Weaned piglets display low gastrointestinal digestion of pea (Pisum sativum L.) lectin and pea...

M. Le Gall, L. Quillien, B. Sève, J. Guéguen and J. P. Lallèslectin and albumin pea albumin 2

Weaned piglets display low gastrointestinal digestion of pea ( Pisum sativum L.)

published online June 12, 2007J ANIM SCI

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1

Running title: Resistance to digestion of pea protein1

2

Weaned piglets display low gastrointestinal digestion of pea (Pisum sativum L.) lectin 3

and albumin pea albumin 214

5

M. Le Gall,†*2 L. Quillien,‡ B. Sève,† J. Guéguen,* and J. P. Lallès†6

7

†INRA, UMR 1079, Systèmes d’Elevage, Nutrition Animale et Humaine, Domaine de la 8

Prise, 35590 Saint-Gilles, France; *INRA, Unité de Recherche Biopolymères, Interactions, 9

Assemblages, Rue de la Géraudière, 44072 Nantes, France; and ‡INRA, Unité de 10

Recherche Génétique et Ecophysiologie des Légumineuses à graines, Domaine d’Epoisses, 11

Bretenières, 21065 Dijon, France12

13

1Financial support for this study was provided by the Regions of Bretagne and Pays 14

de la Loire (Pôle Agronomique Ouest), France. The authors thank P. Touanel for care of 15

experimental animals.16

2Corresponding author: [email protected]

Page 1 of 35 Journal of Animal Science

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ABSTRACT: A study was conducted to investigate the biochemistry of digestion of field 18

pea (Pisum sativum L.) albumins and globulins in the stomach and along the small 19

intestine of weaned piglets with a particular emphasis on the respective roles of these 20

compartments in pea protein digestion. Twenty four piglets were weaned at 28 d of age. 21

They were allocated to 2 diets (control and pea) and 3 slaughter times (3, 6, or 9 h after the 22

last meal) in a 2 x×3 factorial arrangement of treatments in a randomized complete block 23

design. Pea flour provided 30% of total dietary protein in the pea diet. The diets were fed 24

for 2 wk after weaning. After slaughter, gastrointestinal tract compartments were weighed, 25

digesta collected, and pH measured. Digesta from the stomach and proximal, mid, and 26

distal small intestine (SI) were extracted for soluble proteins and analyzed for specific pea 27

proteins using SDS-PAGE, immunoblotting, and mass spectrometry. Tissue weight of the 28

whole gastrointestinal tract (GIT; P = 0.015), cecum (P < 0.001), and colon (P < 0.001) 29

was greater in the pea diet. Digesta pH in the stomach and distal SI was lower (P = 0.02) in 30

the pea diet than the control diet. In the stomach, vicilin, lectin, and pea albumin 2 (PA2) 31

were not digested, while legumin was only partly digested. Legumin and vicilin were 32

totally digested in the SI in less than 3 h. A resistant peptide of 15 kDa located at the N-33

terminus of PA2 was transiently detected at 3 h. A protein band at 20 kDa was consistently 34

identified as lectin. It was present in high intensity in intestinal digesta of pea-fed piglets at 35

all times after the meal compared with those fed the control diet (P < 0.001). Various 36

proteins of, presumably, endogenous origin, displayed differential digestion patterns 37

between the control and the pea-fed piglets (P < 0.05). In conclusion, differences in 38

digestion between specific pea proteins were observed along the GIT of piglets. They 39

could be partly explained by differences in protein digestion in the stomach.40

Key words: albumin, digestion, globulin, pea protein, pig41

Page 2 of 35Journal of Animal Science



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INTRODUCTION42

Field peas can be an alternative to soybean meal in diets for nonruminant animals. 43

