J. Anim Sci. 2007. 85:2972-2981. doi:10.2527/jas.2006-795
© 2007 American Society of Animal Science
Weaned piglets display low gastrointestinal digestion of pea (Pisum sativum L.) lectin and pea albumin 21
M. Le Gall*,
,2,
L. Quillien
,
B. Sève,
J. Guéguen* and
J. P. Lallès
* INRA, Unité de Recherche Biopolymères, Interactions, Assemblages, Rue de la Géraudière, 44072 Nantes, France;
and
INRA, UMR 1079, Systèmes d’Elevage, Nutrition Animale et Humaine, Domaine de la Prise, 35590 Saint-Gilles, France; and and
INRA, Unité de Recherche Génétique et Ecophysiologie des Légumineuses à graines, Domaine d’Epoisses, Bretenières, 21065 Dijon, France
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Abstract
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A study was conducted to investigate the biochemistry of digestion of field pea (Pisum sativum L.) albumins and globulins in the stomach and along the small intestine of weaned piglets with a particular emphasis on the respective roles of these compartments in pea protein digestion. Twenty-four piglets were weaned at 28 d of age. They were allocated to 2 diets (control and pea) and 3 slaughter times (3, 6, or 9 h after the last meal) in a 2 x3 factorial arrangement of treatments in a randomized complete block design. Pea flour provided 30% of total dietary protein in the pea diet. The diets were fed for 2 wk after weaning. After slaughter, gastrointestinal tract (GIT) compartments were weighed, digesta were collected, and pH was measured. Digesta from the stomach and cranial, middle, and caudal small intestine (SI) were extracted for soluble proteins and analyzed for specific pea proteins using SDS-PAGE, immunoblotting, and mass spectrometry. Tissue weight of the whole GIT (P = 0.015), cecum (P <0.001), and colon (P <0.001) was greater in the pea diet. Digesta pH in the stomach and caudal SI was lower (P = 0.02) in the pea diet than the control diet. In the stomach, vicilin, lectin, and pea albumin 2 were not digested, whereas legumin was only partly digested. Legumin and vicilin were totally digested in the SI in less than 3 h. A resistant peptide of 15 kDa located at the N-terminus of pea albumin 2 was transiently detected at 3 h. A protein band at 20 kDa was consistently identified as lectin. It was present in high intensity in intestinal digesta of pea-fed piglets at all times after the meal compared with those fed the control diet (P <0.001). Various proteins of, presumably, endogenous origin displayed differential digestion patterns between the control and the pea-fed piglets (P<0.05). In conclusion, differences in digestion between specific pea proteins were observed along the GIT of piglets. They could be partly explained by differences in protein digestion in the stomach.
Key Words: albumin • digestion • globulin • pea protein • pig
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INTRODUCTION
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Field peas can be an alternative to soybean meal in diets for nonruminant animals. However, diets with high levels of incorporation of field peas (Pisum sativum L.) usually depress growth performance, especially in young animals (Gatel, 1994
). Field peas are rich in Lys but deficient in sulfur AA and Trp. Moreover, the digestibility of plant protein has often been regarded to be lower than that of animal protein, which has generally been attributed to the presence of antinutritional factors, including lectins, protease inhibitors, and tannins (Lallès and Jansman, 1998). Field peas contain the trypsin-chymotrypsin inhibitors of the Bowman-Birk family, which have 14 cysteine residues, all involved in disulfide bridges (Ferrasson et al., 1995). Protease inhibitors form a complex with pancreatic proteases and inactivate them. These proteases are also resistant to digestion, an observation that may contribute to explain the lower digestibility of sulfur AA in pigs (Mariscal-Landin et al., 2002).
Protein hydrolysis of field peas also is influenced by the protein structure. They fall within 2 categories, namely globulins (55 to 65% of CP) and albumins (20 to 25% CP). They are soluble in saline solutions of low ionic strength and water, respectively. Globulins include legumin, vicilin, and convicilin. The albumin fraction consists of lectin, pea albumin 1 and 2 (PA2), PI, and lipoxygenases. Globulins seem to be more digestible than albumins in various animal species (Rubio et al., 1991; Crevieu et al., 1997; Carbonaro et al., 2000). The consumption of field peas increases the ileal output of endogenous proteins in pigs (Le Gall et al., 2005a).
