The Journal of Nutrition
Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions
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
1–3
Marion Oberli,
4,5Agne`s Marsset-Baglieri,
4,5Gheorghe Airinei,
4,5V´eronique Sant´e-Lhoutellier,
6Nadezda Khodorova,
4,5Didier R´emond,
7,8Ang´elique Foucault-Simonin,
4,5Julien Piedcoq,
4,5Daniel Tom´e,
4,5Gilles Fromentin,
4,5Robert Benamouzig,
4,5and Claire Gaudichon
4,5*
4French National Institute for Agricultural Research (INRA) and5AgroParisTech, Research Center for Human Nutrition Ilde de France,
UMR914, Nutrition Physiology and Ingestive Behavior, Paris, France;6French National Institute for Agricultural Research (INRA),
UR370 Quality of Animal Products, Saint Gene`s Champanelle, France;7French National Institute for Agricultural Research (INRA), Unit
of Human Nutrition, Research Center for Human Nutrition Auvergne, Clermont-Ferrand, France; and8Human Nutrition Unit,
Clermont University, University d’Auvergne, Clermont-Ferrand, France
Abstract
Background: Meat protein digestibility can be impaired because of indigestible protein aggregates that form during cooking. When the aggregates are subsequently fermented by the microbiota, they can generate potentially harmful compounds for the colonic mucosa. Objective: This study evaluated the quantity of bovine meat protein escaping digestion in the human small intestine and the metabolic fate of exogenous nitrogen, depending on cooking processes.
Methods: Sixteen volunteers (5 women and 11 men; aged 286 8 y) were equipped with a double lumen intestinal tube positioned at the ileal level. They received a test meal exclusively composed of 120 g of intrinsically15N-labeled bovine
meat, cooked either at 55°C for 5 min (n = 8) or at 90°C for 30 min (n = 8). Ileal effluents and blood and urine samples were collected over an 8-h period after the meal ingestion, and15N enrichments were measured to assess the digestibility of
meat proteins and the transfer of dietary nitrogen into the metabolic pools.
Results: Proteins tended to be less digestible for the meat cooked at 90°C for 30 min than at 55°C for 5 min (90.1% 6 2.1% vs. 94.1%6 0.7% of ingested N; P = 0.08). However, the particle number and size in ileal digesta did not differ between groups. The appearance of variable amounts of intact fibers was observed by microscopy. The kinetics of15N
appearance in plasma proteins, amino acids, and urea were similar between groups. The amount of exogenous nitrogen lost through deamination did not differ between groups (21.2%6 0.8% of ingested N).
Conclusions: Cooking bovine meat at a high temperature for a long time can moderately decrease protein digestibility compared with cooking at a lower temperature for a short time and does not affect postprandial exogenous protein metabolism in young adults. The study was registered at www.clinicaltrials.gov as NCT01685307. J Nutr 2015;145:2221–8.
Keywords:
digestion, stables isotopes, postprandial, protein metabolism, healthy volunteersIntroduction
Meat is an important nutritional source of high-quality proteins
in terms of indispensable amino acid composition, but cooking
processes that act on the macrostructure and microstructure
of meat may alter meat quality by decreasing protein digestion.
During cooking, an accumulation of oxidative damage occurs
in proteins (by an increase in free radical production and an
impairment of the antioxidant protection), leading to protein
aggregation through disulfide and dityrosine bridges and carbonyl
formation (1–6). Interactions between proteins and aldehydic
products of lipid oxidation can also initiate protein aggregation
with the formation of Schiff bases (7, 8).
The formation of protein aggregates may reduce meat protein
proteolysis by digestive enzymes. A dramatic decrease of in
vitro digestibility of myofibrillar proteins from bovine muscles
was observed when cooking time was long (100°C, 45 min), in
1
Supported by a grant from Region Ile de France and the French Agency for Research and Technology (Program Pronutrial). The authors are participants in the EU-funded COST action INFOGEST (COST FA 1005).
2
Author disclosures: M Oberli, A Marsset-Baglieri, G Airinei, V Sant ´e-Lhoutellier, N Khodorova, D R ´emond, A Foucault-Simonin, J Piedcoq, D Tom ´e, G Fromentin, R Benamouzig, and C Gaudichon, no conflicts of interest.
3
Supplemental Figures 1–3 are available from the ‘‘Online Supporting Material’’ link in the online posting of the article and from the same link in the online table of contents at http://jn.nutrition.org.
