrate containing

16% protein, 1% fat, 4% cellulose, and 5% ash (Porcyprima; Sanders Centre Auvergne, France)

distributed in equal portions at 8.00 and 16.00. The pigs had ad libitum access to water.

2.2 Test meals

We tested two solid and two liquid foods: seitan (wheat-based protein), tofu (soya-based

protein), soya milk (soya-based protein), and a pea emulsion (pea-based protein; a soybean oil-

in-water emulsion containing a pea isolate). The seitan, tofu, and soya milk were of commercial

origin. The seitan and tofu were ground (the final median particle sizes were 5.1 ± 0.1 mm for

the tofu and 3.5 ± 0.1 mm for the seitan (n = 4 trials)) and then freeze dried. The soya milk had

undergone ultra-high temperature (UHT) processing. We created the emulsion using a

commercial pea isolate (Pisane M9, Lot: N16231O04, Cosucra, Belgium) and commercial

soybean oil (Emile Noël, France). A pre-emulsion was generated using a homogeniser (T-50

Ultra-Turrax, IKA, Germany) equipped with a 15 G dispersing tool (IKA, Germany) run at

10,000 rpm for 1 min. This pre-emulsion was then homogenised twice using a bench-top

homogeniser (PandaPLUS 2000, GEA, USA) run at 1,000 bar. We added maltodextrine, sugar,

5
and soybean oil to all the foods to ensure they had equal levels of protein (30.0 g), fat (23.1 g),

and calories (980 kcal), in each test-meal (Table S1, supplementary information 1). The test-

meals containing seitan, tofu, soya milk, and pea emulsion weighed 290 g, 435 g, 1,162 g, and

1,175 g, respectively. To estimate the basal flows of endogenous amino acids in the ileum, pigs

were also given a protein-free test-meal, in which the protein source was replaced by 30 g of a

mixture of free amino acids (in proportions resembling those found in meat), assumed to be

completely absorbed from the small intestine. An indigestible transit marker, chromium oxide

(Cr2O3), was added (0.3% of the food’s dry matter content). The characteristics of the different

foods tested are described in Erreur ! Source du renvoi introuvable..

2.3 Sampling and chemical analysis

Each pig was given each of the food types in a randomly determined order. Experimental

trials were separated by at least two days. During each trial, the pigs had ad libitum access to

water. All food types were ingested in less than 15 min.

2.3.1 Portal-vein blood samples

Blood samples (3 ml) were taken from the portal vein before food ingestion and 60, 210,

and 360 min after ingestion. The samples were immediately centrifuged (1,500 G, 10 min, 4°C).

The plasma was removed, frozen in liquid nitrogen, and stored at -80°C. Deproteinisation was

carried out using trichloroacetic acid precipitation. The supernatant was purified using cation

exchange chromatography and free amino acids were then converted into their N-acetyl-propyl

derivatives. It was then possible to measure the 13C-enrichment of free leucine in the plasma

(13C-Leuplasma pv) using gas chromatography-combustion-isotope ratio mass spectrometry (GC-

C-IRMS; IsoPrime, GV Instruments).

2.3.2 Ileal effluent samples

Between 60 min and 540 min post meal ingestion, ileal effluent was collected continuously

in plastic bags (Sachet Whirl-Pak bags; volume: 120 ml, size: 7.5 x 18.5 cm) attached to the

ileal cannula. The bags were regularly renewed based on bursts of digestion. The effluent

6
samples were immediately transferred to pre-weighed aluminium dishes kept at -20°C. We

created pools corresponding to 1 h of sampling, and stored them at -20°C.

The resulting samples were freeze dried (Cryotec AQ 1460). Dry matter (DM) content was

determined after the samples were dried for an additional 24 h in a 60°C oven. The digesta were

finely ground.

To estimate levels of chromium oxide (the indigestible transit marker), the samples were

first subjected to a step of mineralisation (550°C, 6 h), followed by nitric acid dissolution (3

min of boiling in 5% HN03- [w/w]) and filtration (Whatman filter paper: 4–7 µm). They were

then analysed using microwave plasma-atomic emission spectrometry (4210 MP-AES,

Agilent).

Next, we created a sample representative of the entire postprandial period for each animal

for each food type. These samples contained a fixed percentage (based on DM) of each of the

hourly effluent pools, starting from the first-time chromium oxide appeared.

Total nitrogen content (Ncontent) was determined using an elemental analyser (vario

ISOTOPE cube, Elementar). To estimate the amino acid content of food and digesta, we first

performed four separate standardised hydrolysis procedures (AOAC, 2000): sample placement

in 6N HCl for 24 h at 110°C, sample placement in 6N HCl for 48 h at 110°C (for branched-

chain amino acids), sample placement in 6N HCl for 24 h at 110°C after peroxidation with

H2O2 (for sulphur-containing amino acids); and sample placement in 4N Ba(OH)2 for 16 h at

110°C (for tryptophan). A fraction of each hydrolysate was dried and then resuspended in a

dilution buffer to which D-glucosaminic acid had been added as the injection standard. The

levels of the various amino acids (AAcontent) were determined using ion exchange

chromatography and ninhydrin post-column detection (L-8900 high-speed amino acid analyser,

Hitachi).

7
 The level of 13C-leucine enrichment was measured for the hydrolysed samples using gas

chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS; IsoPrime, GV

Instruments), as described for the plasma samples.

2.4 Calculations
Flows are expressed as g of AA or N over the 9-h postprandial period. The digestibility of

crude protein (CP = N x 6.25) is of no great relevance. Whereas the factor 6.25 used to convert

N to CP in food is not perfect but close to reality (Mariotti, Tomé and Mirand, 2008), it does

not apply to ileal contents in which about 30% of N is in non-protein form (urea, ammonia,

creatinine, etc.) (Miner-Williams, Moughan and Fuller, 2009). The CP flows in the present

study were indeed about twice as high as the total AA flows, illustrating the inconsistency of

using the factor of 6.25 for evaluating protein content in ileal samples. Such discrepancies

between CP ant total AA have been observed previously in pigs and minipigs (Hennig et al.,

2004). Thus, the digestibility calculations in this study were performed only for AAs, and the

digestibility of proteins in the text refers to digestibility of total AAs.