However, little is known on the time-course of protein digestion along the gastrointestinal tract (GIT), a factor that might contribute to differences in the degree of hydrolysis among proteins at the ileal level. Therefore, we conducted a study to investigate the postprandial changes in pea protein digestion along the GIT in weaned piglets.
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MATERIALS AND METHODS
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Diets
Two diets were formulated and their composition is given in Table 1. The control diet contained a mixture of skim-milk powder and fish soluble protein concentrate as the sources of CP. Thirty percent of CP from this mixture was replaced by field pea flour in the pea diet. The seeds were ground using a hammer mill prior to being incorporated into the feed. The diets were balanced for CP, indispensable amino acids, with appropriate DL-Met, L-Thr, and L-Trp supplements and NE values (Sève, 1994). The Baccara spring pea cultivar was chosen because of its low content in trypsin inhibitors (Grosjean et al., 2000). It was produced locally at the Station d’Amélioration des Plantes, INRA, Le Rheu, France.
Animals and Feeding
The experiment was conducted under the guidelines of the French Ministry of Agriculture for animal research. The experimental design was a randomized complete block design with a 2 x3 factorial arrangement of 2 diets (control vs. pea) and 3 slaughter times after the last meal (3, 6, and 9 h). Four litters of cross-bred piglets [Pietrain x(Landrace xLarge White)] were weaned at 28 d of age. Four blocks of 6 littermates were formed on the basis of piglet BW, regardless of sex. The initial BW of the piglets in the control and pea group was 8.0 ± 0.8 kg and 8.1 ±0.9 kg, respectively. They were randomly assigned to treatments within litters, housed in individual cages, and fed either of the 2 diets for 2 wk after weaning. The feed was given as a mash (meal to water ratio of 2:1 by weight) twice daily (0900 and 1530) at a rate of 650 kJ· (BW in kg)0.75. Body weights were recorded weekly, and feeding schedules were adjusted accordingly. The piglets had free access to water. The piglets from 2 litters were slaughtered on d 13, and the piglets from the 2 other litters on d 14 after weaning. Each day the slaughter h was randomly assigned within litters and took place at 3, 6, or 9 h after the morning meal. The piglets were killed by electronarcosis and exsanguination.
Digesta Collection
Immediately after slaughter, the GIT was removed, dissected, and divided into 7 segments: stomach, 3 parts of equal length of the small intestine (SI; cranial, middle, and caudal), cecum, and first and second halves in length of the colon. Each segment was weighed and emptied. The total amount of digesta in each segment was collected, mixed thoroughly, and the pH was measured. A solution of preservatives (15 mM EDTA, 1 mM sodium azide, and 2 mM phenylmethylsulfonyl fluoride) was then mixed with the digesta to limit enzyme and bacterial activities. The digesta were then frozen at –20°C, and subsequently freeze-dried, weighed, finely ground, and stored at 4°C until analysis.
Chemical and Biochemical Analyses
The diets were analyzed for DM, minerals, N, and AA as previously described (Le Gall et al., 2005a). The freeze-dried digesta samples were analyzed for DM and N contents. Protein was extracted from the diets and the stomach content by stirring in 16 mM Tris-HCl buffer containing 140 mM SDS with pH 8 for 90 min at room temperature (10 g of diet/L of buffer; 50 g of gastric digesta/L of buffer). Protein was extracted from the digesta from each part of SI by stirring in 100 mM H3BO3, 150 mM NaCl with pH 8 for 90 min at room temperature (200 g of digesta/L of buffer). These buffers were previously shown to maximize the solubilization of proteins (Bush et al., 1992). The samples were then centrifuged at 12,000 xg for 10 min at room temperature. The supernatants were collected and stored at –20°C until SDS-PAGE analysis and immunoblotting. Protein in the supernatants was determined by the bicinchoninic acid micromethod (Pierce, Rockford, IL).