* To whom correspondence should be addressed. E-mail: claire.gaudichon@ agroparistech.fr.
ã 2015 American Society for Nutrition.
Manuscript received May 11, 2015. Initial review completed May 27, 2015. Revision accepted July 16, 2015. 2221
contrast to a short cooking (100°C for 5 min or 270°C for
1 min) (4, 9). Another in vitro digestibility study of myofibrillar
proteins from pig muscles suggested that protein digestion
efficiency and speed may be affected by heat treatments (10).
One in vivo study in minipigs (11) reported that the true ileal
digestibility of meat proteins was not influenced by cooking
temperature, whereas their speed of digestion was modified. To
our knowledge, the only study that has assessed the protein
digestibility of meat in humans was conducted in 5 ileostomized
patients (12). A value of 94% was reported, but it may not be
extrapolated to healthy humans.
The decrease of meat protein digestibility by cooking
pro-cesses may not only reduce the bioavailability of indispensable
amino acids but also increase the quantity of protein that reaches
the large intestine. Proteins that escape digestion are fermented by
the colon microbiota and release potentially harmful metabolites,
such as hydrogen disulfide, ammonia, and phenolic compounds.
These metabolites are believed to have deleterious effects on colon
mucosa (13–15) and could be involved in the suspected role of
red and processed-meat intake in colorectal cancer risk and
overall mortality (9, 16–19), although this relation is controversial
(20–22).
The present study aimed to more precisely evaluate the
im-pact of cooking on meat protein digestibility and metabolic fate.
For this purpose,
15N-labeled bovine meat was cooked
accord-ing to 2 different time and temperature couples: a low
temper-ature and short cooking time to obtain a rare meat (RM)
9and
a high temperature and longer cooking time to obtain a fully
cooked meat (FCM). The fate of
15N-labeled protein
(exoge-nous) products was specifically followed and quantified in the
ileum and different body metabolic pools in healthy volunteers
with the use of an intestinal perfusion technique.
Methods
Subjects.All volunteers were certified as being in good health after routine blood tests and thorough medical examination performed by the medical staff of the Human Nutrition Research Centre of Avicenne Hospital (Bobigny, France). The eligibility criteria were negative serol-ogy for HIV, surface antigen of the hepatitis B virus, and hepatitis C virus; absence of any disease; absence of pregnancy or contraception for women; BMI (in kg/m2) between 18 and 30; and age between 18 and
50 y. All subjects received detailed information on the purpose and potential risks of the protocol and gave written, informed consent for their participation. The study was approved by the Ethical Committee of St-Germain-en-Laye Hospital and was registered at www.clinicaltrials.gov as NCT01685307.
This single-blind study was conducted according to a 2-arm parallel design at the Avicenne Hospital. Twenty-four volunteers were recruited from May 2012 to June 2013, and recruitment was stopped when the groups consisted of at least 8 subjects. Sample size was determined from a power calculation that was based on data in the literature (23–25), using ileal digestibility as the main criteria. The study was eventually performed on 16 subjects because of various difficulties with the toler-ance of the tube (failure of the tube to fit through the nose, pain, vomiting, nonmigration through the pylorus). Subject characteristics are presented in Table 1. Subjects were alternately allocated to 1 of the 2 groups [meat cooked at 55°C for 5 min (RM) or meat cooked at 90°C for 30 min (FCM)] to avoid any random effect because of the season (such as the usual diet of the volunteer). However, the criteria used to assign the last partic-ipants of the study to the RM or FCM group were sex, age, and BMI to have similar subject characteristics in both groups.
Test meals.The protein test meal consisted of 120 g (305 mmol nitro-gen; i.e., 27 g protein) of intrinsically15N-labeled bovine meat
(enrich-ment: 1.39 atom%). The15N-labeled meat was obtained by infusing
(15NH
4)SO4in a calf rumen at the National Institute for Agricultural
Research (UMRH 1213 Herbivores) and which enabled us to differen-tiate between nitrogen that originated from the meal and endogenous nitrogen. Meat was then cooked in a steam oven at either 55°C for 5 min (RM) or 90°C for 30 min (FCM) at the ADIV (technical center for the meat sector Clermont-Ferrand, France), minced, vacuum-packed, and stored at220°C until use. Meat was minced to limit the effect of individual chewing efficiency, which was shown to significantly affect meat protein digestion rate, and postprandial protein metabolism in the elderly (26).
