![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
,**
*
Unité Mixte de Recherche sur le Veau et le Porc, INRA-ENSA de Rennes, 35042 Rennes Cedex, France;
Instituto Superior de Agronomia, 1349-017 Lisboa, Portugal;
**
Instituto de Tecnologia Química e Biológica, 2781-901 Oeiras, Portugal; and
Departamento de Zootecnia, Universidade de Évora, 7002-554 Évora, Portugal
2To whom correspondence should be addressed. E-mail: lalles{at}roazhon.inra.fr.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
KEY WORDS: • piglets • endogenous proteins • serine-protease • proteins • densitometry • sequence analysis
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
). However, this often leads to decreased apparent digestibility of protein and impaired piglet performance. This could be explained in part by the lower intrinsic digestibility of legume proteins because of their compact structure (2
). Other components such as antinutritional factors, including protease inhibitors, lectins, tannins, fiber and other unidentified compounds, may interact with the gut to stimulate endogenous secretion (3
). Ileal losses of endogenous protein have been quantified in pigs fed with diets containing soybean products by several investigators (3
). By contrast, data concerning peas (4
,5
), faba beans (6
), lupin (7
) and chickpeas (8
) are scarce. Endogenous protein could be partitioned into host and bacterial fractions. The former include proteins from desquamated cells, digestive enzymes and mucin, which represents a major contribution (9
,10
). Apart from mucin, most endogenous proteins have been neither quantitated nor biochemically identified at the ileum. The aim of this study was to determine in weaned piglets the influence of legume grains on the ileal flow of some particular endogenous proteins and to attempt identifying them using sodium dodecyl sulfate (SDS)3 polyacrylamide gel electrophoresis (PAGE), densitometry, and N-terminal amino acid sequences.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Experiments were conducted under the guidelines of the Portuguese Ministry of Agriculture for animal research. In expt. 1, 30 crossbred Duroc x Landrace male piglets [8.74 ± 0.25 kg body weight (BW); Universidade de Évora, Portugal] were weaned at 28 d of age. They were divided into five groups of six animals based on their litter origin and average BW at weaning. Similarly, in expt. 2, 18 male piglets (8.23 ± 0.26 kg BW) were divided into three groups of six animals. One piglet from the control group in expt. 2 died 4 d after surgery and was not replaced (8
). Just after weaning, piglets were individually housed in metabolic cages (1.00 m x 0.70 m) where they were fed one of the five (expt. 1) or of the three (expt. 2) experimental diets for 4 wk. The piglets were surgically prepared at d 12 of the experiment with an ileo-rectal anastomosis for ileal digesta collection, according to Bengala Freire et al. (11
).
A control diet (C) with casein as the sole protein source and six experimental diets in which 50% of crude protein (N x 6.25) was supplied by soybean meal (SBM; Glycine max L.), pea (P; Pisum sativum L.), faba bean (FB; Vicia faba L.) or blue lupin (L; Lupinus angustifolius L.) seeds in expt. 1, and white kabuli type (WCP) or black desi type (BCP) chickpea (Cicer arietinum L.) seeds in expt. 2 were formulated (Table 1)
. The legume seeds were analyzed for trypsin inhibitor activity (TIA) according to the method of the American Oil Chemists Society (12
) and faba bean and black chickpea seeds were analyzed for condensed tannins using the vanillin test (13
). Chemical composition of the experimental diets presented in Table 2
was analyzed as described previously (8
). Due to differences in legume seed botanical origin and composition, the experimental diets differed in starch and fiber contents.
|
|
Collection and preparation of ileal digesta samples.