SDS-PAGE Electrophoresis, Densitometry, and Immunoblotting
The SDS-PAGE procedures used were previously described in detail (Le Gall et al., 2005a). Protein extracts from gastric digesta, anterior intestinal digesta (cranial and middle SI), and posterior intestinal digesta (caudal SI) were diluted 10-, 3-, and 2-fold, respectively, with appropriate buffers. The amounts of protein loaded on the gels were 20, 50, and 200 µg per well for the CP extracts, gastric, and SI digesta extracts, respectively. Gels with blue-stained proteins from SI digesta were scanned. Densitometry measurements were performed using image analysis (Amount One, version 4.1, Biorad Laboratories, Hercules, CA) and analyzed as described by Salgado et al. (2002). Densitometry profiles were converted into arbitrary density units and were expressed in arbitrary density units per gram of digesta protein, corresponding to the amount of protein that was deposited on each track on the gel. Because SDS-PAGE patterns of proteins were generally similar among the 4 piglets fed a given diet and slaughtered at the same time after the meal, only 1 representative illustration is given on the figures. However, densitometry analysis was performed for all of the individual digesta at each site of the SI.
Prior to immunoblotting, proteins separated by SDS-PAGE were electro-transferred onto nitrocellulose membranes and were probed with rabbit antibodies for detecting specifically pea legumin, vicilin, lectins, and PA2, as previously described (Le Gall et al., 2005a). Rabbit antibodies were detected with an antirabbit IgG antibody conjugated with an enzyme. The membrane was incubated with the enzyme substrate.
Immunoprecipitation and Sequencing of Undigested PA2 Polypeptides
The protein A technology for purifying proteins using specific antibodies (Ghose et al., 2005) was applied for concentrating digestion polypeptides from PA2 in anterior intestinal digesta (caudal SI). Briefly, some components of digesta that interacted nonspecifically with 4°C under gentle rotation and then centrifugation. The supernatant was incubated overnight at 4°C with the antiPA2 antibodies (also used for immunoblotting) for PA2-anti-PA2 antibody complex formation. Then the protein A beads were added to the mixture and incubated for further 2 h at 4°C under gentle rotation. The resulting PA2-antibody complex adsorbed on the beads was washed extensively with ice-cold phosphate buffer saline (pH 7.4; 136 mM NaCl, 2.68 mM KCl, 1.46 mM KH2PO4, and 8.1 mM Na2HPO4). The complex was eluted in the SDS-PAGE, sample loading buffer prior to being separated by SDS-PAGE and revealed by immunoblotting, as described above for digesta samples.
After transfer to nitrocellulose membranes, the undigested PA2 polypeptides were detected in 1 lane using the same antiPA2 antibodies. The polypeptide chains from the antiPA2 antibodies were revealed in an adjacent lane with a goat anti-rabbit IgG antibodies conjugated to horseradish peroxidase (170 6515, Biorad Laboratories, Hercules, CA) and addition of the peroxidase staining mixture [2.2 M 4-chloro-naphtol and 0.02% hydrogen peroxide in 1:10 (vol/vol) methanol, and phosphate saline buffer].
The specific PA2 bands were then identified on the SDS-PAGE gels and sequenced using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/ MS), as previously reported (Le Gall et al., 2005a). Briefly, bands of interest were excised from the blue-stained gel. Then, they were submitted to digestion with trypsin. Extracted peptides were separated by reverse-phase chromatography and analyzed after fragmentation in MS-MS to be consistent. Mass data collected during LC-MS/MS analysis were processed with Protein Lynx Global Server software (Waters, Milford, MA). Protein identification was performed by searching the peptide masses and MS/MS sequence stretches against the Swiss-Prot (Swiss Institute of Bioinformatics, Switzerland) and National Center for Biotechnology Information (Bethesda, MD) nonredundant sequence databank.
Statistical Analysis
The experimental unit was the piglet. The data were analyzed as a randomized complete block design using the GLM procedure (SAS Inst. Inc., Cary, NC). Variance analysis was performed for testing the effects of diet, time after the last meal, litter, and interaction between diet and time after the meal. Orthogonal contrasts were used to test linear and quadratic effects of time, and the interaction was partitioned into a linear and a quadratic effect of time xdiet.
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RESULTS
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All the piglets were in good health throughout the experiment. The diets were well consumed. No difference in feed intake or BW gain was observed among treatments during the experimental period (Table 2).
The whole GIT was heavier in the piglets fed the pea diet compared with the control (P = 0.015; Table 3). The difference was accounted for by the weight of the cecum (P <0.001) and the large intestine (P <0.001).