Oxidative and conformational protein modifications were measured in cooked meat. The carbonyl content is indicative of protein oxidation. Carbonyl groups are formed from the modification of secondary amine function of basic amino acids (lysine, arginine, and histidine) and can decrease the ability of digestive enzymes to recognize proteins. The protein surface hydrophobicity, an indicator of protein denaturation, is related to aggregation. Both were evaluated by spectrophotometry as previ-ously described (14). The carbonyl content was similar between groups (5 nmol 2,4-dinitrophenylhydrazine/mg protein). In contrast, the hydro-phobicity was significantly higher in FCM (35–37mg bound chromophore bromophenol blue) than in RM (29–30mg bound bromophenol blue) (P = 0.02).
Before the experiment, meat was defrosted in plastic bags under water at 40°C for 30 min. Fifty milligrams of13C-labeled inulin,
enriched at 97% (Isolife), and 450 mg nonenriched inulin were incor-porated into the meat as a nonabsorbable marker of the meal and 0.5 g salt. Meat was then finally warmed in a pan for 15 s on each side. Clinical protocol.During the week before the experiment, the subjects had to follow a standard diet adapted to their body weight to control their protein intake (1.4 g protein/kg body weight), as previously described (27).
The subjects arrived at the hospital the morning before the ex-periment in a fasted state. A double lumen intestinal tube was passed through the nose under local anesthesia and was allowed to progress through the digestive tract for 24 h as previously described (28). The subjects were given meals at 1200 and 2000, and then they fasted overnight. On the day of the experiment, the position of the intestinal tube was checked by radiography to verify its location at the terminal ileum. A catheter was inserted in the forearm vein for blood sampling. A saline solution that contained polyethylene glycol 4000 (PEG-4000; 20 g/L), used as a nonabsorbable marker of the intestinal flow, was perfused into the ileum at a flow rate of 1 mL/min to calculate the flow rate of the intestinal effluents. After a basal blood and urine sampling and a basal collection of ileal effluents performed for 30 min, subjects had 10 min to eat their meal and were not allowed to ingest food until the end of the experiment. They were given a glass of water every hour. The postprandial sampling period lasted for 8 h after the meal ingestion. The intestinal content was continuously collected over ice by aspiration, 20 cm distally from the PEG perfusion site, and pooled every 30 min, treated with diisoropylfluorophosphate as a protease inhibitor, frozen at 220°C, and freeze-dried until analysis. Blood was sampled every 30 min for 4 h and hourly thereafter. Blood samples were immediately centrifuged, and the supernatant fluid was frozen at220°C until analysis. Total urine
TABLE 1 Characteristics of young adult subjects who ingested RM or FCM1 55°C, 5 min (RM) 90°C, 30 min (FCM) Sex, F/M 2/6 3/5 Age, y 31.0 6 9.9 24.9 6 4.1 BMI, kg/m2 23.7 6 3.4 23.2 6 3.8 TBW, L 40.8 6 7.3 38.3 6 8.4
1Values are means6 SDs, n = 8. FCM, fully cooked meat; RM, rare meat; TBW, total body water.
9
Abbreviations used: APE, atom percent excess; FCM, fully-cooked meat; GIP, glucose-dependent insulinotropic peptide; NPPU, net postprandial protein utilization; PEG, polyethylene glycol; RID, real ileal digestibility; RM, rare meat.
was collected every 2 h. Urine was divided into aliquots that were either treated with thymol oil and liquid paraffin as preservatives and kept at 4°C or directly frozen at 220°C until analysis. Body composition was predicted with the use of bioelectrical impedance analysis.
Analytical methods.The concentration of PEG-4000 in the ileal samples was determined by a turbidimetric method (28, 29) to determine the ileal flow rate.
Plasma glucose was assayed by a glucose oxidase method (Glucose RTU kit; BioM´erieux). Urea concentrations were measured in plasma and urine by using an urease-glutamate dehydrogenase technique (urea kit; BioM´erieux).
Plasma insulin and glucagon, which are known to respond to protein intake (30), and glucose-dependent insulinotropic peptide (GIP) con-centrations were simultaneously determined with the use of an endo-crine kit panel (Bio-Plex Pro Assay; Bio-Rad Laboratories Inc.) on a Bioplex 200 system (Bio-Rad Laboratories Inc.). GIP is an insulinotropic factor and can also be involved in gastric emptying control (31).