Ileal digesta were collected from anastomosed piglets over 7 d during wk 4 of the experimental period for determination of ileal nutrient digestibility, as described previously (8
). Briefly, ileal digesta were individually collected, at 
4-h intervals, using plastic trays placed under the cages. Each 4-h fraction was mixed with sodium benzoate and phenylmethylsulfonyl fluoride (10 and 0.37 g/kg digesta, respectively) to minimize further protein breakdown and immediately stored at -20°C until the end of the collection period. Afterwards, representative individual samples (600 g/piglet) of digesta from the 7-d period were prepared, freeze-dried and ground in a Retsch mill (Retsch 5657; Haan, Germany) through a 1-mm mesh screen.
Soluble protein was extracted from ileal digesta samples by stirring in borate buffer (100 mmol/L H3BO3, 150 mmol/L NaCl, pH 8.0) for 1.5 h (300 g digesta/L buffer) and centrifuged at 12000 x g for 10 min at room temperature. The supernatants were collected and stored at -20°C until electrophoresis analysis. Soluble protein was measured using the method of Lowry et al. (14
).
SDS-PAGE and densitometry measurements.
Electrophoresis procedure for densitometric analysis was carried out using a mini-gel apparatus (Touzart & Matignon AE-6450; Vitry-sur-Seine, France) in a 125 g/L acrylamide separating gel and a 45 g/L acrylamide stacking gel, according to the method of Laemmli (15
). The samples were dissolved in 1 mol/L Tris-HCl buffer (pH 6.8) containing 2.7 mol/L glycerol, 0.139 mol/L SDS and 0.4 mmol/L mercaptoethanol, and heated to 100°C for 3 min, for reduction of disulfide bonds. The samples were loaded onto the gel, with equivalent amounts of soluble proteins (200 µg) deposited in each well. Molecular mass standards (14.4–97.0 kDa; 17-0446-01; Pharmacia, Uppsala, Sweden) were also loaded in a separate well. Electrophoresis was performed in 62.5 mmol/L Tris-HCl buffer with 3.4 mmol/L SDS for 2 h. The electric field conditions were 220V and 40mA and were set in a Touzart & Matignon unit (AE-8350) connected to an Atto-Touzard vat. Protein electrophoresis was monitored by Coomassie blue R-250 staining.
Gels with blue-stained proteins were scanned using a phosphor imager (Quantum Appligene, Version 2.03; Illkirch, France). Densitometry measurements were performed using image analysis (ImageQuaNT, Version 4.2a; Molecular Dynamics, Sunnyvale, CA). The distance of protein migration along the gel was converted into a molecular mass scale using the standard proteins run in the same gel. The molecular mass of particular bands easily distinguishable visually was estimated by linear regression from the migration curves of the standard proteins run in the same gel.
First, densitometry profiles were converted into arbitrary density unit (ADU) concentrations, taking account of ileal digesta protein solubility in the extraction buffer. Second, ADU flow was calculated multiplying ADU concentrations by the ileal flow (g/d) of soluble protein determined from ileal digestibility data (7
,8
). Third, ileal ADU flow was expressed on a BW basis [ADU/(d · kg BW)].
Immunodetection of legume proteins in ileal digesta.
Hyperimmune plasmas were prepared in New Zealand white rabbits (CEGAV, Saint-Mars d’Egrenne, France) against the crude protein extracts of each legume seed, according to Kilshaw and Sissons (16
).
SDS-PAGE of crude protein extracts and ileal digesta samples was performed as described above. Protein loads were 40 and 200 µg per well for the crude protein extracts and ileal digesta samples, respectively. Separated proteins were transferred from the gel to a nitrocellulose membrane (NC; Millipore Corporation, Bedford, MA) following a semidry protocol with a transblot apparatus (Pharmacia). The gel and corresponding membrane were held in a buffer containing 3.4 mmol/L SDS and 200 mL/L methanol for 45 min. Protein transfer on NC was monitored by Ponceau red staining (P-7170; Sigma, Saint Quentin Fallavier, France). The membranes were saturated using 50 g/L skim milk powder in 150 mmol/L Tris-HCl buffer (pH 7.6) for 1 h and incubated overnight with hyperimmune plasmas at a 1:500 dilutions in 150 mmol/L Tris-HCl buffer (pH 7.6) + 50 g/L skim milk powder. After two washings with Tris-Tween 20 buffer (0.5 mL/L), the membranes were incubated with the goat anti-rabbit IgG labeled with peroxidase (dilution 1:1000) for 2 h. Finally, binding was revealed using diaminobenzidine (D-5905; Sigma) as the substrate in 150 mmol/L Tris-HCl buffer (pH 7.6) containing 7 mL/L nickel chloride and 1 mL/L H2O2.
