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* INRA, Unité de Recherche sur les Protéines Végétales et leurs Interactions (URPVI), 44072 Nantes Cedex 03;
INRA- E.N.S.A.R., Unité Mixte de Recherche sur le Veau et le Porc, 35590 Saint-Gilles; and
** INRA, Unité de Recherches en Génétique et Ecophysiologie des Légumineuses, Domaine d’Epoisses, 21110 Bretenieres
3To whom correspondence should be addressed. E-mail: quillien{at}epoisses.inra.fr.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • endogenous protein • pea protein • pigs • digestion • grinding
In recent years, there has been growing interest in the production of grain legumes (lupins, faba beans, chickpeas, peas) as alternative protein sources; these legumes would replace commonly incorporated ingredients such as soybean meal in pig diets. Pea seeds, containing an average of 20–30% crude protein (CP)4 (1) and a high level of starch (35–45%), are valuable protein and energy resources for pigs. Pea proteins are rich in lysine but can be deficient in sulfur amino acids (AAs) and tryptophan for nonruminant animals.
In peas, a large variation in CP and AA content among cultivars is observed (2), and this variation is affected by both genetic and environmental factors (3,4). The protein digestibility of legume seeds is highly variable and is generally lower than that of other diets based on soybean or casein (5–7). This may be explained in part by the lower intrinsic digestibility of legume proteins due to their compact structure (8). Pea proteins are commonly classified into 2 groups on the basis of their characteristics in solution. The albumin fraction, 20–25% of the seed protein content, is made up of water-soluble proteins, lipoxygenases, protease inhibitors, lectins, and 2 pea albumins (PA1 and PA2). Globulins, salt-soluble proteins, which represent 55–65% of seed protein content, are composed of 2 major groups: legumin (11S) and vicilin (7S). The globulin fraction is more digestible than the albumin fraction (9–11). Antinutritional factors (ANF) including protease inhibitors, lectins, tannins, and fibers may limit the protein digestibility (12–14). These components may act as physical barriers to proteolytic enzymes; they could form complexes with proteolytic enzymes, which become inactive, or they may interact with the gut to stimulate endogenous secretion and disturb the absorption of peptides and AA. Endogenous proteins could be partitioned into host and bacterial fractions, including proteins from salivary and gastric secretions, pancreatic and bile secretions, small intestinal secretions, desquamated cells, and mucin, which represent a major contribution (15). Quantification of endogenous nitrogen losses at the terminal ileum has been determined by the isotope dilution method, but the composition of this fraction in terms of proteins and peptides is largely unknown.
The objectives of this study were to quantify and identify proteins at the terminal ileum in growing pigs. We compared diets based on different pea cultivars (Madria, Solara and Eiffel) and used 2 different particle sizes for the Solara-containing diet. All soluble ileal proteins were separated using PAGE. To determine their protein origin (endogenous or dietary), 3 methods were used: 1) correlations between (endogenous, dietary or total) ileal flows previously determined (16) and soluble ileal protein flows were assessed; 2) the resistant peptides of pea proteins (legumin, vicilin, lectin, PA2, or PA1b) were identified by immunoblotting; and 3) all proteins detected using PAGE were analyzed by LC-MS-MS spectrometry.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Diet and digesta collection.
Three pea cultivars (Pisum sativum L.), Solara, Eiffel, and Madria, were harvested in Dijon (INRA, Unité de recherche en Génétique et Ecophysiologie des Légumineuses). In each field, a restricted area (
40 m2) was treated with labeled ammonium nitrate (15N2, 11.5%, Cambridge Isotope Labs).
The seeds were ground through a 2.5-mm mesh screen before mixing; 55 kg of unlabeled and 15 kg of labeled Solara seeds were finely ground to a particle size of 25–50 µm in diameter. This was termed the micro-grinding treatment (Tecaliman). A protein-free diet and diets containing pea cultivars each as the sole protein source (15%) were formulated. Labeled diets were formulated by substitution of labeled for unlabeled pea, and 0.3% chromic oxide was substituted for starch as shown in the supplemental diet composition table.