2.4.1 Total flows of N and amino acids in the ileum

The flows of N and amino acids (AA) were determined using the chromium oxide data and

the following equation: Xi = Xcontent × DM × Crintake/Crcontent, where X is N or an AA, DM is the

total dry matter in the ileal sample, Xcontent is the amount of nitrogen or a specific amino acid in

the ileal sample, and Crintake and Crcontent are the quantity of chromium oxide in the food type

and ileal sample, respectively.

2.4.2 Flows of endogenous crude protein and amino acids in the ileum
The flow of endogenous leucine in the ileum (Leuendo) was calculated using the following

equation: Leuendo = Leui x (13C-Leucontent - 13

C-Leufood)/(13C-Leuplasma pv - 13
C-Leufood), where
13 13 13
Leui is the quantity of leucine in the ileal sample and C-Leucontent, C-Leufood, and C-
13
Leuplasma pv are the levels of C-leucine enrichment in the ileal sample, food type, and portal

vein plasma, respectively.

8
 The flows of other endogenous amino acids in the ileum (AAendo) were determined using

the following equation: AAendo = Leuendo x ([AA]/[Leu])PFcontent, where ([AA]/[Leu])PFcontent is

the ratio between the levels of a given AA and leucine in the ileal samples from the trial in

which the pigs were fed the protein-free meal.

2.4.3 Ileal digestibilities of crude protein and amino acids

2.4.3.1 Apparent ileal digestibility of crude protein and amino acids

The apparent ileal digestibilities (AIDs) of nitrogen and the amino acids were expressed as

percentages and determined with the equation AID(X) = 100 x ([Xmeal – Xi]/ Xmeal), where

X is N or an AA.

2.4.3.2 Standardised ileal digestibility of crude protein and amino acids

Basal flows of endogenous nitrogen and amino acids (Xbasal endo) were defined as the flows

of N and AAs in the ileum following the ingestion of the protein-free meal (PF).

The standardised ileal digestibilities (SID) of nitrogen and amino acids were expressed as

percentages and determined with the equation: SID(AA) = 100 x (Xmeal – [Xi – Xbasal endo]/Xmeal),

where X is N or an AA.

2.4.3.3 True ileal digestibility of amino acids

The true ileal digestibilities (TID) of amino acids were expressed as percentages and

determined with the equation TID(AA) = 100 x (AAintake – [AAi – AAendo]/AAintake).

2.5 Data and statistical analysis

Data were analysed using an analysis of variance (ANOVA) using protein source and

animal as fixed effects (GLM procedure of SAS®). Comparisons of the means were performed

using Duncan’s multiple range test at a 95% confidence level. The paired t-test was used for

certain specific comparisons between tofu and soya milk. Data are presented as means ± SEM.

9
3 Results

3.1 Amino acid profiles of the foods

The amino acid profiles of the different foods tested are presented in Erreur ! Source du

renvoi introuvable.. As expected, the legume-based foods—the tofu, pea emulsion, and soya

milk—were relatively rich in lysine. Their lysine content accounted for 5.6 to 7.0 % of CP

content. In contrast, for seitan, the cereal-based food, Lysine accounted for only 1.4 % of CP

content. Conversely, seitan contained higher levels of sulphur-containing amino acids (SAAs)

than did the other foods (3.8 vs 1.4 to 2.9 % of CP content, for the other foods). Compared

with the reference amino acid profile for adults (FAO, 2013), seitan had lower than

recommended levels of lysine and threonine, and pea emulsion had lower than recommended

levels of SAAs. The soya-based foods (tofu and soya milk) had similar, well-balanced amino

acid profiles; individual IAA content met or exceeded the reference amino acid profile.

3.2 Apparent ileal digestibility

Although we observed significant differences between the foods tested regarding the AIDs

of some of the amino acids (Lys, Met, Asp/Asn, Cys, Glu/Gln) (Table 2), the AIDs for the total

AAs were very similar (77–84%). The AID of lysine was lower for seitan (54%) than for the

other food types (range: 83–87%). The AID of methionine (49%) was lower for the pea

emulsion than for soya milk and seitan (90%), while tofu had an intermediate value (69%). The

AID of cysteine was lower for tofu and pea emulsion (36 and 44%, respectively) than for seitan

and soya milk (74 and 79%, respectively).

3.3 Standard ileal digestibility

The protein free test meal showed that the contribution of the basal endogenous flow to the

total amino acid flow at the ileum averaged 3.7 ± 1.0 g over the postprandial period studied.

The amino acid pattern of this endogenous flow is given in Table S2 (supplementary

information 2). Standard ileal digestibility (SID; Table 3) takes into account this basal

10
endogenous flow. It is the value of ileal digestibility most often reported in the literature. The

SIDs of the total AAs were 91, 92, 89, and 87% for the seitan, pea emulsion, soya milk, and

tofu, respectively.

3.4 True ileal digestibility

Based on leucine enrichments, the contribution of endogenous protein secretions to total

protein flow at the ileum was about 2/3 for seitan, pea emulsion and tofu, but it was only 1/2

for soya milk (Table 4). Although numerically greater for the solids than for the liquids,

endogenous leucine flow was not significantly different between the 4 foods tested. The use of
13
C-leucine enrichments allowed taking into account not only basal endogenous flow, but also

endogenous losses specific to the food tested. Thus, whereas basal endogenous leucine flow

was estimated to be 0.23 ± 0.06 g, total endogenous leucine flows were 0.37 ± 0.11, 0.48 ±

0.09, 0.31 ± 0.03, and 0.26 ± 0.06 for seitan, tofu, soya milk and pea emulsion respectively.