However, diets with high levels of incorporation of field peas (Pisum sativum L.) usually 44

depress growth performance, especially in young animals (Gatel et al., 1994). Field peas 45

are rich in Lys but deficient in sulfur AA and Trp. Moreover, the digestibility of plant 46

protein has often been regarded to be lower than that of animal protein, which has 47

generally been attributed to the presence of anti-nutritional factors, including lectins, 48

protease inhibitors, and tannins (Lallès and Jansman, 1998). Field peas contain the trypsin-49

chymotrypsin inhibitors of the Bowman-Birk family, which have 14 cysteine residues, all 50

involved in disulfide bridges (Ferrasson et al., 1995). Protease inhibitors (PI) form a 51

complex with pancreatic proteases and inactivate them. These proteases are also resistant 52

to digestion, an observation that may contribute to explain the lower digestibility of sulfur 53

AA in pigs (Mariscal-Landin et al., 2002).54

Protein hydrolysis of field peas also is influenced by the protein structure. They fall 55

within 2 categories, namely globulins (55 to 65% of CP) and albumins (20 to 25% CP). 56

They are soluble in saline solutions of low ionic strength and water, respectively. 57

Globulins include legumin, vicilin, and convicilin. The albumin fraction consists of lectin, 58

pea albumin 1 and 2 (PA1 and PA2), PI, and lipoxygenases. Globulins seem to be more 59

digestible than albumins in various animal species (Carbonaro et al., 2000; Crevieu et al., 60

1997; Rubio et al., 1991). The consumption of field peas increases the ileal output of 61

endogenous proteins in pigs (Le Gall et al., 2005a). 62

However, little is known on the time-course of protein digestion along the 63

gastrointestinal tract (GIT), a factor that might contribute to differences in the degree of 64

hydrolysis among proteins at the ileal level. Therefore, we conducted a study to investigate 65

the post-prandial changes in pea protein digestion along the GIT in weaned piglets.66




4

MATERIALS AND METHODS67

Diets68

Two diets were formulated and their composition is given in Table 1. The control 69

(C) diet contained a mixture of skim-milk powder and fish soluble protein concentrate as 70

the sources of CP. Thirty percent of CP from this mixture was replaced by field pea flour 71

in the pea diet (P). The seeds were ground using a hammer mill prior to being incorporated 72

into the feed. The diets were balanced for CP, indispensable amino acids, with appropriate 73

DL-Met, L-Thr, and L-Trp supplements and NE values (Sève, 1994). The Baccara spring 74

pea cultivar was chosen because of its low content in trypsin inhibitors (Grosjean et al., 75

2000). It was produced locally at INRA (Station d'Amélioration des Plantes, Le Rheu, 76

France). 77

Animals and Feeding78

The experiment was conducted under the guidelines of the French Ministry of 79

Agriculture for animal research. The experimental design was a randomized complete 80

block design with a 2 × 3 factorial arrangement of 2 diets (control vs. pea) and 3 slaughter 81

times after the last meal (3, 6, and 9 h). Four litters of crossbred piglets [ Pietrain ×82

(Landrace × Large White)] were weaned at 28 d of age. Four blocks of 6 littermates were 83

formed on the basis of piglet BW, regardless of gender. The initial BW of the piglets in the 84

control and pea group was 8.0 ± 0.8 kg and 8.1 ± 0.9 kg, respectively. They were randomly 85

assigned to treatments within litters, housed in individual cages, and fed either of the 286

diets for 2 wk after weaning. The feed was given as a mash (meal to water ratio of 2:1 by 87

weight) twice daily (0900 and 1530) at a rate of 650 kJ · (BW in kg)0.75. Body weights 88

were recorded weekly, and feeding schedules were adjusted accordingly. The piglets had 89

free access to water. The piglets from 2 litters were slaughtered on d 13 and the piglets 90

from the 2 other litters on d 14 after weaning. Each day the slaughter h was randomly 91




5

assigned within litters. It took place at 3, 6, or 9 h after the morning meal. The piglets were 92

killed by electronarcosis and exsanguination. 93

Digesta Collection94

Immediately after slaughter, the GIT was removed, dissected, and divided into 795

segments: stomach, 3 equal parts in length of the small intestine (SI; proximal, mid, and96

distal), cecum, and first and second halves in length of the colon. Each segment was 97

weighed and emptied. The total amount of digesta in each segment was collected, mixed 98

thoroughly, and the pH was measured. A solution of preservatives [15 mM EDTA, 1 mM99

sodium azide, and 2 mM phenylmethylsulfonyl fluoride] was then mixed with the digesta 100

to limit enzyme and bacterial activities. The digesta were then frozen at -20°C, and101

subsequently freeze-dried, weighed, finely ground, and stored at 4°C until analysis.102