The pigs were fed 80 g of dry matter (DM) intake per metabolic body weight [(BW, kg)0.75] mixed with water (1:2) twice daily in equal amounts at 800 and 1530 h. Each pig was fed the experimental diets successively for 4 consecutive weeks according to a 4 x 4 Latin square design. After 4 d of adaptation to an experimental diet, the ileal digesta that appeared over 48 h, or 3 d when pigs were fed the protein-free diet, were collected quantitatively twice daily, after feeding, into 0.5 L of 0.36 mol/L H2SO4. Pigs were fed the unlabeled diets except on the morning of d 7 when they were fed the chromic oxide (3 g/kg) and 15N2 labeled diet. The second meal of that day (1530 h) was not labeled. The digesta were collected every hour in plastic bags for 9 h, and the remainder was collected the next morning. Immediately after collection, the bags containing ileal digesta were stored at –20°C then freeze-dried, weighed, finely ground, and stored at 4°C until further analysis. During wk 5 of the experiment, pigs were fed the protein-free diet. During that time period, there was no test meal and the total collection period was 3 d.
Chemical and biochemical analyses. Diets and freeze-dried ileal digesta collected for 2 d were analyzed for nitrogen (N) contents. The digesta collected each hour (per pig and per diet) were analyzed for DM, N, chromic oxide, total 15N2 enrichments (18), and soluble protein contents. The digestibility coefficients of N were calculated as described by Hess et al. (18).
Soluble proteins were extracted from ileal digesta samples by stirring in borate buffer (0.1 mol/L H3BO3, 0.15 mol/L NaCl, pH 8) for 90 min at room temperature (270 g digesta/L buffer) and centrifuging at 12,000 x g for 10 min at room temperature. The supernatants were collected and stored at –20°C until electrophoresis analyses and immunoblotting.
Pea proteins used as a control in immunoblotting were extracted from pea-containing diets by stirring in SDS-PAGE sample loading buffer for 90 min at room temperature (10 g diet/L buffer) and centrifuging at 12,000 x g for 10 min at room temperature.
Electrophoretic analyses of ileal proteins. The soluble protein extracts were diluted 2-fold with Tris-HCl buffer (0.16 mol/L, pH 8.8) containing 0.120 mol/L sucrose, 0.140 mol/L SDS, and 0.02 g/L bromophenol blue. Protein disulfide bridges were reduced with ß-mercaptoethanol for 2 min at 100°C. Proteins were electrophoresed in polyacrylamide gels (15%) according to Laemmli (19). Protein loads were 20 and 270 µg per well for the diet protein extracts and ileal digesta samples, respectively. Molecular weight (MW) standards (14.4–97.0 kDa; 17–0446-01, Pharmacia) were also loaded in a separate well. Proteins were fixed using trichloroacetic acid (0.75 mol/L) and stained with Coomassie blue G 250 (2 mg/L).
Gels with blue-stained proteins were scanned. Densitometry measurements were performed using image analysis (Amount One, Version 4.1) and analyzed as described by Salgado et al. (7). First, densitometry profiles were converted into arbitrary density unit (DU) concentrations, taking into account the ileal digesta protein solubility in the extraction buffer. Second, DU flow was calculated; l DU flow was expressed per dry matter ingested (DU/kg DMI).
LC-MS-MS analyses. Bands of interest were excised from Coomassie blue-stained gel, reduced and alkylated using dithiothreitol and iodoacetamide, respectively, then subjected to digestion with trypsin (Promega). Extracted peptides were dried and stored at –80°C until use. Peptides for LC-MS-MS analyses were dissolved in buffer A (H2O:acetonitrile:formic acid, 96:4:0.1, by vol). Nanoscale capillary LC-MS-MS analyses of the digested proteins were performed using an UltiMate capillary LC system (LC packings/Dionex) coupled to a hybrid quadrupole orthogonal acceleration time-of-flight MS-MS (Q-TOF Global, Waters). Chromatographic separations were conducted on a reversed-phase capillary column (Pepmap C18, 75 µm i.d., 15 cm length, LC Packings) with a flow rate of 200 nL/min. The gradient profile used consisted of a linear decrease from 100% to 45% B (H2O:acetonitrile:formic acid, 10:90:0.085, by vol) in 50 min, followed by a linear increase to 100% B in 10 min. Mass data acquisitions were performed by MassLynx software (Waters) using automatic switching between MS and MS-MS modes ("survey scan" mode). Peptides eluted from the chromatographic column were detected for 1 s; when their signal reached a user-defined threshold (8 counts/s), they were selected for fragmentation in MS-MS. Mass data collected during the LC-MS-MS analysis were processed and searched for protein identification against the National Center for Biotechnology Information (NCBI) protein databank using the Protein Lynx Global Server 2.0 software (Waters). Proteins were identified on the basis of the correlation of their in-silico predicted tryptic peptides with experimentally measured peptides, considering both the mass of the intact peptide (MS data) and the masses of the corresponding MS-MS fragments. Tolerance for matching masses was set at 0.1 Da, for both MS and MS-MS measurements. Most of the proteins reported were identified with at least 2 of their predicted tryptic peptides matching the experimental data. A further inspection of the identified peptides was performed manually to validate the results.