The difference of specific endogenous leucine flow between tofu and soya milk was particularly

noteworthy (0.25 ± 0.09 vs 0.08 ± 0.03, respectively, P = 0.05). In our calculations the basal

and total endogenous flows of the other AA relied on the same profile of endogenous AA (that

observed with the PF meal), as the same differences were observed for them.

True ileal digestibility (TID; Table 4) takes into account total endogenous flows (basal +

specific). The TID of the total AAs were very high, and did not differ significantly between the

foods tested (92–97%). The TIDs for all the individual amino acids were also very high.

Although the soya milk and the tofu had the same protein source, they displayed small

differences in the TIDs of their amino acids: the TID of methionine was lower for tofu, and,

conversely, the TID of leucine and isoleucine was lower for soya milk.

3.5 Nutritional protein quality of the foods

The digestible indispensable amino acid content of the food is presented in Table 5. DIAAS

(FAO and WHO, 1991; FAO, 2013) of the four foods tested was calculated for the three age

11
groups (< 6 months; 6 months to 36 months; older child, adolescent, and adult) (Table 6). The

digestible amino acids that were limiting did not differ between score types or age groups,

except for the soya milk, for which leucine was the limiting amino acid for newborns, whereas

it was lysine for children below 3 years old, and valine above 3 years old.

4 Discussion

TID is considered as the best predictor of the bioavailability of dietary protein amino acids.

However, it is difficult to measure. In this study, we used a miniature pig model as an animal

model for humans, pig being considered as the animal model best adapted for the study of

digestion in the upper part of the gut (stomach + small intestine) (Rowan et al., 1994). We used

the approach of a single test meal in animals otherwise receiving a standard diet. This means

that the animals were not subjected to a period of continuous adaptation to the meal being tested.

They were exposed to the meals just once before the TID measurement (at least one week

before), to check the palatability of the meals. The TID obtained with this standardized

approach reflects more what happens in humans, who are characterized by the diversity of their

diet. Indeed, it does not exacerbate the potential negative effect of chronic exposure to the

potential presence of antinutritional factors, such as lectins that may significantly affect

endogenous secretions and gut health (Vasconcelos and Oliveira, 2004).

For the TID measurement, it is necessary to distinguish between residual dietary proteins

and endogenous proteins within the chyme collected at the end of the small intestine. Since

targeted foods with isotopic intrinsic labelling of proteins were not at our disposal, we opted

for the 13C-leucine labelling of endogenous proteins. This method is less subject to the biases

associated with rapid amino acid recycling than is the labelled dietary protein method.

However, it does not allow the direct measurement of the endogenous flow of each amino acid,

in contrast with the use of labelled proteins (Hess et al., 2000). In the present study, the

endogenous flow of leucine was extrapolated to the other amino acids, using the amino acid

12
pattern of the ileal chyme collected after a protein free meal (thus containing only endogenous

secretion). Endogenous secretions can be separated into basal secretions (those observed with

the protein free meal) and the specific endogenous losses in response to the food tested. For the

TID calculation in the present study, we postulated a similar pattern of amino acids between

basal and specific endogenous secretions. This is an approximation; in fact, the amino acid

composition of the specific losses can slightly differ from that of the basal secretions. For

instance, some foods containing polyphenols can specifically increase salivary proline-rich

proteins (PRP) secretion (Jansman, Frohlich and Marquardt, 1994), which are low in

indispensable amino acids and hardly digested by digestive enzymes. In addition, some peptides

released during digestion can specifically increase mucin secretions (threonine-rich proteins)

(Plaisancié et al., 2013).

There were no data in the literature on the digestibility of plant-based protein foods.

Common understanding is that proteins in plant food feature lower digestibility than that in

animal products, but evidence is lacking to support this for protein-rich plant foods that are

commercially available. Indeed, most digestibility measurements have been performed on plant

protein isolates, or raw materials. This study is the first to characterise the true ileal digestibility

(TID) of proteins in seitan, tofu, and soya milk; foods commonly consumed by humans. We

found that their TIDs were high: 98, 95, and 92%, respectively. Although slightly higher, these

values are consistent with those seen for wheat protein isolates (93%) (De Vrese et al., 2000)

and soybean protein isolates (91.5–96%) (Bos et al., 2003; Rutherfurd et al., 2015). The TID

of the protein in pea emulsion (94%) resembled the TIDs previously observed for pea isolates

(90–98%) (Mariotti et al., 2001; Rutherfurd et al., 2015). Furthermore, the TIDs of the protein

of the plant food we tested are comparable to those of animal proteins: 91–98% for meat (Oberli

et al., 2015; Hodgkinson et al., 2018; Kashyap et al., 2018) and 94–95% for milk (Bos et al.,

1999; Rutherfurd et al., 2015).

13
 Whatever the structure of the food, solid or liquid, the endogenous secretions were only

slightly affected and the protein digestibility remained high. The differences between soy milk

and tofu were however especially noteworthy, because they clearly illustrated the so-called

matrix effect (Thorning et al., 2017): different digestion of food products deriving from the

same raw material. Indeed, based on leucine flows, it can be estimated that the coagulation

process of soya milk in tofu production leads to an increase in specific endogenous losses. In a

previous study with the same soya milk and tofu (Reynaud et al., 2020), we observed a very

different evolution of gastric pH over the postprandial period. Apart from the potential

difference in gastric emptying due to difference in nutrient solubility of the two protein sources

(Schop et al., 2019) or to the difference in test-meal volume, this difference may have an impact

on the gastric pre-digestion of soya proteins and modify the nature of the peptides released

during intestinal digestion, which in turn could affect endogenous ileal losses (Hodgkinson et

al., 2000). On the other hand, tofu making slightly increased branched chain amino acids and

nearly all amino acids, whereas it decreased the digestibility of sulphur-containing amino acids.