Plasma concentrations of amino acids were determined by ion ex-change chromatography on deproteinized samples, with the addition of norleucine as an internal standard (Hitachi L8900; ScienceTec).
For isotopic determination, urinary urea and ammonia and plasma urea and free amino acids were isolated with the use of a sodium and potassium form of a cation exchange resin (Biorad Dowex AG50-X8, mesh 100–200; Interchim) as described previously (23, 24).
Total nitrogen and15N enrichment of samples were determined by
using isotope ratio MS (Isoprime; GV Instrument) coupled with an elemental nitrogen analyzer (Vario L3; Elementar), with atropine and glutamic acid as elemental and isotopic standards, respectively, as previously described (32). Enrichment was expressed as atom percent excess (APE). Total carbon content and13C enrichment of ileal
efflu-ents were similarly measured, with cyclohexanone and glutamic acid as elemental and isotopic standards, respectively.
Granulometry measurements on the effluents were performed with the Sysmex flow particle image analyzer (FPIA-3000; Malvern Instru-ments Ltd.) as previously described (3) to assess aggregation in the ileal effluents. Approximately 50 mg lyophilized ileal samples were rehy-drated for 1 h in 3 mL Tris-HCl 0.1 M (pH = 8) and homogenized. Then, 750mL of sample was injected into the granulometer where it was dispersed in an electrolytic sheath solution, then submitted to a light pulse to take images of all particles. Two measurements were completed for each sample. Particle shapes (circularity), numbers, and sizes were thus evaluated with an automated imaging technique, as previously described (3). We also performed a qualitative analysis of the images from subjects with the highest digestibility in the RM group and the lowest digestibility in the FCM group, by selecting the 100 biggest particles in each sample, to assess the number of undigested meat fibers. Calculation.The flow rate in the ileum was evaluated for each 30-min period from the dilution of PEG-4000 between the perfusion solution and the ileal effluents collected through the intestinal tube and was corrected for the perfusion flow rate (1 mL/min), as previously described (32).
The total nitrogen flow rate (Ntot-ileum, in mmol) in the ileum for each
period was calculated as follows (equation1): Ntot-ileum¼N
sð%Þ 3 DMs3 F
143 10 ð1Þ
whereNs(%) is the nitrogen percentage measured in the freeze-dried
ileal sample, DMsis the dry matter of the sample (g/100 g),F is the flow
rate in the ileum, and 14 is the nitrogen molar mass.
The exogenous nitrogen flow rate (Nexo-ileum, in mmol) was evaluated
as follows (equation2):
Nexo-ileum¼ Ntot-ileum3
APEeffluents
APEmeal ð2Þ
where APEeffluents and APEmeal are the15N enrichment excess in the
effluents and in the meal, respectively.
Endogenous nitrogen (Nendo-ileum, in mmol), mainly digestive
en-zymes, mucoproteins, and desquamated epithelial cells (33), was con-sequently calculated as follows:Nendo-ileum=Ntol-ileum2 Nexo-ileum.
The real ileal digestibility (RID; in % of ingested nitrogen) was then calculated from the cumulated exogenous nitrogen collected in the ileum over 8 h and normalized for the recovery of the nonabsorbable marker (13C inulin) as follows (equation3):
RID¼ ðNing2ð+Nexo-ileumÞ=R13C3 100Þ=Ning3 100 ð3Þ
whereNingis the quantity of nitrogen in the meal (in mmol),SNexo-ileum
is the cumulative amount of exogenous nitrogen collected in the ileum over 8 h (mmol), andR13Cis the percentage of recovery of13C inulin,
used as a nonabsorbable marker, in the ileum over 8 h.
The time course of exogenous nitrogen incorporation [Nexo(t),
expressed as a percentage of the nitrogen ingested amount/30 min] into the monitored nitrogen pools (serum proteins, body urea, and urinary urea) was derived from the following formula (equation4):
NexoðtÞ ¼ NtotðtÞ 3
APEsðtÞ
APEmeal3 Ningested3 100 ð4Þ
whereNtot(t) is the total nitrogen content of the pool (in mmol) at each
time pointt, APEs(t) is the15N enrichment above baseline of the sample
at timet, APEmealis the15N enrichment excess of the meal, andNingested
is the nitrogen content of the meal (in mmol).