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Table 3. Relative weight (g/kg of BW) of the entire empty gastrointestinal tract and its segments in piglets fed the control and the pea diet
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The residual DM present in the stomach of piglets at slaughter decreased linearly (P <0.001) and quadratically with time (P = 0.05; Table 4). Three hours after the meal, 51 and 56% of DM intake was emptied from the stomach, and 15 and 12% of DM intake was still present in the stomach 9 h after the last meal for control and pea groups, respectively. No interaction between the type of diet and the digestion time was observed, and there was no effect of the diet. In contrast, gastric pH was decreased in the pea than with the control diet (P = 0.02). It decreased linearly (P <0.001) with time. There was a tendency (P = 0.06) for a time xdiet interaction for digesta pH in the cranial SI, which was caused by a sharp decrease with time in the control group compared with the pea group (linear effect of time xdiet, P = 0.02). The pH in the middle SI was more variable and not affected by the tested factors. Digesta pH in the caudal SI was decreased with the pea as compared with the control diet (P = 0.02) without effect of time.
SDS-PAGE, Densitometry, and Immunoblotting Analyses of Digesta
Digestion in the Stomach.
The SDS-PAGE gels stained for proteins showed quite different banding patterns between the control and the pea diets (Figure 1A). Low numbers of protein bands were detected in gastric digesta after 9 h compared with those present in the diet extract. Many of them were not present in the feed, so they were most probably hydrolyzed dietary proteins or of endogenous origin.
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Figure 1. Protein digestion in the stomach of piglets fed a control (C) diet or a diet containing pea (P) as revealed by (A) SDS-PAGE in reducing conditions and Coomassie Blue staining; and (B) immunoblotting with antibodies against pea vicilin (a), legumin (b), lectin ß subunit (c), and albumin PA2 (d) for the diet extracts and the digesta extracts from the piglets fed the pea diet (no antibody reacted with proteins in the controls). The digesta were collected by the slaughter technique at 3, 6, and 9 h after the meal and extracted for proteins before analysis. Individual tracks represent individual samples. M = molecular mass markers in kDa on the left.
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Western blotting showed the presence of vicilin and convicilin in the pea diet, but not in the control (Figure 1Ba
). Vicilin proteins of molecular weight (MW) of 50 and 33 kDa, and smaller vicilin polypeptides were transiently observed at 3 h, but not afterward. The band of convicilin was still present at 3 h in the digesta of some pigs, but it disappeared thereafter. Legumin was revealed in the pea diet as 2 major bands (
-major and ß polypeptides) of MW of 40 and 20 kDa, respectively (Figure 1Bb). These bands and a cleaved -polypeptide were still clearly observed in gastric digesta 3 h after the meal. By 6 h, only legumin polypeptides of MW equal to or smaller than 30 kDa were detected. These were no longer present at 9 h after the meal. Only the ß subunit of pea lectin was seen at a MW of 17 kDa in the pea diet and in gastric digesta 3 h after the meal but not thereafter (Figure 1Bc). The subunit of lectin was not detected by antibodies. Pea albumin 2 was detected at a MW of 26 kDa in the pea diet and corresponding digesta at 3 h after the meal but not thereafter (Figure 1Bd). Cleaved PA2 peptides of MW between 17 and 25 kDa were transiently observed at 3 h.
Digestion in the Small Intestine.
The patterns of proteins observed in the cranial SI were characterized by a high number of protein bands (Figure 2A). Densitometry data with statistically significant effects of time or diet or both are presented in Table 5. At cranial SI, the density of 4 bands (noted d1 to d4) was affected by the diet. The density of band d1 was decreased (P = 0.003), whereas band d2 was greater in the pea group compared with the control (P = 0.006) without effect of time. Band d3 was virtually absent in the control piglets regardless of time, whereas its density decreased sharply between 3 and 6 h over time in the pea-fed piglets (time linear xdiet, P = 0.002; time quadratic xdiet, P = 0.036). Band d4 was absent in the control group, whereas its density was high in the pea group (P = 0.001) regardless of time. The resistance of lectin to digestion in the cranial SI was confirmed by immunoblotting (Figure 2Bb). Legumin was detected as the ß-polypeptide only at 3 h and only in 1 piglet (Figure 2Ba). Pea albumin 2 was not evidenced by immunoblotting in the cranial SI (Figure 2Bc). Finally, vicilin or digestion fragments were not detected in the cranial SI (data not shown).