Whereas the heat treatment applied during tofu making (70-85°C) may have decreased

antiprotease activity and increased protein digestibility, the coagulation process could explain

the negative effect on sulphur-containing amino acids. Indeed, it seems that sulphur-containing

amino acids mainly occur inside the molecular aggregates of the gels and are surrounded by

hydrophilic subunits (Peng, Ren and Guo, 2016), which could make them less accessible to

digestive enzymes, and explain their lower digestibility.

Seitan had the lowest nutritional protein quality (DIAAS range: 20–31%) due to its low

lysine content. This result concurs with what has been seen for wheat protein in bread (DIAAS:

20%) (Han et al., 2018). Lysine has also been found to be a limiting amino acid in whole-grain

wheat, but the DIAASs calculated for whole-grain wheat are generally higher (30–60%)

(Cervantes-Pahm, Liu and Stein, 2014; Mathai, Liu and Stein, 2017). This difference may be

14
attributable to seitan’s lower lysine levels—they were only half the lysine levels of whole-grain

wheat. The DIAASs of the pea emulsion were intermediate (42–64%), and SAAs were limiting

across all age groups. These values are consistent with those observed for a pea isolate (45–

84%) (Mathai, Liu and Stein, 2017).

Because the seitan and the pea emulsion were almost completely digested, the nutritional

protein quality of these two food types is essentially defined by their amino acid profiles, which

were unbalanced. In contrast, the tofu and soya milk had similar, well-balanced profiles. Thus,

the differences in the nutritional quality of their proteins is essentially determined by the

digestibility of their amino acids. Indeed, because soya milk has a well-balanced amino acid

composition, with high digestibility, its DIAAS is high (78% to 116%), and the limiting amino

acid evolves according to the age-related reference profile, whereas for tofu, the reduced

digestibility of the sulphur-containing amino acids makes them limiting, whatever the reference

profile, and leads to a 15% decrease in DIAAS values in comparison to soya milk. Whatever

the case, in agreement with the results previously reported for soya isolates (Mathai, Liu and

Stein, 2017)(Rutherfurd et al., 2015), the DIAAS of soya milk and tofu proteins, for the adult

population, are close to those reported for milk and meat (Mathai, Liu and Stein,

2017)(Hodgkinson et al., 2018), indicating the good nutritional quality of the proteins of these

products.

4.1 Conclusions
The four plant-based protein food studied here—seitan, pea emulsion, soya milk, and

tofu—displayed similar, high values of true ileal digestibility of amino acids. For the tofu and

soya milk (both soya-based foods), the difference in nutritional protein quality was primarily

driven by differences in AA digestibility, because the foods’ AA profiles were well balanced.

For the seitan and the pea emulsion, the lower nutritional protein quality was essentially

15
explained by the degree of amino acid imbalance and the amounts of limiting amino acids

relative to standard profiles.

Acknowledgements

We would like to thank Julien Hermet, the head of the animal care department, and Philippe

Lhoste and Yohan Delorme, both animal care workers. We are also grateful to Jessica Pearce

for correcting the English of the manuscript and to Denys Durand for the help he provided in

performing the surgery with Didier Rémond.

Author Contributions

DR designed the study. CB, BC, MV, YR, and DR performed the experiments, and NH

conducted the amino acid dosages. DR, CB, and YR analysed the data. DR and YR drafted the

manuscript, and all the authors helped revise it.

16
References

AOAC (2000) Official Methods of Analysis, 17th edn. Association of Official Analytical

Chemists, Washington, DC.

Barbé, F. et al. (2013) ‘The heat treatment and the gelation are strong determinants of the

kinetics of milk proteins digestion and of the peripheral availability of amino acids’, Food

Chemistry. Elsevier Ltd, 136(3–4), pp. 1203–1212. doi: 10.1016/j.foodchem.2012.09.022.

Bax, M. L. et al. (2012) ‘Cooking temperature is a key determinant of in vitro meat protein

digestion rate: Investigation of underlying mechanisms’, Journal of Agricultural and Food

Chemistry, 60(10), pp. 2569–2576. doi: 10.1021/jf205280y.

Bos, C. et al. (1999) ‘Assessment of net postprandial protein utilization of 15N-labelled

milk nitrogen in human subjects’, British Journal of Nutrition, 81(3), pp. 221–226.

Bos, C. et al. (2003) ‘Human Nutrition and Metabolism Postprandial Kinetics of Dietary

Amino Acids Are the Main Determinant of Their Metabolism after Soy or Milk Protein

Ingestion in Humans’, Journal of nutrition, 133(5), pp. 1308–1315.

Cervantes-Pahm, S. K., Liu, Y. and Stein, H. H. (2014) ‘Digestible indispensable amino

acid score and digestible amino acids in eight cereal grains’, Brit. J. Nutr., 111, pp. 1663–1672.

doi: 10.1017/S0007114513004273.

FAO (2013) Report of an FAO Expert Consultation: Dietary protein quality evaluation in

human nutrition.

FAO and WHO (1991) Joint FAO/WHO Expert Consultation on Protein Quality

Evaluation (1989: Bethesda, Maryland).

Le Feunteun, S. et al. (2014) ‘Impact of the Dairy Matrix Structure on Milk Protein

Digestion Kinetics: Mechanistic Modelling Based on Mini-pig In Vivo Data’, Food and

Bioprocess Technology, 7(4), pp. 1099–1113. doi: 10.1007/s11947-013-1116-6.

Gaudichon, C. et al. (2002) ‘Ileal Losses of Nitrogen and Amino Acids in Humans and

17
Their Improtance to the Assessment of Amino Acid Requirements’, Gastroenterology, 123, pp.

50–59. doi: 10.1053/gast.2002.34233.