Chemical and Biochemical Analyses103

The diets were analyzed for DM, minerals, N, and AA as previously described (Le 104

Gall et al., 2005a). The freeze-dried digesta samples were analyzed for DM and N 105

contents. Protein was extracted from the diets and the stomach content by stirring in 16 106

mM Tris-HCl buffer containing 140 mM SDS with pH 8 for 90 min at room temperature 107

(10 g diet/L buffer; 50 g gastric digesta/L buffer). Protein was extracted from the digesta 108

from each part of SI by stirring in 100 mM H3BO3, 150 mM NaCl with pH 8 for 90 min at 109

room temperature (200 g digesta/L buffer). These buffers were previously shown to 110

maximize the solubilization of proteins (Bush et al., 1992). The samples were then 111

centrifuged at 12,000 × g for 10 min at room temperature. The supernatants were collected 112

and stored at -20°C until SDS-PAGE analysis and immunoblotting. Protein in the 113

supernatants was determined by the bichinchonimic acid micromethod (Pierce, Rockford, 114

IL).115

SDS-PAGE Electrophoresis, Densitometry, and Immunoblotting116




6

The SDS-PAGE procedures used were previously described in detail (Le Gall et al., 117

2005a). Protein extracts from gastric digesta, anterior intestinal digesta (proximal and mid118

SI), and posterior intestinal digesta (distal SI) were diluted 10-, 3-, and 2-fold, respectively, 119

with appropriate buffers. The amounts of protein loaded on the gels were 20, 50, and 200 120

µg per well for the CP extracts, gastric , and SI digesta extracts, respectively. Gels with 121

blue-stained proteins from SI digesta were scanned. Densitometry measurements were 122

performed using image analysis (Amount One, version 4.1, Biorad Laboratories, Hercules, 123

CA) and analyzed as described by Salgado et al. (2002). Densitometry profiles were 124

converted into arbitrary density unit (DU) and were expressed in DU/g of digesta protein 125

corresponding to the amount of protein, which was deposited on each track on the gel.126

Because SDS-PAGE patterns of proteins were generally similar among the 4 piglets fed a 127

given diet and slaughtered at the same time after the meal, only 1 representative illustration 128

is given on the figures. However, densitometry analysis was performed for all the 129

individual digesta at each site of the SI.130

Prior to immunoblotting, proteins separated by SDS-PAGE were electro-transferred 131

onto nitrocellulose membranes and were probed with rabbit antibodies for detecting 132

specifically pea legumin, vicilin, lectins, and PA2, as previously described (Le Gall et al., 133

2005a). Rabbit antibodies were detected with an anti-rabbit IgG antibody conjugated with 134

an enzyme. The membrane was incubated with the enzyme substrate.135

Immunoprecipitation and Sequencing of Indigested PA2 Polypeptides136

The protein A technology for purifying proteins using specific antibodies (Ghose et 137

al., 2005) was applied for concentrating digestion polypeptides from PA2 in anterior 138

intestinal digesta (distal SI). Briefly, some components of digesta that interacted 139

nonspecifically with protein A were removed after incubating protein A beads (20333; 140

Pierce) with soluble proteins from distal SI digesta (prepared as explained above) for 2 h at 141




7

4°C under gentle rotation and then centrifugation. The supernatant was incubated 142

overnight at 4°C with the anti-PA2 antibodies (also used for immunoblotting) for PA2-143

anti-PA2 antibody complex formation. Then the protein A beads were added to the mixture 144

and incubated for further 2 h at 4°C under gentle rotation. The resulting PA2-antibody 145

complex adsorbed on the beads was washed extensively with ice-cold phosphate buffer 146

saline (pH 7.4; 136 mM NaCl, 2.68 mM KCl, 1.46 mM KH2PO4, and 8.1 mM Na2HPO4). 147