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Figure 2. Protein digestion in the cranial small intestine (SI1) of piglets fed a control (C) diet or a diet containing pea (P) as revealed by (A) SDS-PAGE in reducing conditions and Coomassie Blue staining; and (B) immunoblotting with antibodies against (a) pea legumin, (b) lectin ß subunit, and (c) albumin PA2 for the diet extracts and the digesta extracts from the piglets fed the pea diet (no antibody reacted with proteins in the controls). The digesta were collected by the slaughter technique at 3, 6, and 9 h after the meal and extracted for proteins before analysis. Individual tracks represent individual samples. M = molecular mass markers in kDa on the left. The numbered bands in A refer to those further analyzed by densitometry.
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Table 5. Densitometry of protein bands in digesta of the cranial, middle, and caudal segments of the small intestine (densitometric unit ·g–1 of protein)
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After SDS-PAGE of proteins in digesta of the middle SI, 6 protein bands (noted j1 to j6; Table 5) with visually contrasted staining densities (Figure 3A) had their intensity influenced by the diet. An effect of time was noted for bands j4 and j5 (P = 0.023 and P = 0.054, respectively). The intensity decreased linearly from 3 to 9 h for band j4 (P = 0.024), but the variation was quadratic with a greater value at 6 h for j5 (P = 0.07). The protein band j6 was identified by immunoblotting as lectin (Figure 3Aa). There was a time xdiet interaction in band j6 intensity (time linear xdiet, P = 0.011; time quadratic xdiet, P = 0.031), which was caused by the absence of this band in digesta from the control diet and by its high intensity at 3 and 6 h after the meal and a decrease between 6 and 9 h in the pea diet. Immunoblotting of digesta from the middle SI confirmed the presence of lectin at all times (3, 6, and 9 h) after the meal in the pea-fed piglets (Figure 3Ba; Table 5). The whole PA2 molecule was not detected, but a polypeptide of MW of 15 kDa was revealed at 3 h (Figure 3Bb). Neither vicilin nor legumin were detected in the digesta of the middle-SI (data not shown).
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Figure 3. Protein digestion in the middle small intestine (SI2) of piglets fed a control (C) diet or a diet containing pea (P) as revealed by (A) SDS-PAGE in reducing conditions and Coomassie Blue staining; and (B) immunoblotting with antibodies against (a) lectin ß subunit and (b) albumin PA2 for the diet extracts and the digesta extracts from the piglets fed the pea diet (no antibody reacted with proteins in the controls). The digesta were collected by the slaughter technique at 3, 6, and 9 h after the meal and extracted for proteins before analysis. Individual tracks represent individual samples. M = molecular mass markers in kDa on the left. The numbered bands in A refer to those further analyzed by densitometry.
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After SDS-PAGE separation of proteins in digesta of the caudal SI, 6 protein bands (noted i1 to i6; Table 5) with visually contrasted stainings (Figure 4A) were further analyzed by densitometry. The staining density was decreased in the digesta of the pea-fed piglets compared with the controls for bands i1 to i4 (P = 0.041 to <0.001). In contrast, bands i5 and i6 were more densely stained in the pea-fed piglets (P <0.001). There was a trend for a time xdiet interaction for band i2 intensity. The interaction was caused by a linear decrease in the control group and a quadratic increase with time in the pea group (time linear xdiet, P = 0.085; time quadratic xdiet, P = 0.088). The intensity of band i3 was influenced by the time xdiet interaction (time linear xdiet, P = 0.007), which was caused by a linear decrease with time in the control digesta but constantly low yet a linear increase in the pea diet digesta. There was a time xdiet interaction for band i4 (time linear xdiet, P = 0.034), which was caused by the absence of this band in digesta from the pea diet and by a decrease of its intensity with time in the control digesta. The protein band i5 was not identified by immunoblotting, although its intensity was high in the pea-fed piglets (P <0.001) and there was no effect of time. The protein band i6 was identified by immunoblotting as lectin (Figure 4Aa). There was a time xdiet interaction for this band (time linear xdiet, P = 0.087; time quadratic xdiet, P = 0.021), which was caused by the absence of the band in digesta from the control diet and by an increase between 3 and 6 h and plateaued at 9 h after the meal in the pea-fed pigs.