The total nitrogen content in the serum protein pool was determined as the product of the nitrogen concentration of this pool and the serum volume, estimated from NadlerÕs equation for blood volume (34) and the subjectÕs hematocrit (35). The total body urea nitrogen pool was assessed from total body water (assessed with Watson equations), urea concen-trations in plasma, and a correction factor of 92% to take into account the water content of blood, as previously described (23).
The net postprandial protein utilization (NPPU, in % of ingested nitrogen) after 8 h, which is the amount of exogenous nitrogen retained in the body, was determined as follows (equation5):
NPPU¼Ningested2
+
Nexo-ileum2+Nexo urinary urea2Nexo body urea
Ningested 3100 ð5Þ
whereSNexo urinary ureais the cumulative amount of exogenous nitrogen
excreted in urinary urea andNexo body urea is the residual amount of
exogenous nitrogen in body urea at 8 h.
AUCs for plasma amino acids, urea, glucose, and hormones were calculated with the trapezoidal rule.
Statistical analysis.Data are expressed as means6 SEMs. The effect of treatment was analyzed with at test with the TTEST procedure of SAS 9.1 (SAS Institute). For digestibility, a Wilcoxon test was used because of the non-normal distribution [P = 0.003 (Shapiro test) and P = 0.01 (Kolmogorov test)] and a trend for a nonhomogeneity of variance [P = 0.06 (Levene test)]. Kinetics were analyzed with a mixed model with the group as simple factor and time as repeated factor, with the MIXED procedure of SAS 9.1 (SAS Institute). Changes over time of variables in comparison with the baseline value were tested with contrast analysis within the mixed model. Differences were considered statistically significant atP # 0.05.
Results
Nitrogen kinetics and RID of
15N-labeled meat.
The effluent
flow rates in the terminal ileum varied in time (P = 0.03) but
were similar between the RM and FCM groups (Figure 1A). The
total volume of effluents passing through the ileum over the 8-h
period after meat ingestion did not differ between groups and
reached 653
6 190 mL for RM and 747 6 156 mL for FCM.
The use of
15N-labeled meat enabled us to differentiate
between exogenous and endogenous nitrogen in the ileal
efflu-ents. Exogenous nitrogen rapidly reached the ileum after meal
ingestion as quickly as 30 min, and the maximum flow rate was
observed between 1 and 3.5 h after meat ingestion (Figure 1B).
For both groups, flow rates observed between 1 and 3.5 h were
significantly different from the basal value (
P < 0.0001–0.04,
depending on the time point). Over the postprandial period, it
was higher in the FCM group than in the RM group (cooking
effect:
P = 0.04), especially because of a higher flow rate in the
first 3 h.
The flux of endogenous nitrogen also significantly varied with
time (P < 0.0001) and peaked at a mean of 1 h after meat
inges-tion. It then decreased and stayed stable during the remaining
postprandial period. Over the kinetic period, significantly more
endogenous nitrogen was found in the FCM than in the RM group
(P = 0.03).
The cumulative amounts of
15N recovery at the terminal
ileum over 8 h (Figure 2) were 17.6
6 2.1 mmol for the RM
group and 30.8
6 6.6 mmol for the FCM group (cooking effect:
P = 0.08).
Finally, taking into account the quantity of nitrogen ingested
(305 mmol nitrogen), the RID was 94.1%
6 0.7% for RM and
90.1%
6 2.1% for FCM (P = 0.08). The cooking at 90°C for
30 min tended to increase the variance of digestibility (
P = 0.06).
Qualitative analysis of ileal effluents.
The analysis by
granulometry revealed no effect of cooking and time on the
number (4847
6 241), the diameter (10.3 6 0.08 mm), or the
circularity of the particles between the 2 groups (Supplemental
Figure 1). Nevertheless, the examination of particles by
micro-scopy revealed different profiles, depending on the time after
the ingestion and on the subjects. Basal effluents were
charac-terized by the presence of filamentous particles (Figure 3A).
After meat ingestion, structured particles in the form of rods
or blocks appeared at a variable frequency, depending on
subjects and samples. These particles were identified as
un-digested meat fibers. However, we did not observe any
aggre-gates (Figure 3B). In many effluents collected far after meat
ingestion (>4 h), less-structured particles appeared (Figure 3C). The
number of fibers counted over the 8-h sampling period was 100 in
the subject with lowest digestibility (79.2%) and 30 in the subject
with highest digestibility (96.4%).