The complex was eluted in the SDS-PAGE sample loading buffer prior to being separated 148

by SDS-PAGE and revealed by immunoblotting as described above for digesta samples. 149

After transfer to nitrocellulose membranes, the indigested PA2 polypeptides were detected 150

in 1 lane using the same anti-PA2 antibodies. The polypeptide chains from the anti-PA2 151

antibodies were revealed in an adjacent lane with a goat anti-rabbit IgG antibodies 152

conjugated to horseradish peroxidase (170 6515; Biorad Laboratories, Hercules, CA) and 153

addition of the peroxidase staining mixture (2.2 M 4-chloro-naphtol and 0.02% hydrogen 154

peroxide in 1:10 methanol, and phosphate saline buffer). The specific PA2 bands were then 155

identified on the SDS-PAGE gels and sequenced using liquid chromatography-mass 156

spectrometry/mass spectrometry (LC-MS/MS) as previously reported (Le Gall et al., 157

2005a). Briefly, bands of interest were excised from blue-stained gel. Then, they were 158

submitted to digestion with trypsin. Extracted peptides were separated by reverse phase 159

chromatography and analyzed after fragmentation in MS-MS to be consistent. Mass data 160

collected during LC-MS/MS analysis were processed with Protein Lynx Global Server 161

software (Waters; Milford, MA). Protein identification was performed by searching the 162

peptide masses and MS/MS sequence stretches against the Swiss-Prot (Swiss Institute of 163

Bioinformatics, Switzerland) and National Center for Biotechnology Information 164

(Bethesda, MD) non-redundant sequence databank.165

Statistical Analysis166




8

The experimental unit was the piglet. The data were analyzed as a randomized 167

complete block design using the GLM procedure (SAS Inst. Inc., Cary, NC). Variance 168

analysis was performed for testing the effects of diet, time after the last meal, litter, and 169

interaction between diet and time after the meal. Orthogonal contrasts were used to test 170

linear and quadratic effects of time, and the interaction was partitioned into a linear effect 171

of time × diet and a quadratic effect of time x diet.172

RESULTS173

All the piglets were in good health throughout the experiment. The diets were well 174

consumed. No difference in feed intake or BW gain was observed among treatments during 175

the experimental period (Table 2).176

The whole GIT was heavier in the piglets fed the pea diet compared to the control 177

(P = 0.015; Table 3). The difference was accounted for by the weight of the cecum (P < 178

0.001) and the large intestine (P < 0.001).179

The residual DM present in the stomach of piglets at slaughter decreased linearly (P180

< 0.001) and quadratically with time (P = 0.05; Table 4). Three hours after the meal, 51 181

and 56% of DM intake was emptied from the stomach and 15 and 12% of DM intake was 182

still present in the stomach 9 h after the last meal for control and pea groups, respectively. 183

No interaction between the type of diet and the digestion time was observed, and there was 184

no effect of the diet. In contrast, gastric pH was decreased in the pea than with the control 185

diet (P = 0.02). It decreased linearly (P < 0.001) with time. There was a tendency (P = 186

0.06) for a time × diet interaction for digesta pH in the proximal SI, which was caused by a 187

sharp decrease with time in the control group compared with the pea group (linear effect of 188

time × diet, P = 0.02). The pH in the mid SI was more variable and not affected by the 189

tested factors. Digesta pH in the distal SI was decreased with the pea as compared to the190




9

control diet (P = 0.02) without effect of time.191

SDS-PAGE, Densitometry, and Immunoblotting Analyses of Digesta192

Digestion in the Stomach. The SDS-PAGE gels stained for proteins showed quite 193

different banding patterns between the control and the pea diets (Figure 1A). Low numbers 194

of protein bands were detected in gastric digesta after 9 h as compared to those present in 195

the diet extract. Many of them were not present in the feed, so they were most probably 196

hydrolyzed dietary proteins or of endogenous origin.197

Western blotting showed the presence of vicilin and convicilin in the pea diet, but 198

not in the control (Figure 1Ba). Vicilin proteins of molecular weight (MW) of 50 and 33 199

kDa, and smaller vicilin polypeptides were transiently observed at 3 h, but not afterward. 200