Gausserès, N. et al. (1996) ‘The gastro-ileal digestion of 15N-labelled pea nitrogen in adult

humans’, British Journal of Nutrition, 76, pp. 75–85. doi: 10.1079/BJN19960010.

Gausserès, N. et al. (1997) ‘[15 N]-Labeled Pea Flour Protein Nitrogen Exhibits Good Ileal

Digestibility and Postprandial Retention in Humans’, Nutrient Metabolism, 127(6), pp. 1160–

1165.

GEPV (2019) Les protéines végétales en plein essor !

Gerbens-leenes, P. W., Nonhebel, S. and Krol, M. S. (2010) ‘Food consumption patterns

and economic growth . Increasing affluence and the use of natural resources’, Appetite, 55, pp.

597–608. doi: 10.1016/j.appet.2010.09.013.

German, B., Damodaran, S. and Kinsella, J. E. (1982) ‘Thermal Dissociation and

Association Behavior of Soy Proteins’, Journal of Agricultural and Food Chemistry, 30(5), pp.

807–811. doi: 10.1021/jf00113a002.

Gilani, G. S., Xiao, C. X. and Cockell, K. A. (2012) ‘Impact of Antinutritional Factors in

Food Proteins on the Digestibility of Protein and the Bioavailability of Amino Acids and on

Protein Quality’, British Journal of Nutrition, 108(S2), pp. S315–S332. doi:

10.1017/S0007114512002371.

González, A. D., Frostell, B. and Carlsson-Kanyama, A. (2011) ‘Protein efficiency per unit

energy and per unit greenhouse gas emissions : Potential contribution of diet choices to climate

change mitigation’, Food Policy, 36, pp. 562–570. doi: 10.1016/j.foodpol.2011.07.003.

Han, Fei et al. (2018) ‘Digestible indispensable amino acid scores of nine cooked cereal

grains’, Brit. J. Nutr. doi: 10.1017/S0007114518003033.

Hennig, U. et al. (2004) ‘Relative ileal amino acid flows and microbial counts in intestinal

effluents of Goettingen Minipigs and Saddleback pigs are not different’, Journal of animal

18
science, 82(7), pp. 1976–1985.

Hess, V. et al. (2000) ‘Comparison of the isotope dilution method for determination of the

ileal endogenous amino acid losses with labelled diet and labelled pigs’, British Journal of

Nutrition, 83(2), pp. 123–130.

Hodgkinson, S. M. et al. (2000) ‘The effect of dietary peptide concentration on endogenous

ileal amino acid loss in the growing pig’, Brit. J. Nutr., 83, pp. 421–430.

Hodgkinson, S. M. et al. (2018) ‘Cooking Conditions Affect the True Ileal Digestible

Amino Acid Content and Digestible Indispensable Amino Acid Score (DIAAS) of Bovine Meat

as Determined in Pigs’, The Journal of Nutrition, 148, pp. 1564–1569. doi: 10.1093/jn/nxy153.

Jansman, A. J., Frohlich, A. A. and Marquardt, R. R. (1994) ‘Production of proline-rich

proteins by the parotid glands of rats is enhanced by feeding diets containing tannins from faba

beans (Vicia faba L.)’, The Journal of Nutrition, 124(2), pp. 249–258.

Kashyap, S. et al. (2018) ‘Ileal digestibility of intrinsically labeled hen’s egg and meat

protein determined with the dual stable isotope tracer method in Indian adults’, The American

Journal of Clinical Nutrition. Oxford University Press, 108(5), pp. 980–987.

Li, S., Sauer, W. C. and Caine, W. R. (1998) ‘Response of Nutrient Digestibilities to

Feeding Diets with Low and High Levels of Soybean Trypsin Inhibitors in Growing Pigs’, J

Sci Food Agric, 76, pp. 357–363.

Mariotti, F. et al. (2001) ‘The Influence of the Albumin Fraction on the Bioavailability and

Postprandial Utilization of Pea Protein Given Selectively to Humans’, The journal of nutrition,

131(6), pp. 1706–1713.

Mariotti, F., Tomé, D. and Mirand, P. P. (2008) ‘Converting nitrogen into protein - Beyond

6.25 and Jones’ factors’, Critical Reviews in Food Science and Nutrition, 48(2), pp. 177–184.

doi: 10.1080/10408390701279749.

Mathai, J. K., Liu, Y. and Stein, H. H. (2017) ‘Values for digestible indispensable amino

19
acid scores (DIAAS) for some dairy and plant proteins may better describe protein quality than

values calculated using the concept for protein digestibility-corrected amino acid scores

(PDCAAS)’, Brit. J. Nutr., 117, pp. 490–499. doi: 10.1017/S0007114517000125.

Miner-Williams, W., Moughan, P. J. and Fuller, M. F. (2009) ‘Endogenous Components of

Digesta Protein from the Terminal Ileum of Pigs Fed a Casein-Based Diet’, Journal of

Agricultural and Food Chemistry, 57(5), pp. 2072–2078.

Oberli, M. et al. (2015) ‘High true ileal digestibility but not postprandial utilization of

nitrogen from bovine meat protein in humans is moderately decreased by high-temperature,

long-duration cooking’, The Journal of nutrition, 145(10), pp. 2221–2228. doi:

10.3945/jn.115.216838.contrast.

Peng, X., Ren, C. and Guo, S. (2016) ‘Particle formation and gelation of soymilk: Effect of

heat’, Trends in Food Science and Technology. Elsevier Ltd, 54, pp. 138–147. doi:

10.1016/j.tifs.2016.06.005.

Plaisancié, P. et al. (2013) ‘A novel bioactive peptide from yoghurts modulates expression

of the gel-forming MUC2 mucin as well as population of goblet cells and Paneth cells along

the small intestine ☆’, The Journal of Nutritional Biochemistry. Elsevier Inc., 24(1), pp. 213–

221. doi: 10.1016/j.jnutbio.2012.05.004.