SDS-PAGE and amino acid sequence.
One representative ileal digesta sample from a piglet fed the black chickpea diet was found, after electrophoresis was conducted as described for all samples above, to be particularly rich in a group of proteins that were also over-represented in the pigs fed the legume-based diets compared with the controls. This sample was subjected to additional electrophoresis on a full-sized slab gel (17 cm x 19 cm) according to the method of Yuen et al. (17
) before amino acid sequencing of protein bands of interest. All the solutions used in the gels and the electrophoresis buffers were freshly prepared and filtered (Whatman 3MM). The SDS used was either ultra-pure or twice recrystallized from ethanol and water. The gels were subjected to a pre-electrophoresis (30 min at 200 V) to prevent blockage of the amino terminals of digesta polypeptides (18
). Also, 50 µmol/L glutathione was added to the upper buffer to avoid degradation of tryptophan and methionine residues (18
). Electrophoresis was performed after addition of 0.1 mmol/L thioglycolic acid on the upper buffer. This was done to remove charged impurities, noncharged reactive species like acrylamide monomers and other reactive substances and to reduce peroxides and residual radicals (18
).
Three separated proteins were blotted from the gel to Millipore Immobilon-P [poly-vinylidene difluoride polymer (PVDF)] transfer membranes (0.45 µm; Millipore Corporation). The buffer used for electroblotting was a 10 mmol/L solution of 3-[cyclohexamide]-1-propanosulfonic acid in 100 mL/L methanol. Electroblotting was performed for 1 h and 15 min at 15 V and 4°C, following a semidry protocol with a transblot apparatus (Pharmacia). Protein transfer on PVDF membranes was monitored by Ponceau red staining (P-7170; Sigma). Proteins immobilized on the membranes were used for N-terminal sequencing according to Hewick et al. (19
).
The N-terminal sequences of the major blue-stained proteins in digesta were determined by Edman degradation on an automatic sequencer (model cLC Procise Sequencing System) from Applied Biosystems (Foster City, CA). The PVDF-blotted proteins were loaded on the top of a polybrene-coated, precycled, glass fiber filter. The manufacturer’s pulsed liquid Edman chemistry program was used to degrade the proteins. The sequence analysis was determined for the first 10–11 N-terminal amino acids from each protein.
Identification of sequences and statistical analysis.
The amino acid sequences were compared and identified using the Fasta database program of the European Bioinformatics Institute (Cambridge, UK) (20
).
Statistical analyses of BW, dry matter intake (DMI) and ileal flow of soluble protein data were carried out with SAS statistical software package SAS/STAT (Version 6; SAS Institute, Cary, NC), using the general linear models procedure (GLM). When a significant effect was found (P < 0.05), the means were compared using the 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, Berkeley, CA). Differences among groups were identified using the distribution-free multiple comparisons based on Kruskal-Wallis rank sums (21
). Differences were considered significant at P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
At the end of expt. 2, pigs fed the legume-containing diets were heavier (P < 0.05) than the controls (Table 3)
. Those pigs fed the legume-containing diets had a dry matter intake (DMI) that was (P < 0.05) or tended to be (P < 0.10) higher than the controls, in expt. 2 and expt. 1 (Table 4)
, respectively. However, DMI per kilogram of BW did not differ among treatment groups (P > 0.05). The ileal flow of soluble proteins was greater in the pigs fed the legume-containing diets than in the controls in both experiments.