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Figure 4. Protein digestion in the caudal small intestine (SI3) of piglets fed a control (C) diet or a diet containing pea (P) as revealed by (A) SDS-PAGE in reducing conditions and Coomassie Blue staining; and (B) immunoblotting with antibodies against (a) lectin ß subunit and (b) albumin PA2 for the diet extracts and the digesta extracts from the piglets fed the pea diet (no antibody reacted with proteins in the controls). The digesta were collected by the slaughter technique at 3, 6, and 9 h after the meal and extracted for proteins before analysis. Individual tracks represent individual samples. M = molecular mass markers in kDa on the left. The numbered bands in A refer to those further analyzed by densitometry.
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The 15-kDa polypeptide from PA2 was also detected in ileal digesta at 3 and 6 h but not at 9 h after the meal (Figure 4Bb). The PA2 polypeptides undigested in the caudal SI were further characterized after concentration by immunoprecipitation. Three bands of MW of 22, 17, and 15 kDa were identified by immunoblotting as originating from PA2 (Figure 5). Their identity was confirmed by LC-MS/MS sequencing and the resistant peptides of PA2 were essentially localized on the AA sequence of the PA2 molecule in the N- terminus (data not shown).
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Figure 5. Separation by SDS-PAGE (A) and identification by immunoblotting (B) of pea albumin PA2 in the pea flour (P) and of PA2 polypeptides present in ileal (SI3) digesta of piglets fed the pea diet. These undigested polypeptides were first immunoprecipitated from digesta using protein A technology. Track 1 = molecular weight markers in kDa; track 2 = pea proteins (A) and PA2 (B) in the pea flour extract; track 3 = Rabbit IgG antibodies (Ac) specific for PA2; track 4 = immunoprecipitated (Immunoprec.) proteins including the antiPA2 rabbit antibodies and the undigested PA2 polypeptides (A) and immunoblotting revelation with the antiPA2 antibodies; and track 5 = similar to track 4 for (A) and revelation with an antirabbit IgG antibody alone (5). The undigested PA2 polypeptides specifically detected and analyzed by mass spectrometry were obtained by difference between the band patterns present on track 4 and those present on track 5 of the immunoblot (B); they are identified with black arrowheads on panel A.
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DISCUSSION
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The present results confirm our previous observations in growing pigs: 1) pea globulins are well digested, 2) PA2 is only partially digested, 3) a PA2-resistant polypeptide of MW of 15 kDa is present, and 4) lectin is totally resistant to GIT digestion (Le Gall et al., 2005a). The major point of this work is that such differences in intestinal digestion between pea proteins originates in the stomach.
Digestion of Pea Protein Along the Gastrointestinal Tract of Piglets
The gastric digestion seems to be the important step for the pea protein hydrolysis, in accordance with the results obtained after in vitro hydrolysis of different pea cultivars (Perrot et al., 1999). Indeed, peptides from PA2 digestion were detected in intestinal digesta a short time (less than 3 h) after the meal. During this short period, gastric pH did not seem to be adequate for protein digestion. It was high (pH >4.5) compared with the optimal pH for pepsin action (pH 2 to 4; Beynon and Bond, 1989). This may reflect the fact that gastric pH of piglets soon after weaning is greater than in older pigs (Makkink et al., 1994). The amounts of gastric secretion are the greatest only 1.5 to 2.5 h after the meal (Cranwell, 1985), and the inefficiency of pepsin hydrolysis may be responsible for the low digestion of pea proteins. Indeed, the dissociation-association behavior of the polypeptides from pea legumin is influenced by pH. Between pH 3.5 and 5.8, most of the protein is in the form of aggregates preventing pepsin access. Below pH 3.4, legumin is completely dissociated and its polypeptide chains are unfolded (Guéguen et al., 1988). These conditions were met after 6 h of gastric digestion, and the ß-polypeptides forming the hydrophobic core of legumin were not protected. Their greater resistance to hydrolysis compared with that of the -polypeptides may also be due to their highly ordered structure (Subirade et al., 1994) and greater hydrophobicity.