Exogenous nitrogen transfer to plasma and urinary pools.
The transfer of exogenous nitrogen to proteins and plasma-free
amino acids are shown in Figure 4A and B. The
incorpora-tion kinetics of exogenous nitrogen into plasma proteins were
identical for both cooking processes, and exogenous nitrogen
incorporation increased (P < 0.0001) during the 8-h
postpran-dial period. Eight hours after the ingestion of the meal, the mean
amount of exogenous nitrogen incorporated into plasma
pro-teins was 15.4
6 1.0 mmol, representing ;5% of ingested
nitrogen (Figure 4A). The transfer of exogenous nitrogen to
plasma-free amino acids significantly increased (P < 0.0001) as
soon as 1 h and peaked 2 h after ingestion of RM and 3 h for
FCM, without any significant difference between the groups.
Then, it progressively decreased but remained higher than the
basal value 8 h after the meal (Figure 4B). The transfer of
15N to
body urea was not different between the 2 groups (Figure 4C). It
significantly increased (P < 0.0001) 1 h after meat ingestion,
peaked between 3 and 4 h, and then slowly declined. The
residual amounts of exogenous nitrogen into the body urea pool
8 h after the ingestion of the meal were similar, representing
;11% of ingested nitrogen.
The cumulative amounts of exogenous nitrogen excreted in
urinary urea during the 8-h postprandial period were similar
between the groups (29.8
6 2.9 vs. 31.3 6 2.3 mmol for RM and
FCM, respectively), representing
;10% of total ingested
nitro-gen (data not shown). The postprandial exonitro-genous nitronitro-gen
deamination was calculated as the sum of the total
15N amount
excreted during 8 h and the residual amount of
15N in the urea
body pool. Thus, the quantities of exogenous nitrogen that were
lost through deamination did not differ between groups and
reached 66.6
6 2.9 mmol for RM and 62.2 6 3.7 mmol for
FCM, representing 22.1%
6 0.87% and 20.3% 6 1.39%,
respectively, of total ingested nitrogen.
NPPU of meat protein nitrogen.
The NPPU, calculated from
the RID and the nitrogen percentage ingested retained in the body
over the 8-h postprandial period, was not significantly different
FIGURE 1 Ileal flow rates of effluents (A), and endogenous andexogenous nitrogen (B) in young adults over 8 h after ingestion of RM and FCM. Values are means6 SEMs, n = 8. FCM, fully cooked meat; RM, rare meat.
FIGURE 2 Cumulated recovery of exogenous nitrogen in the ileal effluents of young adults over 8 h after ingestion of RM or FCM15
N-labeled meat. Values are means6 SEMs, n = 8. FCM, fully cooked meat; RM, rare meat. Downloaded from https://academic.oup.com/jn/article/145/10/2221/4590108 by guest on 24 June 2021
between the groups and reached 71.9%
6 1.1% of ingested
nitrogen for RM and 70.2%
6 2.1% for FCM.
Plasma amino acids, glucose, and urea concentrations.
Concentrations of plasma-free amino acids, urea, and glucose
were similar between the RM and FCM groups (Supplemental
Figure 2). A significant effect of time was observed for all
amino acids except taurine, but there were no significant differences
in plasma amino acid concentrations between the RM and FCM
groups. The 8-h AUCs for indispensable amino acids and dispensable
amino acids were similar between the groups (P = 0.73 and P = 0.88,
respectively) (data not shown). Plasma urea concentration
signifi-cantly increased between 2 and 7 h after meal ingestion. Plasma
glucose concentration increased after meal ingestion with a peak
after 2 h, then decreased, and was significantly lower than the initial
value for the last 3 h. The 8-h AUCs for plasma glucose and urea
were identical between the RM and FCM groups (
P = 0.97 and P =
0.67, respectively).
Insulin, glucagon, and GIP concentrations.
Concentrations
of gastrointestinal and pancreatic hormones were also measured in
the blood (Supplemental Figure 3). An effect of time was found
for the 3 hormones, but insulin, glucagon, and GIP secretions after
the meal ingestion were not significantly different between the
RM and FCM groups, and the 8-h AUCs were similar (insulin:
P =
0.77; glucagon:
P = 0.44; GIP: P = 0.41).