|
|
Two representative SDS-PAGE gels of ileal digesta soluble protein extracts after Coomassie R-250 blue staining show the MW diversity of stained digesta proteins, including the strong staining of three protein bands between 25 and 30 kDa (Fig. 1
).
|
), and 2 (Fig. 2
B) the legume-based diets generated a sharp increase in average ADU flow in the molecular mass range of 25 to 30 kDa compared with the control diet. The protein bands located at molecular masses of 25 and 30 kDa had an ADU ileal flow that was significantly higher (P < 0.05) in all the groups of pigs fed the legume-based diets compared with the controls (Tables 5
and 6)
. This was also true for the protein band at 27 kDa of molecular mass in expt. 2 (Table 6
, P < 0.05). A similar tendency (P < 0.10) was observed for the 27-kDa protein in expt. 1 (Table 5)
. In contrast, the ADU flows of protein bands located at molecular masses of 34, 44, 58 and 103 kDa were not affected by treatment.
|
|
|
The hyperimmune plasmas raised against crude extracts of proteins from legume seeds recognized a large array of protein bands in these extracts (Fig. 3
lanes 2 and 3). By contrast, no staining with these antibodies was observed in the molecular mass range of 25 to 30 kDa in ileal digesta of pigs fed peas (lane 5) or black chickpeas (lane 7). Similar results were observed for ileal digesta samples from piglets fed the other legume-containing diets (data not shown).
|
Eleven N-terminal amino acids of the 25-kDa protein in ileal digesta were identified by N-terminal sequencing (Table 7)
. Ten amino acid residues were identified for the 27- and 30-kDa proteins. The 27-kDa protein band yielded a mixture of two proteins with the N-terminal sequence reported in Table 7
corresponding to the major sequence signal. The 30-kDa protein band of pig ileal digesta had an N-terminal sequence with eight residues in common with the 27-kDa protein.
|
. The N-terminal amino acid sequence of the 25-kDa protein was similar to the N-terminal sequence of porcine trypsin, except for the N-terminal residue that was valine for our 25-kDa protein, instead of isoleucine for trypsin (22
,23
). The 27-kDa protein had a sequence similar to that of pig coagulation factor IX between residues 45 and 54 (24
), except that valine at location 8 in our 27-kDa protein corresponded to a lysine residue at position 52 of coagulation factor IX. The 27-kDa protein N-terminal sequence was also 80% identical with porcine chymotrypsin homolog p31 between residues 16 and 25 (25
). Glycine and asparagine residues at position 3 and 6 of the 27-kDa protein N-terminal sequence corresponded to asparagine and glutamic acid residues at positions 18 and 21, respectively, of chymotrypsin. The N-terminal sequence of the 30-kDa protein band of pig ileal digesta was closer to coagulation factor IX than to chymotrypsin (80 and 60% identity, respectively). The 30-kDa protein had valine and isoleucine residues in positions 1 and 8, instead of isoleucine and valine, respectively, for the 27-kDa protein. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
strongly support an endogenous origin for these proteins. However, they all differed from the 57-kDa endogenous protein found in chickens fed raw peas (26
).
Trypsin and chymotrypsin are the major pancreatic serine proteases dumped into the duodenum during the digestion process. An increase in their concentration at the ileum could reflect enhanced pancreatic secretion and/or decreased digestion along the small intestine. Pancreatic enzyme secretion increases through a cholecystokinin-mediated regulation pathway, in response to soybean or trypsin inhibitor intake, at least in rats and birds (27
). This does not seem to be true in pigs. Dietary inclusion of soybean products with different levels of trypsin inhibitor (38 and 9 mg TIA/g) (28
) or peas (29
) had minor effects on pancreatic protease secretion. Here, trypsin inhibitor activity varied from 0.2 to 7.0 mg TIA/g in blue lupin and white chickpeas, respectively. This influenced neither pancreas relative weight nor trypsin- or chymotrypsin-specific and total activities in the gland (7
,8
). Thus, it is unlikely that pancreatic secretion was enhanced in this study.