Reynaud, Y. et al. (2020) ‘Temporal changes in postprandial intragastric pH: comparing

measurement methods, food structure effects, and kinetic modelling’, Food Research

International. Elsevier Ltd. doi: 10.1016/j.foodres.2019.108784.

Rowan, A. M. et al. (1994) ‘Comparison of the ileal and faecal digestibility of dietary amino

acids in adult humans and evaluation of the pig as a model animal for digestion studies in man’,

Br. J. Nutr., 71, pp. 29–42.

Rutherfurd, S. M. et al. (2015) ‘Protein Digestibility-Corrected Amino Acid Scores and

Digestible Indispensable Amino Acid Scores Differentially Describe Protein Quality in

20
Growing Male Rats’, The Journal of Nutrition, 145(2), pp. 372–379. doi:

10.3945/jn.114.195438.fecal.

Schop, M. et al. (2019) ‘Increasing intake of dietary soluble nutrients affects digesta

passage rate in the stomach of growing pigs’, British Journal of Nutrition, 121(5), pp. 529–537.

Thorning, T. K. et al. (2017) ‘Whole dairy matrix or single nutrients in assessment of health

effects: current evidence and knowledge gaps’, The American journal of clinical nutrition,

105(5), pp. 1033–1045. doi: 10.3945/ajcn.116.151548.Am.

Vasconcelos, I. M. and Oliveira, T. A. (2004) ‘Antinutritional properties of plant lectins’,

Toxicon, 44, pp. 385–403. doi: 10.1016/j.toxicon.2004.05.005.

De Vrese, M. et al. (2000) ‘Protein-Bound D -Amino Acids , and to a Lesser Extent

Lysinoalanine, Decrease True Ileal Protein Digestibility in Minipigs as Determined with 15N-

labeling’, The journal of nutrition, 130(8), pp. 2026–2031.

21
Table 1 Dry matter (DM), crude protein (CP), and amino acid (AA) contents in seitan, tofu, soya milk and
pea emulsion.
Pea
Seitan Tofu Soya milk
emulsion
Dry matter (%; as-fed basis) 76.72 46.03 17.49 15.99
Crude protein (N x 6.25) (% DM) 20.93 23.71 21.83 16.79
Indispensable amino acids (% DM)
Histidine 0.47 0.63 0.61 0.53
Isoleucine 0.76 1.13 1.07 0.80
Leucine 1.42 1.85 1.69 1.44
Lysine 0.29 1.34 1.22 1.18
Methionine 0.39 0.27 0.29 0.08
Phenylalanine 1.03 1.24 1.12 0.94
Threonine 0.46 0.85 0.82 0.65
Tryptophan 0.57 0.55 0.57 0.51
Valine 0.78 1.12 1.04 0.81
Total indispensable AAs (% DM) 6.16 8.97 8.42 6.95
Dispensable amino acids (% DM)
Arginine 0.26 0.51 0.55 0.45
Alanine 0.54 0.93 0.83 0.71
Aspartic acid/Asparagine 0.61 2.52 2.36 1.91
Cysteine 0.40 0.30 0.35 0.15
Glutamic acid/Glutamine 6.76 4.09 3.81 2.55
Glycine 0.63 0.92 0.85 0.66
Proline 2.48 1.31 1.28 0.90
Serine 0.83 1.06 0.97 0.79
Tyrosine 0.49 0.66 0.60 0.53
Total dispensable AAs (% DM) 13.00 12.29 11.60 8.65
Total AAs (% DM) 19.16 21.26 20.02 15.60
Lysine (% CP) 1.39 5.65 5.59 7.03
Methionine + cysteine (% CP) 3.78 2.40 2.93 1.37

22
Table 2 Apparent ileal digestibility (AID) of amino acids in seitan, tofu, soya milk and pea emulsion in
minipigs. Values are means ± SEM, n = 6.a
Pea
Seitan Tofu Soya milk P
emulsion
N, % 74.1 ± 4.6 56.5 ± 6.4 71.3 ± 2.5 68.0 ± 3.5 NS
Indispensable amino acids, %
Histidine 82.1 ± 4.7 81.3 ± 2.9 82.7 ± 3.7 81.8 ± 2.5 NS
Isoleucine 83.6 ± 3.4 81.0 ± 2.4 81.0 ± 3.4 84.7 ± 2.0 NS
Leucine 84.7 ± 3.2 80.4 ± 3.0 79.3 ± 3.1 84.8 ± 2.0 NS
Lysine 54.3b ± 8.9 83.0a ± 3.1 84.8a ± 2.9 86.6a ± 2.0 0.001
Methionine 90.1a ± 1.9 69.4c ± 5.6 89.7a ± 2.8 49.3b ± 10.2 0.001
Phenylalanine 85.2 ± 3.0 81.1 ± 3.0 82.3 ± 2.9 84.7 ± 1.7 NS
Threonine 49.8 ± 10.6 58.5 ± 6.4 67.9 ± 4.8 66.0 ± 3.7 NS
Tryptophan 75.3 ± 8.5 77.4 ± 2.4 84.3 ± 2.4 83.6 ± 2.6 NS
Valine 78.9 ± 4.3 75.4 ± 3.3 78.1 ± 3.5 80.3 ± 3.2 NS
Dispensable amino acids, %
Arginine 55.7 ± 9.4 61.9 ± 6.0 67.1 ± 8.2 75.4 ± 3.0 NS
Alanine 69.6 ± 6.9 72.4 ± 4.6 74.6 ± 3.8 79.1 ± 2.6 NS
b a a a
Aspartic acid/Asparagine 55.1 ± 9.9 78.0 ± 3.8 80.1 ± 3.9 75.7 ± 3.2 0.024
Cysteine 74.1a ± 3.9 35.8b ± 10.2 78.5a ± 3.8 44.4b ± 11.3 0.004
Glutamic acid/Glutamine 94.9a ± 1.0 86.6b ± 2.0 85.8b ± 3.6 84.3b ± 2.5 0.019
Glycine 45.0 ± 9.7 52.6 ± 5.5 68.5 ± 5.2 56.9 ± 6.5 NS
Proline 89.6 ± 3.2 79.7 ± 4.4 81.9 ± 4.0 82.7 ± 2.8 NS
Serine 80.4 ± 3.9 74.1 ± 4.4 77.6 ± 3.6 78.8 ± 2.4 NS
Tyrosine 79.4 ± 4.9 76.1 ± 3.8 79.0 ± 3.6 86.0 ± 3.1 NS
Total Amino Acids 83.7 ± 3.4 77.1 ± 3.3 80.2 ± 3.6 79.9 ± 2.5 NS
a
Different uppercase letter in the same line show significant differences (P < 0.05) among the different
food types for nireogen (N) and amino acids.