Immunoblotting. Rabbit polyclonal serums against legumin, vicilin, lectins, PA1b, and PA2 were obtained by s.c. immunization with the purified protein. Each injection contained 1 mg of protein in PBS (0.010 mol/L Na2HPO4, 0.15 mol/L NaCl, pH7.2), emulsified with complete (first injection), or incomplete (boost injections) Freund’s adjuvant. Boosting was repeated every 15 d and serum collected after the 4th injection.
Proteins separated by electrophoresis were electroblotted onto nitro-cellulose sheets (pore diameter 0.2 µm) according to Towbin et al. (20). After quenching of the free sites by skim milk powder (50 g/L) in PBS buffer, pH 7.4, for 1 h, sera diluted in PBS buffer were used to reveal specific bands of pea proteins. The final revelation was obtained after incubation with goat-anti rabbit IgG conjugated to horseradish peroxidase and addition of the peroxidase staining mixture [2.2 mmol/L 4-chloro-naphtol, 0.02% hydrogen peroxide in methanol:PBS (1:10, v:v)].
Statistical analyses of DMI and ileal flow of soluble protein data were carried out with the SAS statistical software package SAS/STAT (version 6; SAS Institute), using the general linear models procedure. When a significant effect was found (P < 0.1), the means were compared using Duncan’s Multiple Range test. Densitometry data for the major separated protein bands were analyzed by a Kruskal-Wallis test using the StatView software package (version 4.5; Abacus Concepts). Differences among groups were identified using the distribution-free multiple comparisons based on Kruskal-Wallis rank sums. Differences were considered significant at P < 0.1.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Pea proteins extracted from the 4 diets and separated by SDS-PAGE were identified by immunoblotting (Fig. 1). The main constituent polypeptides of legumin, vicilin, lectin, and PA2 were noted on the gel. The 
major (L1), the minor (L2), and the ß polypeptides of legumin, of MW 40, 25 and 20 kDa, respectively, were detected by the specific legumin antibodies. All of the vicilin constitutive polypeptides of 50 (V2), 33 (V3), 30 (V4), 19 (V5), and 16.5 kDa MW (V6) were detected except that at 12.5 kDa. Convicilin (70 kDa) (V1) cross-reacting with vicilin antibodies was also detected. Lectin (only the ß polypeptides) (ß-L) and PA2 were detected by the specific antibodies at 17 and 26 kDa, respectively. By SDS-PAGE analysis, no quantitative or qualitative difference was visible among the 4 pea-containing diets. By immunoblotting, the specific antibodies against the 4 main pea proteins recognized these proteins with the same intensity in the different diets.
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Albumin PA2 (26 kDa) was not detected except in 1 pig fed the Solara-containing diet. A 15-kDa band corresponding to a cleaved peptide from PA2 was identified by immunoblotting (Fig. 3A) in soluble protein extracts from pooled ileal digesta of pigs fed the Solara or Eiffel-containing diets. The detection levels differed according to the pea cultivar contained in the diet, suggesting variations in the amounts of resistant peptide. Resistant peptide from PA2 of the Madria cultivar-containing diet was detected in smaller amounts than the peptide of Solara- or Eiffel-containing diets. Micro-grinding improved the PA2 susceptibility to digestion, i.e., less resistant peptide was detected in the Solara micro-grinding pea-containing diet.