Other antinutritional factors present in legume seeds that could stimulate pancreas secretion are tannins and lectins. Tannins from faba beans provided as seeds (29
) or hulls (6
) did not increase the volume of secretion of pancreatic juice and total activities of trypsin and chymotrypsin in young pigs. There is little information concerning the effect of lectins on pancreas secretion (30
). Legume grains also are known to differ in the type and amount of fiber. Effects of fiber on the exocrine pancreas remain unclear. As reviewed by Jakob et al. (31
), pectin, cellulose and straw meal had no effect on pancreas total protease output, contrary to wheat bran or potato fiber, which increased trypsin secretion in some studies. Nevertheless, the antinutritional factors mentioned above (32
) as well as pea inner fiber (but not wood cellulose) (4
) and neutral detergent fiber (33
)clearly increased ileal losses of endogenous proteins in pigs. Also, a four- to six-fold increase in the ileal flow of active trypsin was demonstrated after 4 wk of consumption of a milk replacer containing raw peas in preruminant calves (34
). Phaseolin, a 7S kidney bean (Phaseolus vulgaris) storage protein, fed in a purified form also dramatically increased fecal losses of endogenous proteins in rats (35
). This protein, which was found to bind intestinal walls, was, therefore, considered by these authors to act as a natural secretagogue. Legume storage proteins resistant to gastrointestinal digestion have been found in pigs fed kidney beans (36
), calves fed heated soybean flour (37
) and chickens fed raw peas (26
). Whether these dietary proteins bear secretagogue properties in these animal species is unknown.
A second hypothesis for increased ileal loss of trypsin and chymotrypsin is reduced intestinal protein hydrolysis and amino acid absorption. While protease inhibitors bind trypsin and/or chymotrypsin (27
), tannins complex proteins (including enzymes) flowing along the gastrointestinal tract, regardless of their endogenous or dietary origin (38
). Altogether, this would contribute to reducing the efficiency of endogenous protein digestion. It is estimated to be above 75% before the terminal ileum of pigs in normal circumstances (39
,40
). Dietary inclusion of the legume seeds and soybean meal studied here increased the flow of undigested nutrients at the ileum of piglets (7
,8
). More undigested components along the small intestine could have contributed further to the protection of pancreatic enzymes from degradation because intraluminal nutrients have been shown to enhance survival of pancreatic enzyme activities in the small intestine (41
). Finally, due to differences in antinutritional activities and fiber type and contents of the legumes studied here, it is difficult to attribute the increased ileal flow of the 25-, 27- and 30-kDa proteins to a particular factor. Rather, all these factors may have contributed to the observed phenomenon.
A third hypothesis for increased ileal flow of specific endogenous proteins as observed here is the assault of the gut lining with dietary constituents, thus, leading to enhanced protein secretion or loss from the gastrointestinal tract and leakage from the blood. Greer and Pusztai (42
) demonstrated that feeding kidney beans to rats increased vascular permeability and gut luminal accumulation of plasma protein. It was suggested that lectin was partly responsible for this leakage. In this study, legume seeds and soybean meal consumption resulted in altered morphology and brush border enzyme activities in the duodenum (7
,8
).
Inclusion of legume grain or soybean meal into diets of weaned piglets enhanced ileal losses of three proteins of 25, 27 and 30 kDa. These have been identified as having N-terminal amino acid sequences close to endogenous proteins of the serine protease family. Future investigations will be aimed at purifying, characterizing and specifically assaying these proteins to determine their contribution to ileal losses of essential amino acids in pigs.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
3 Abbreviations used:ADU, arbitrary density units; BCP, black chickpea; BW, body weight; C, control; DMI, dry matter intake; FB, faba bean; L, blue lupin; NC, nitrocellulose; P, pea; PAGE, polyacrylamide gel electrophoresis; PVDF, poly-vinylidene difluoride polymer; SBM, soybean meal; SDS, sodium dodecyl sulfate; TIA, trypsin inhibitor activity; WCP, white chickpea.