23
Table 3 Standard ileal digestibility (SID) of amino acids in seitan, tofu, soya milk and pea emulsion in
minipigs. Values are means ± SEM, n = 6. a
Pea
Seitan Tofu Soya milk P
emulsion
N x 6.25, % 94.0 ± 4.6 76.0 ± 6.4 92.2 ± 2.5 97.4 ± 3.5 NS
Indispensable amino acids, %
Histidine 90.1 ± 4.7 87.9 ± 2.9 89.5 ± 3.7 90.2 ± 2.5 NS
Isoleucine 91.2 ± 3.4 86.6 ± 2.4 86.9 ± 3.4 93.1 ± 2.0 NS
Leucine 91.9 ± 3.2 86.6 ± 3.0 86.0 ± 3.1 93.2 ± 2.0 NS
Lysine 76.4 ± 8.9 88.2 ± 3.1 90.5 ± 2.9 93.0 ± 2.0 NS
Methionine 94.0a ± 1.9 75.7b ± 5.5 95.5 ± 2.8a 71.3b ± 10.2 0.013
Phenylalanine 93.8 ± 3.0 89.0 ± 3.0 91.0 ± 2.9 95.8 ± 1.7 NS
Threonine 74.7 ± 10.6 73.5 ± 6.4 83.1 ± 4.8 86.9 ± 3.7 NS
Tryptophan 87.3 ± 8.5 91.3 ± 2.4 97.5 ± 2.4 99.7 ± 2.6 NS
Valine 89.7 ± 4.3 83.8 ± 3.3 87.0 ± 3.5 92.5 ± 3.2 NS
Dispensable amino acids, %
Arginine 80.9 ± 9.4 76.1 ± 6.0 80.2 ± 8.2 92.5 ± 3.0 NS
Alanine 85.7 ± 6.9 82.7 ± 4.6 86.0 ± 3.8 93.5 ± 2.6 NS
Aspartic acid/Asparagine 78.3 ± 9.9 84.3 ± 3.8 86.7 ± 3.9 84.5 ± 3.2 NS
Cysteine 92.1a ± 3.9 62.8b ± 10.2 101.2a ± 3.8 101.9a ± 11.3 0.011
Glutamic acid/Glutamine 97.8 ± 1.0 92.0 ± 2.0 91.6 ± 3.6 93.5 ± 2.5 0.019
Glycine 74.8 ± 9.7 75.0 ± 5.5 92.6 ± 5.2 90.5 ± 6.5 NS
Proline 94.2 ± 3.2 89.5 ± 4.4 91.7 ± 4.0 97.8 ± 2.8 NS
Serine 91.4 ± 3.9 83.7 ± 4.4 88.0 ± 3.6 92.5 ± 2.4 NS
Tyrosine 88.5 ± 4.9 83.6 ± 3.8 87.0 ± 3.6 95.9 ± 3.1 NS
Total Amino Acids 92.3 ± 3.4 86.0 ± 3.3 89.3 ± 3.6 92.6 ± 2.5 NS
a
Different uppercase letter in the same line show significant differences (P < 0.05) among the different
food types for protein (N x 6.25) and amino acids.