Convicilin is easily hydrolyzed by pepsin, even at pH >4.5, but vicilin is resistant under these conditions. This differential susceptibility to hydrolysis between convicilin and vicilin, shown in vitro (Le Gall et al., 2005b), is also observed in the chicken (Crevieu et al., 1997). In the current study, vicilin had disappeared after 6 h of gastric digestion. In the digesta collected 3 h after the meal, vicilin was probably quite insoluble due to the greater pH (5.0 to 6.0; Rangel et al., 2003). Then, it may have become totally soluble during the gastric phase at pH <3.0. Consequently, vicilin may have been more easily emptied from the stomach with the liquid phase and would no longer be detected in the stomach.
Pea protein digestion in the SI seemed to be more efficient in pigs than in other animal species. Indeed, in rats and chicken, only the -polypeptides of legumin were degraded, the ß-polypeptides being totally resistant (Aubry and Boucrot, 1986; Crevieu et al., 1997). The albumin PA2 was shown to resist trypsin hydrolysis in vitro (Gruen et al., 1987; Perrot et al., 1999) and SI digestion in chickens (Crevieu et al., 1997). In young piglets used in the current study and growing pigs used in an earlier study (Le Gall et al., 2005a), we observed a cleaved peptide of MW of 15 kDa in digesta of the middle and caudal SI. These data indicate that a part of PA2 is susceptible to digestion in the gut of mammals, as opposed to birds.
Endogenous Protein
Many proteins present in GIT digesta are of endogenous origin and mainly consist of enzymes and antibodies according to the literature. These proteins themselves could be partly or totally degraded and reabsorbed during the digestion process. Proteins of greater MW may correspond to glucoamylase or pancreatic alpha-amylase, or both, as previously identified at the terminal ileum of growing pigs (Le Gall et al., 2005a). Proteins of intermediary MW may be fragments of antibodies (Le Gall et al., 2005a) and proteins of the pancreatic serine protease family (trypsin and chymotrypsin; Salgado et al., 2002). Surprisingly, many of these endogenous proteins were present less in digesta after pea consumption, but this may be explained by our analytical method. Indeed, the same amount of undigestible soluble protein was deposited in each track of gels. Therefore, a lower proportion of endogenous protein in the digesta did not indicate that the ileal flow of endogenous protein was lower. A greater proportion of dietary undigested protein, like lectins in the pea digesta, will result in a lower proportion of endogenous protein. Indeed, based on the published tables (INRA-AFZ, 2004), the ileal digestibility of pea protein seems to be lower than of milk and fish protein. These differences are due to the presence of PI and lectin, which are known to increase the ileal flow of dietary protein, and the same components and fiber increase the endogenous losses. At the ileum, only 1 band (i5) was present in digesta after pea consumption. This band was previously identified as corresponding to proteins of the pancreatic serine protease family (Salgado et al., 2002). Greater endogenous losses with pea-based diets have been ascribed to cotyledon cell-wall fiber displaying a high water holding capacity (Leterme et al., 1998).
In this study, the major storage pea proteins, vicilin and legumin, were rapidly broken down in the stomach and were subsequently digested in the SI of piglets. Lectin remained virtually intact throughout the stomach and SI. The pea albumin PA2 transiently survived as a 15-kDa peptide located at the N-terminus of the molecule. This may contribute to the lower digestibility and availability of sulfur AA in pigs fed diets based on field peas because pea albumins are rich in these AA. To improve the use of field peas in pig diets, cultivars with a high globulin to albumin ratio should be used.
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Footnotes
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1 Financial support for this study was provided by the Regions of Bretagne and Pays de la Loire (Pôle Agronomique Ouest), France. The authors thank P. Touanel for care of experimental animals. 
2 Corresponding author: Maud.LeGall{at}rennes.inra.fr
Received for publication December 5, 2006.
Accepted for publication June 6, 2007.
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LITERATURE CITED
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Bush, R. S., R. Toullec, I. Caugant, and P. Guilloteau. 1992. Effects of raw pea flour on nutrient digestibility and immune response in the preruminant calf. J. Dairy Sci. 75:3539–3552.[Abstract]
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Abstract
INTRODUCTION