Manuscript received 9 November 2001. Initial review completed 4 January 2002. Revision accepted 26 February 2002.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Gatel, F. (1994) Protein quality of legume seeds for non-ruminant animals: a literature review. Anim. Feed Sci. Technol. 45:317-348.
2. Nielsen, S. S., Deshpande, S. S., Hermodson, M. A. & Scott, M. P. (1988) Comparative digestibility of legume storage proteins. J. Agric. Food Chem. 36:896-902.
3. Nyachoti, C. M., De Lange, C.F.M., MacBride, B. W. & Schulze, H. (1997) Significance of endogenous gut nitrogen losses in the nutrition of growing pigs: a review. Can. J. Anim. Sci. 77:149-163.
4. Leterme, P., Froidmont, E., Rossi, F. & Théwis, A. (1998) The high water-holding capacity of pea inner fibers affects the ileal flow of endogenous amino acids in pigs. J. Agric. Food Chem. 46:1927-1934.
5. Hess, V., Thibault, J. N. & Sève, B. (1998) The 15N amino acid dilution method allows the determination of the real digestibility and of the ileal endogenous losses of the respective amino acid in pigs. J. Nutr. 128:1969-1977.
6. Jansman, A.J.M., Verstegen, M.W.A., Huisman, J. & Van den Berg, J.W.O. (1995) Effects of hulls of faba bean (Vicia faba L.) with a low content of condensed tannins on the apparent ileal and fecal digestibility of nutrient and the excretion of endogenous protein in ileal digesta and faeces of pigs. J. Anim. Sci. 73:118-127.[Abstract]
7. Salgado, P., Freire, J.P.B., Mourato, M., Cabral, F., Toullec, R. & Lallès, J. P. (2001) Comparative effects of different legume protein sources in weaned piglets: nutrient digestibility, intestinal morphology and digestive enzymes. Livest. Prod. Sci. 74:191-202.
8. Salgado, P., Lallès, J. P., Toullec, R., Mourato, M., Cabral, F. & Freire, J.P.B. (2001) Nutrient digestibility of chickpea (Cicer arietinum L.) seeds and effects on the small intestine of weaned piglets. Anim. Feed Sci. Technol. 91:197-212.[Medline]
9. Lien, K. A., Sauer, W. C. & Fenton, M. (1997) Mucin output in ileal digesta of pigs fed a protein-free diet. Z. Ernahrungswiss 36:182-190.[Medline]
10. Montagne, L., Toullec, R., Formal, M. & Lallès, J. P. (2000) Influence of dietary protein level and origin on the flow of mucin along the small intestine of the preruminant calf. J. Dairy Sci. 83:2820-2828.[Abstract]
11. Bengala Freire, J. P., Hulin, J. C., Peiniau, J. & Aumaitre, A. (1988) Determination of ileal digestibility by shunt technique in the early-weaned pig: methodological aspects and utilisation of starch-rich diets. Livest. Prod. Sci. 20:233-247.
12. American Oil Chemists Society (1983) Trypsin inhibitor activity. Official and Tentative Methods of the American Oil Chemists Society Ba Official Methods 1983:12-75 American Oil Chemists Society Washington, DC. .
13. Price, M. L., Scoyoc, S. & Van Butler, L. G. (1978) A critical evaluation of the vanillin reaction as an assay for tannin in sorghum grain. J. Agric. Food Chem. 26:1214-1218.
14. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.