The lectin (17 kDa) present in control pea protein had the same MW in the ileal digesta (Fig. 3B). The amount detected was less for the Madria and Solara micro-grinding containing-diets than for Solara- and Eiffel-containing diets.
The lower levels of lectin and of the cleaved peptide from PA2 in the ileal soluble protein extracts of pigs fed the Madria-containing diet may be explained by the lower proportion of these dietary proteins in these samples because this diet induced the greatest endogenous losses. The lower levels of lectin and of the cleaved peptide from PA2 in the ileal soluble protein extracts of pigs fed the micro-ground Solara-containing diet may be explained by the lower amount of the fraction ranging from 10 to 95 kDa (analyzed by the electrophoretic method), balanced by the higher amount of the fraction with MW < 10 kDa (not analyzed by this method).
Albumin PA1b (3.7 kDa) was detected in the same amounts in digesta and in pea protein control (Fig. 3C) for the 4 analyzed soluble extracts.
The 2 methods used to identified the indigestible peptides were complementary. The LC-MS-MS was particularly suited for identifying proteins of endogenous origin because this method did not require specific knowledge of the proteins that were present at the terminal ileum. In contrast, in immunoblotting, antibodies to specific predefined proteins are required. However, immunodetection was more sensitive than LC-MS-MS. The cleaved peptide from PA2 not detected by the Coomassie blue could not be analyzed by LC-MS-MS. From the results obtained with these 2 techniques, we could conclude that the ileal proteins that were soluble under the chosen extraction conditions and of MW between 15 and 95 kDa under reducing conditions were quantitatively of endogenous origin; however, some dietary proteins, especially from the albumin fraction, were present.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Identification of endogenous losses. At the terminal ileum, the principal soluble protein losses ranging from 15 to 95 kDa were mainly of endogenous protein origin. In accordance with the literature, endogenous gut protein losses consist mainly of enzymes and antibodies, and significant differences were observed between pea cultivars. Secretion and reabsorption of endogenous gut proteins were influenced by factors such as body weight, dietary fiber content, DMI, and dietary ANF content. In our study, the 3 pea cultivars chosen had similar trypsin inhibitor content but different fiber contents. The Madria cultivar had a higher fiber content than the Eiffel and Solara cultivars and induced the highest endogenous protein losses (3.75 g/kg of DMI for Madria vs. 2.88 for Eiffel vs. g/kg DMI 2.41 for Solara).
Effect of micro-grinding. In agreement with Lahaye et al. (21), the ileal endogenous nitrogen flow was not modified by a reduction in particle size due to micro-grinding of Solara as previously reported (16); according to the present data, the ileal soluble protein flow, although considered to be primarily of endogenous origin, decreased (Solara vs. micro-ground Solara). However, with our method, only a part of the nitrogen losses was analyzed. Although the same amount of soluble protein of each digesta was put on each track of the gel, the fraction analyzable by electrophoresis (proteins with MW > 15kDa) compared with the proportion that could not be analyzed (peptides < 15kDa) could change from one digesta to another due to the diet. The low digestibility of a diet may be explained by the low degradation of its proteins or by the low absorption of peptides after degradation by digestive enzymes. The access of dietary proteins to enzymes was improved with the particle size reduction. Consequently, dietary proteins were more rapidly degraded, increasing the endogenous protein degradation and the production of small peptides. Because the proportion of small peptides vs. proteins increased after micro-grinding, we hypothesized that reabsorption was a limiting step due to a saturation of absorption sites. In general, the reduction of diet particle size improves N and AA digestibilities (22–24). PA2 hydrolysis was improved by the reduction in particle size because the smaller amounts of the residues from this protein were detected in the digesta of pigs fed finely ground meal (Solara vs. micro-ground Solara diets). These results may explain in part the gain in real nitrogen digestibility measured in the experiment reported previously (16). Indeed, micro-grinding improved the real digestibility of the Solara containing diet by 8.2 points. Cell walls may be important barriers for protein digestion and, with their disruption, the protein content becomes more accessible to enzymes (25).
Identification of remaining dietary proteins.