24
Table 4 True ileal digestibility (TID) of amino acids in seitan, tofu, soya milk and pea emulsion in
minipigs. Values are means ± SEM, n = 6. a
Pea
Seitan Tofu Soya milk P
emulsion
Leuendo/Leutotal ileum (%)b 71.6a ± 7.7 67.0a ± 7.0 46.1c ± 4.8 62.4a,b ± 5.3 0.025
Leuendo/Leuintake (%)c 11.7 ± 3.4 12.8 ± 2.0 9.0 ± 0.8 9.7 ± 1.8 NS
TID, %
Indispensable amino acids, %
Histidine 95.6 ± 1.1 95.7 ± 2.1 92.2 ± 3.2 91.8 ± 1.2 NS
Isoleucine 96.1a ± 1.3 92.9a,b ± 1.7 89.1b ± 2.9 94.6a ± 1.0 0.035
Leucine 96.4a ± 1.0 93.3a ± 1.9 88.3b ± 2.6 94.5a ± 0.9 0.009
Lysine 90.1 ± 5.3 93.9 ± 2.1 92.5 ± 2.4 93.9 ± 1.2 NS
Methionine 97.8a ± 1.1 85.6b ± 6.0 99.3a ± 2.5 80.3b ± 7.7 0.034
Phenylalanine 99.9 ± 2.0 98.6 ± 2.2 94.7 ± 2.3 98.3 ± 1.3 NS
Threonine 94.1 ± 5.3 93.0 ± 4.5 90.6 ± 3.9 92.4 ± 2.7 NS
Tryptophan 92.2 ± 4.6 102.6 ± 2.4 99.8 ± 2.1 99.6 ± 3.5 NS
Valine 96.8 ± 1.9 93.3 ± 2.2 90.4 ± 2.9 94.8 ± 1.4 NS
Dispensable amino acids, %
Arginine 94.7 ± 4.8 90.4 ± 4.3 84.0 ± 7.1 94.2 ± 1.5 NS
Alanine 96.7 ± 3.1 94.7 ± 3.2 90.6 ± 3.1 96.3 ± 1.3 NS
Aspartic acid/Asparagine 92.3 ± 3.4 91.0 ± 2.9 88.9 ± 3.5 85.7 ± 2.0 NS
Cysteine 99.0 ± 5.6 83.8 ± 11.7 104.6 ± 4.2 100.8 ± 9.9 NS
Glutamic acid/Glutamine 99.6 ± 0.6 97.7 ± 1.5 93.4 ± 3.2 94.8 ± 1.0 NS
Glycine 87.9 ± 10.2 94.3 ± 4.6 97.4 ± 4.3 91.3 ± 7.1 NS
Proline 98.2 ± 1.1 103.2 ± 3.4 97.1 ± 3.2 102.7 ± 3.0 NS
Serine 99.8 ± 2.5 95.9 ± 3.4 92.8 ± 3.0 96.0 ± 1.6 NS
Tyrosine 94.4 ± 1.5 92.0 ± 2.8 90.1 ± 3.0 97.7 ± 2.5 NS
Total Amino Acids 97.5 ± 1.5 95.0 ± 2.3 92.3 ± 3.0 94.2 ± 1.3 NS
a
Different uppercase letter in the same line show significant differences (P < 0.05) among the different
food types for amino acids.
b
Percent of leucine of endogenous origin in leucine flowing at the ileum
c
Endogenous leucine flowing at the ileum relative to ingested leucine

25
Table 5 Digestible indispensable amino acids (g/ 100 AA) in seitan, tofu, soya milk and pea emulsion
in minipigs. Values are means ± SEM, n = 6. a
Pea
Seitan Tofu Soya milk P
emulsion
Histidine 1.34c ± 0.03 5.91b ± 0.06 5.65b ± 0.10 7.10a ± 0.04 0.001
Isoleucine 3.79b ± 0.05 4.94a ± 0.09 4.74a ± 0.16 4.85a ± 0.05 0.001
Leucine 7.15c ± 0.07 8.13b ± 0.17 7.46c ± 0.22 8.74a ± 0.08 0.001
Lysine 1.34c ± 0.08 5.91b ± 0.13 5.65b ± 0.15 7.10a ± 0.09 0.001
Methionine 2.00a ± 0.02 1.07c ± 0.07 1.42b ± 0.04 0.42d ± 0.04 0.001
Cysteine 2.08a ± 0.12 1.17b ± 0.16 1.82a ± 0.07 0.96b ± 0.09 0.001
Phenylalanine 5.37b ± 0.11 5.76a ± 0.13 5.30b ± 0.13 5.95a ± 0.08 0.001
Tyrosine 2.40c ± 0.04 2.84b ± 0.09 2.70b ± 0.09 3.30a ± 0.08 0.001
Threonine 2.27b ± 0.13 3.71a ± 0.18 3.73a ± 0.16 3.86a ± 0.11 0.001
Tryptophan 2.73b ± 0.13 2.63b ± 0.06 2.82b ± 0.06 3.23a ± 0.11 0.002
b a a a
Valine 3.95 ± 0.08 4.90 ± 0.12 4.70 ± 0.15 4.95 ± 0.07 0.001
a
Different uppercase letter in the same line show significant differences (P < 0.05) among the different
food types for amino acids.

26
Table 6 Digestible indispensable amino acid scores (DIAAS, %), and limiting amino acid for seitan,
tofu, soya milk and pea emulsion.
Seitan Tofu Soya milk Pea emulsion
Infant (birth to 6 months) 19 68 78 42
(Lys) (SAAs) (Leu) (SAAs)
Child (6 months to 3 years) 24 83 99 51
(Lys) (SAAs) (Lys) (SAAs)
Older child, adolescent, adult 28 97 117 60
(Lys) (SAAs) (Val) (SAAs)
Scores were calculated using the recommended amino acid scoring patterns for three age groups
(FAO, 2013).

27
 Table S1 Composition of test-meals (g, as fed)

Soya milk Pea emulsion

Seitan meal Tofu meal
meal meal
Seitan (dehydrated) 42.1 - - -
Tofu (dehaydrated) - 68.1 - -
Soya milk - - 1010.1 -
Pea emulsion - - - 1010.1
Maltodextrine 129.6 132.6 120.0 133.0
Sugar 30 30 30 30
Soybean oil 20.8 2.1 2.1
Water 67.4 204.6 - -

28
Table S2 N and amino acids ileal flows observed in minipigs (n = 6) receiving a protein-free
diet. Values are means ± SEM.
Ileal flow, g
N (Dumas method) 1.82 ± 0.51
Indispensable amino acids
Histidine 0.09 ± 0.02
Isoleucine 0.13 ± 0.04
Leucine 0.24 ± 0.06
Lysine 0.14 ± 0.04
Methionine 0.04 ± 0.01
Phenylalanine 0.22 ± 0.06
Threonine 0.29 ± 0.09
Tryptophan 0.14 ±0.04
Valine 0.20 ± 0.05
Dispensable amino acids
Arginine 0.14 ± 0.04
Alanine 0.38 ± 0.10
Aspartic acid/Asparagine 0.31 ± 0.09
Cysteine 0.14 ± 0.04
Glutamic acid/Glutamine 0.45 ± 0.12
Glycine 0.38 ± 0.10
Proline 0.30 ± 0.08
Serine 0.23 ± 0.06
Tyrosine 0.10 ± 0.03
Total amino acids 3.72 ± 1.01

29