15. Laemmli, U. K. (1970) Cleavage of structural proteins during the heat of bacteriophage T4. Nature 227:680-685.[Medline]
16. Kilshaw, P. J. & Sissons, J. W. (1979) Gastrointestinal allergy to soya bean protein in preruminant calves: antibody production and digestive disturbances in calves fed heated soya bean flour. Res. Vet. Sci. 27:361-365.[Medline]
17. Yuen, S., Hunkapiller, N. W., Wilson, K. J. & Yuan, R. M. (1986) SDS-PAGE electroblotting. User Bulletin 25 1986 Applied Biosystems, Inc Foster City, CA. .
18. Moos, M., Jr., Nguyen, N. Y. & Liu, T. Y. (1988) Reproducible high yield sequencing of proteins electrophoretically separated and transferred to an inert support. J. Biol. Chem. 263:6005-6008.
19. Hewick, R. M., Hunkapiller, M. W., Hood, L. E. & Dryer, W. J. (1981) A gas-liquid solid phase peptide and protein sequenator. J. Biol. Chem. 256:7990-7997.
20. Pearson, W. R. & Lipman, D. J. (1988) Improved tools for biological sequence comparison. Proc. Nat. Acad. Sci. USA 85:2444-2448.
21. Kruskal, W. H. & Wallis, W. A. (1952) Use of ranks in one criterion variance analysis. J. Am. Stat. Assoc. 47:614-617.
22. Charles, M., Rovery, M., Guidoni, A. A. & Desnuelle, P. (1963) Trypsinogen and trypsin of pig. Biochim. Biophys. Acta 69:115-129.
23. Hermodson, M. A., Ericsson, L. H., Neurath, H. & Walsh, K. A. (1973) Determination of the amino acid sequence of porcine trypsin by sequenator analysis. Biochemistry 12:3146-3153.[Medline]
24. Sarkar, G., Koeberl, D. D. & Sommer, S. S. (1990) Direct sequencing of the activation peptide and the catalytic domain of the factor IX gene in six species. Genomics 6:133-143.[Medline]
25. Gorr, S.-U., Hamilton, J. W. & Cohn, D. V. (1992) Regulated, but not constitutive, secretory proteins bind porcine chymotrypsinogen. J. Biol. Chem. 267:21595-21600.
26. Crévieu, I., Carré, B., Chagneau, A. M., Quillien, L., Guéguen, J. & Bérot, S. (1997) Identification of resistant pea (Pisum sativum L.) proteins in the digestive tract of chickens. J. Food Agric. Chem. 45:1295-1300.
27. Liener, I. E. (1994) Implications of antinutritional components in soybean foods. Crit. Rev. Food Sci. Nutr. 34:31-67.[Medline]
28. Li, S., Sauer, W. C. & Caine, W. R. (1998) Response of nutrient digestibilities to feeding diets with low and high level of soybean trypsin inhibitors in growing pigs. J. Sci. Food Agric. 76:357-363.
29. Gabert, V. G., Sauer, W. C., Li, S. & Fan, M. Z. (1996) Exocrine pancreatic secretions in young pigs fed diets containing faba beans (Vicia faba) and peas (Pisum sativum): concentrations and flows of total, protein-bound and free amino acids. J. Sci. Food Agric. 70:256-262.
30. Sauer, W. C. & Mosenthin, R. (1999) Anti-nutritionnal factors and exocrine pancreatic secretion in pigs. Pierzynowsky, S. G. Zabielski, R. eds. Biology of the Pancreas in Growing Animals 1999:371-380 Elsevier Sciences BV Amsterdam, The Netherlands. .
31. Jakob, S., Mosenthin, R. & Sauer, W. C. (1999) Carbohydrates and exocrine pancreatic secretion in pigs. Pierzynowsky, S. G. Zabielski, R. eds. Biology of the Pancreas in Growing Animals 1999:361-370 Elsevier Sciences BV Amsterdam, The Netherlands. .
32. Lallès, J. P. & Jansman, A.J.M. (1998) Recent progress in the understanding of the mode of action and effects of antinutritional factors from legume seeds in non-ruminant farm animals. Jansman, A.J.M. Hills, G. D. Huisman, J. Van der Poel, A.F.B. eds. Recent Advances of Research in Antinutritional Factors in Legume Seeds and Rapeseeds 1998:219-232 Wageningen Pers Wageningen, The Netherlands. .