Lectin was shown to be highly resistant to proteolytic breakdown in vivo (10,26–28) and to represent an important part of the ileal soluble proteins. Pea lectin, made of 2 , ß subunits, of 6 and 17 kDa, respectively, has a high content in ß sheet regions and a compact structure (29). This protein has the capacity to form complexes with sugars. Even though pea lectin was shown to be innocuous for the pig gastrointestinal tract (12), this protein may form a complex with dietary sugars, for example, and become inaccessible to digestive enzymes. These structural and behavioral characteristics may explain pea lectin resistance. Albumin PA1b was resistant to the pig gastric and small intestinal enzymes and also in the sheep rumen (30,31). The resistance to hydrolysis of this albumin fraction at the terminal ileum of pigs may explain in part the small amount of cysteine obtained by digestion of pea protein in pigs (32). PA1 in peas represents <10% of total pea proteins but was reported to contribute about half of the total sulfur content in the mature seed (33). The major legume proteins, the globulins, were well degraded in the pig gastric and small intestine. Therefore, the lower digestibility of pea or other legume proteins compared with animal proteins such as casein is not due to the globulin fraction, which appears to be more digestible than the albumin fraction in many animal species, such as chickens, rats, or even humans (10,34,35). However, differences in susceptibility to hydrolysis for the 2 major proteins were observed depending on the animal species. For pigs (results presented here) and for piglets (28), the degradation of and ß polypeptides of legumin was complete, but for chickens (10), rats (26), or in the sheep rumen, only the polypeptides were degraded, and the ß polypeptides were resistant. These results may be explained by the legumin structure and the gastric pH. The 11S fraction was shown to be destabilized at low pH (36), but in chickens or sheep, the time in the stomach is limited or even nonexistent. By contrast, in pigs, the gastric pH is low and may destabilize the globular structure, permitting the ß polypeptides to become accessible to digestive enzymes.
Vicilin was totally hydrolyzed in the pig gastric and small intestine, whereas polypeptides of 50 and 19 kDa were still weakly detected at the terminal ileum (28) in weaned piglets. In chickens and rats, the pea 7S fraction was also degraded (10,26). Others 7S proteins, such as ß conglycinin of soybean or ß conglutin of lupin, were well degraded, whereas phaseolin, the 7S protein of kidney bean was shown to be resistant (37,38). Indeed, in contrast to native phaseolin, whose degradation by the major gastrointestinal enzymes was restricted to a vulnerable central region of its constituent polypeptides (39), it was shown that vicilin may be hydrolyzed by trypsin (40). This higher susceptibility to hydrolysis of vicilin compared with phaseolin may be explained by the lower level of vicilin glycosylation and by its heterogeneous peptide composition due to post-translational peptide cleavages (41).
In conclusion, at the pig terminal ileum, the correlations between dietary or endogenous ileal protein flows and the nitrogen flow allowed us to attribute the band in the range of 5–100 kDa to proteins of dietary or endogenous origin. Proteins in the range of 25–100 kDa were endogenous proteins, whereas those with a smaller molecular weight were degraded dietary proteins. Identification by immunoblotting and LC-MS-MS analyses of the ileal digesta proteins confirmed these results. The major pea storage proteins were well degraded, whereas the albumin proteins (lectin, PA1b, and PA2) were resistant in porcine gastric and small intestine. Particle size reduction improved PA2 susceptibility to hydrolysis and decreased the endogenous losses in the MW range from 15 to 95 kDa. As shown by many authors, it is difficult to establish the relations among pea cultivar composition, the presence of ANF, and variation in digestibility. The genotypic variability of the protein composition, especially of resistant proteins (albumin fraction, i.e., lectin, PA2, and PA1b), will not greatly affect the ileal flow of soluble proteins because this fraction contains essentially endogenous proteins.
To explain the variations in ileal loss, we must consider the low peptide fraction, which was not analyzed in our present work. Quantitative variations in this fraction could be due to limited hydrolysis of the dietary proteins or limited adsorption of the peptides, hypotheses that remain to be verified.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by the regions Bretagne and Pays de la Loire (PAO, Pôle Agronomique Ouest).
4 Abbreviations used: AA, amino acid; ANF, antinutritional factors; BW, body weight; CP, crude protein; DM, dry matter; DMI, dry matter ingested; DU, density unit; MW, molecular weight; PA, pea albumin.
Manuscript received 29 September 2004. Initial review completed 14 November 2004. Revision accepted 13 January 2005.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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