The band of convicilin was still present at 3 h in the digesta of some pigs, but it 201

disappeared thereafter. Legumin was revealed in the pea diet as 2 major bands (α-major 202

and β polypeptides) of MW of 40 and 20 kDa, respectively (Figure 1Bb). These bands and 203

a cleaved α-polypeptide were still clearly observed in gastric digesta 3 h after the meal. By 204

6 h, only legumin polypeptides of MW equal to or smaller than 30 kDa were detected. 205

These were no longer present at 9 h after the meal. Only the β subunit of pea lectin was 206

seen at a MW of 17 kDa in the pea diet and in gastric digesta 3 h after the meal but not 207

thereafter (Figure 1Bc). The α subunit of lectin was not detected by antibodies. Pea 208

albumin 2 was detected at a MW of 26 kDa in the pea diet and corresponding digesta at 3 h 209

after the meal but not thereafter (Figure 1Bd). Cleaved PA2 peptides of MW between 17 210

and 25 kDa were transiently observed at 3 h. 211

Digestion in the Small Intestine. The patterns of proteins observed in the proximal 212

SI were characterized by a high number of protein bands (Figure 2A). Densitometry data213

with statistically significant effects of time or diet or both are presented in Table 5. At 214

proximal SI, the density of 4 bands (noted d1 to d4) was affected by the diet. The density 215




10

of band d1 was decreased (P = 0.003), while band d2 was greater in the pea group 216

compared to the control (P = 0.006) without effect of time. Band d3 was virtually absent in 217

the control piglets regardless of time, while its density decreased sharply between 3 and 6 h 218

over time in the pea-fed piglets (time linear × diet, P = 0.002; time quadratic × diet, P = 219

0.036). Band d4 was absent in the control group while its density was high in the pea group220

(P = 0.001) regardless of time. The resistance of lectin to digestion in the proximal SI was 221

confirmed by immunoblotting (Figure 2Bb). Legumin was detected as the β-polypeptide 222

only at 3 h and only in one piglet (Figure 2Ba). Pea albumin 2 was not evidenced by 223

immunoblotting in the proximal SI (Figure 2Bc). Finally, vicilin or digestion fragments 224

were not detected in the proximal SI (data not shown). 225

After SDS-PAGE of proteins in digesta of the mid SI, 6 protein bands (noted j1 to 226

j6; Table 5) with visually contrasted staining densities (Figure 3A) had their intensity227

influenced by the diet. An effect of time was noted for bands j4 and j5 (P = 0.023 and P = 228

0.054, respectively). The intensity decreased linearly from 3 to 9 h for band j4 (P = 0.024), 229

but the variation was quadratic with a greater value at 6 h for j5 (P = 0.07). The protein 230

band j6 was identified by immunoblotting as being lectin (Figure 3Aa). There was a time ×231

diet interaction in band j6 intensity (time linear × diet, P = 0.011; time quadratic × diet, P = 232

0.031), which was caused by the absence of this band in digesta from the control diet and 233

by its high intensity at 3 and 6 h after the meal and a decrease between 6 and 9 h in the pea 234

diet. Immunoblotting of digesta from the mid SI confirmed the presence of lectin at all 235

times (3, 6, and 9 h) after the meal in the pea-fed piglets (Figure 3Ba; Table 5). The whole 236

PA2 molecule was not detected but a polypeptide of MW of 15 kDa was revealed at 3 h 237

(Figure 3Bb). Neither vicilin nor legumin were detected in the digesta of the mid-SI (data 238

not shown). 239




11

After SDS-PAGE separation of proteins in digesta of the distal SI, 6 protein bands 240

(noted i1 to i6; Table 5) with visually contrasted stainings (Figure 4A) were further 241

analyzed by densitometry. The staining density was decreased in the digesta of the pea-fed 242

piglets compared to the controls for bands i1 to i4 (P = 0.041 to < 0.001). In contrast, 243

bands i5 and i6 were more densely stained in the pea-fed piglets (P < 0.001). There was a 244

trend for a time × diet interaction for band i2 intensity. The interaction was caused by a 245

linear decrease in the control group and a quadratic increase with time in the pea group 246