33. Schulze, H., Van Leeuwen, P., Verstegen, M.W.A. & Van den Berg, J.W.O. (1995) Dietary level and source of neutral detergent fiber and ileal endogenous nitrogen flow in pigs. J. Anim. Sci. 73:441-448.[Abstract]
34. Lallès, J. P. & Toullec, R. (1994) Trypsin loss at the ileum of calves fed milk replacers containing legume protein. Ann. Zootech. 43:263.
35. Santoro, L. G., Grant, G. & Pusztai, A. (1999) In vivo degradation and stimulating effect of phaseolin on nitrogen secretion in rats. Plant Foods Hum. Nutr. 53:223-236.[Medline]
36. Begbie, R. & Ross, A. W. (1993) Resistance of the kidney bean reserve protein phaseolin to proteolysis in the porcine digestive tract. J. Sci. Food Agric. 61:301-307.
37. Lallès, J. P., Tukur, H. M., Salgado, P., Mills, E.N.C., Morgan, M.R.A., Quillien, L., Levieux, D. & Toullec, R. (1999) Immunochemical studies on gastric and intestinal digestion of soybean glycinin and ß-conglycinin in vivo. J. Agric. Food Chem. 47:2797-2806.[Medline]
38. Jansman, A.J.M. & Longstaff, M. (1993) Nutritional effects of tannins and vicin/convicin in legume seeds. Van der Poel, A.F.B. Huisman, J. Saini, H. S. eds. Recent Advances of Research in Antinutritional Factors in Legume Seeds and Rapeseeds 1993:301-316 Wageningen Pers Wageningen, The Netherlands. EAAP publication 70.
39. Souffrant, W. B. (1991) Endogenous nitrogen losses during digestion in pigs. Verstegen, M.W.A. Huisman, J. Den Hartog, L. A. eds. Proceedings of the Vth International Symposium on Digestive Physiology in Pigs 1991:147-166 Pudoc Wageningen, The Netherlands. EAAP Publication 54.
40. Krawielitzki, K., Kreienbring, F., Zebrowska, T., Schadereit, R. & Kowalczyk, J. (1994) Estimation of N absorption, secretion and reabsorption in different intestinal sections of growing pigs using the 15N-dilution method. Souffrant, W. B. Hagemeister, H. eds. Proceedings of the VIth International Symposium on Digestive Physiology in Pigs 1994:79-82 Dummerstorf, Germany. EAAP Publication 80.
41. Holtmann, G., Kelly, D. G., Sternby, B. & Dimagno, E. P. (1997) Survival of human pancreatic enzymes during small bowel transit: effect of nutrients, bile acids, and enzymes. Am. J. Physiol. 273:553-558.
42. Greer, F. & Pusztai, A. (1985) Toxicity of kidney bean (Phaseolus vulgaris) in rats: changes in intestinal permeability. Digestion 32:42-46.[Medline]
This article has been cited by other articles:
|
|
 |
|
  M. Le Gall, L. Quillien, B. Seve, J. Gueguen, and J. P. Lalles Weaned piglets display low gastrointestinal digestion of pea (Pisum sativum L.) lectin and pea albumin 2 J Anim Sci, November 1, 2007; 85(11): 2972 - 2981. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Le Gall, L. Quillien, J. Gueguen, H. Rogniaux, and B. Seve Identification of Dietary and Endogenous Ileal Protein Losses in Pigs by Immunoblotting and Mass Spectrometry J. Nutr., May 1, 2005; 135(5): 1215 - 1222. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. S. Gilani and E. Sepehr Protein Digestibility and Quality in Products Containing Antinutritional Factors Are Adversely Affected by Old Age in Rats J. Nutr., January 1, 2003; 133(1): 220 - 225. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