Discussion
The aim of this study was to assess bovine meat protein
di-gestibility in healthy volunteers with the use of 2 cooking
treatments that differed in temperature and in time (90
°C for
30 min and 55°C for 5 min) and to assess the metabolic fate
of absorbed exogenous nitrogen. With the use of a precise
tech-nique (ileal tubes and intrinsic labeling of meat with
15N), we
observed high digestibility and nitrogen retention of meat
pro-tein and a trend toward a lower digestibility with cooking at a
high temperature for a long time, without affecting the
subse-quent postprandial utilization of exogenous nitrogen.
Digestibility of meat proteins has rarely been evaluated,
except in 1 study in ileostomized patients (12) that reported a
digestibility of 94%, for meat saut´eed for a short time (8 min).
This value is consistent with what we found and what was
reported in cannulated minipigs (11). Compared with other
protein sources, the digestibility of meat proteins is as high as
milk proteins (28). All other proteins that were evaluated with
similar methods [e.g., eggs (36), soy (37), pea (38), wheat (24)]
had lower ileal digestibility values of
;90–91%.
Cooking meat at a higher temperature for a longer period of
time increased exogenous protein flux in the ileum. As a
con-sequence, it tended to decrease digestibility, but the lowering of
4% was not significant because of an insufficient statistical
power for a summary measure (digestibility) compared with
repeated measure (kinetic of exogenous nitrogen recovery). An a
posteriori analysis shows that the statistical power for
digest-ibility is 0.7 and that 4 additional subjects would have been
necessary to obtain a power of 0.8, but, given the limited
amount of labeled meat and the limited storage time of meat
meals, it was not possible to include more subjects. Moreover,
we found a trend for a higher dispersion of the digestibility value
in the FCM group, which is likely to reflect an impairment of
digestibility. For instance, the study conducted by Bos et al. (23)
on rapeseed protein digestibility revealed that the low
digest-ibility (84%) was associated with a high SD (8.8%), whereas
the SD was lower for other sources of proteins (1% for milk
and 2–4% for soy, pea, and wheat). A similar observation was
reported on raw egg proteins (36), for which the lower
digest-ibility was associated with a higher SD (10%) than for cooked
egg (0.8%).
Although it cannot be directly extrapolated to in vivo
diges-tion, static in vitro studies showed a decrease of myofibrillar
protein digestibility by gastric and pancreatic proteases for long
cooking times, whereas short cooking times did not have any
effect (4). Sant´e-Lhoutellier et al. (4) reported a cumulative
reduction of digestibility (measured after digestion by pepsin
and then by pancreatic proteases), reaching 75% with cooking
at 100
°C for 45 min, whereas digestibility with cooking at
270°C for 1 min was unchanged. Wen et al. (39) also reported a
lower in vitro digestibility with trypsin, and to a higher extent
FIGURE 3 Images of particles in the ileal effluents collected in young adults before the ingestion of the meal (A), 30 min (arrows indicate meat fibers) (B), and 6 h (C) after the ingestion of the meal.with pepsin, when pork meat was cooked at 60°C for 12 min
compared with 100
°C for 3 h. It is not excluded that combining
a high temperature and a long cooking time exacerbated the
differences and thus supports our results. Moreover, the higher
hydrophobicity we observed in the highly cooked meat could
have been involved in the formation of protein aggregates, thus
reducing the susceptibility to protease digestion (14).
To our knowledge, the present study is the first to attempt to
characterize the size of the residual exogenous particles that
reach the colon. The qualitative examination by microscopy
interestingly revealed the appearance of meat fibers in variable
frequency, depending on the time point and the subject. In the
subject with the lowest digestibility, the number of fibers
counted in the 100 biggest particles was the highest at each
time point. This observation suggests that the loss of
digest-ibility in some subjects is due to the early arrival of intact fibers in
the terminal ileum. However, this approach could not be systematic
among subjects and time points because a specific staining of meat
fibers would have been useful to ascertain the identification that
was unclear in several samples. In particular, it is possible that a
prolonged stay in the stomach in the presence of protease partially
destroyed the fibers and prevented a clear identification. The study
by Coll et al. (40) could support this hypothesis, because they found
that the addition of a proteolytic mix to a meat meal revealed
the infiltration of proteases inside the muscle fibers. In contrast to
the microscopy analysis, the granulometry measurements did not
allow detecting any cooking process effect on particle characteristics
in the ileal effluents. In vitro studies revealed a decrease in the
FIGURE 4 Time course of dietary nitrogenin plasma proteins (A), plasma amino acids (B), and body urea (C) in young adult over 8 h after ingestion of15N-labeled RM or FCM. Values are means 6 SEMs, n = 8. APE, atom percent excess; FCM, fully cooked meat; RM, rare meat.
diameter of particles and an increase in their circularity after a
higher-temperature cooking intensity, this being due to the
aggregation of myofibrils (3, 11). These modifications observed
in the cooked meat seem to have no effect on the structure of
residual particles that reach the colon, because the qualitative
examination in microscopy did not reveal the presence of
aggregates.
The effect of cooking meat on ileal digestibility was
previ-ously assessed in minipigs (11). In that study, bovine meat was
cooked at various temperatures (60°C, 75°C, or 95°C) but for a
single duration (30 min). Under these cooking conditions, which
differ from those in our study, the investigators observed no
significant effect of meat preparation on protein digestibility in
the small intestine but did observe a modulation of kinetics, a
slower digestion being reported with the highest cooking
tem-perature (95
°C). Conversely, in our study, the digestion speed of
meat proteins was not affected by cooking conditions. The effect
of cooking temperature was reported to be nonlinear, according
to a bell-shaped evolution from RM to FCM, the highest rate of
digestion being observed for a cooking temperature of
;75°C
(11). This was explained by a gradual denaturation of proteins
for cooking temperatures up to 75°C, followed by formation of
molecular aggregates at higher temperatures because of the
oxidation process (10). These phenomena, respectively, lead to
an increase and then a decrease in the accessibility of digestive
enzymes to their cleavage sites within the proteins, thus
affect-ing digestion speed. Doneness of the meat relies both on the
temperature and the time of cooking. In the present study, both
variables were modified to obtain contrasting cooking
condi-tions. We can suggest that RM or FCM is at the extremes of the
bell-shaped evolution of the effect of cooking temperature on
the protein digestion speed, and thus probably explains the lack
of effect on digestion kinetic in the present study.
The digestion speed is an important variable because it was
shown to affect the postprandial utilization of exogenous amino
acids, depending on the physiologic status of the subjects. For
instance, in young adults, slowly digested proteins are more
beneficial because they are less deaminated than fast proteins
(41), whereas with anabolic resistance such as in very old
subjects, rapidly digested proteins are more beneficial to restore
the postprandial protein anabolism (42). This study also
ex-plored the metabolic fate of exogenous nitrogen after its
absorp-tion in the small intestine. The cooking process had no effect
on digestive kinetics and on postprandial exogenous nitrogen
metabolism. We did not report any impact of cooking at 90°C
for 30 min, neither on exogenous nitrogen incorporation into
plasma amino acids and proteins nor on its incorporation into
body urea and excretion into urinary urea. This result is in line
with the absence of any effect on digestion kinetics, which were
shown to play major roles in the postprandial metabolism of
exogenous proteins (41, 43). Moreover, the decrease of protein
digestibility was too moderate to have significant impact on
exogenous nitrogen utilization by the body. Finally, the NPPU of
meat was 70–72%, this value being the value of casein and
cooked eggs (27, 36, 41).
This study showed that meat proteins have a high true ileal
digestibility and net postprandial nitrogen utilization in
hu-mans. Cooking at a high temperature for a long time tended to
decrease protein digestibility by
;4%, with an associated
increase of variability. This loss can be considered as
moder-ate, resulting only in an additional 1 g of proteins reaching the
colon for an intake of 25–30 g protein from meat. Whether
this has deleterious effects on colon mucosa remains to be
further investigated.
Acknowledgments
We thank Marie Claude Amar from the Volunteer Research
Centre at Avicenne Hospital for recruiting the subjects and
collecting the samples and Noredine Hafnaoui from the UNH
of Clermont-Ferrand for performing the plasma amino acid
measurements. AM-B, VS-L, DR, and CG designed the
re-search; MO, AM-B, GA, VS-L, NK, DR, AF-S, JP, RB, and CG
conducted the research; MO, VS-L, and CG analyzed the data;
MO, AM-B, DT, GF, and CG wrote the paper; and CG had
primary responsibility for its final content. All authors read and
approved the final manuscript.
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