(time linear × diet, P = 0.085; time quadratic × diet, P = 0.088). The intensity of band i3 247

was influenced by the time × diet interaction (time linear × diet, P = 0.007), which was 248

caused by a linear decrease with time in the control digesta but constantly low yet a linear 249

increase in the pea diet digesta. There was a time × diet interaction for band i4 (time linear250

× diet, P = 0.034), which was caused by the absence of this band in digesta from the pea 251

diet and by a decrease of its intensity with time in the control digesta. The protein band i5 252

was not identified by immunoblotting, although its intensity was high in the pea-fed piglets253

(P < 0.001) and there was no effect of time. The protein band i6 was identified by 254

immunoblotting as being lectin (Figure 4Aa). There was a time × diet interaction for this 255

band (time linear × diet, P = 0.087; time quadratic × diet, P = 0.021), which was caused by 256

the absence of the band in digesta from the control diet and by an increase between 3 and 6 257

h and plateaued at 9 h after the meal in the pea-fed pigs.258

The 15 kDa polypeptide from PA2 was also detected in ileal digesta at 3 and 6 h 259

but not at 9h after the meal (Figure 4Bb). The PA2 polypeptides undigested in the distal SI 260

were further characterized after concentration by immunoprecipitation. Three bands of 261

MW of 22, 17, and 15 kDa were identified by immunoblotting as originating from PA2 262

(Figure 5). Their identity was confirmed by LC-MS/MS sequencing and the resistant 263




12

peptides of PA2 were essentially localized on the AA sequence of the PA2 molecule in the 264

N- terminus (data not shown).265

DISCUSSION266

The present results confirm our previous observations in growing pigs: 1) pea 267

globulins are well digested, 2) PA2 is only partially digested, 3) a PA2 resistant 268

polypeptide of MW of 15 kDa is present, and 4) lectin is totally resistant to GIT digestion 269

(Le Gall et al., 2005a). The major point of this work is that such differences in intestinal 270

digestion between pea proteins originate in the stomach. 271

Digestion of Pea Protein along the Gastrointestinal Tract of Piglets272

The gastric digestion seems to be the important step for the pea protein hydrolysis, 273

in accordance with the results obtained after in vitro hydrolysis of different pea cultivars274

(Perrot et al., 1999). Indeed, peptides from PA2 digestion were detected in intestinal 275

digesta a short time (less than 3 h) after the meal. During this short period, gastric pH did 276

not seem to be adequate for protein digestion. It was high (pH > 4.5) compared to the 277

optimal pH for pepsin action (pH 2 to 4; Beynon and Bond, 1989). This may reflect the 278

fact that gastric pH of piglets soon after weaning is greater than in older pigs (Makkink et 279

al., 1994). The amounts of gastric secretion are the greatest only 1.5 to 2.5 h after the meal280

(Cranwell, 1985), and the inefficiency of pepsin hydrolysis may be responsible for the low 281

digestion of pea proteins. Indeed, the dissociation-association behavior of the polypeptides 282

from pea legumin is influenced by pH. Between pH 3.5 and 5.8, most of the protein is in 283

the form of aggregates preventing pepsin access. Below pH 3.4, legumin is completely 284

dissociated and its polypeptide chains are unfolded (Guéguen et al., 1988). These 285

conditions were met after 6 h of gastric digestion, and the β-polypeptides forming the 286

hydrophobic core of legumin were not protected. Their greater resistance to hydrolysis 287




13

compared to that of the α-polypeptides may also be due to their highly ordered structure288

(Subirade et al., 1994) and greater hydrophobicity. 289

Convicilin is easily hydrolyzed by pepsin, even at pH > 4.5, but vicilin is resistant 290

under these conditions. This differential susceptibility to hydrolysis between convicilin and 291

vicilin, shown in vitro (Le Gall et al., 2005b), is also observed in the chicken (Crevieu et 292

al., 1997). In the present study, vicilin had disappeared after 6 h of gastric digestion. In the 293

digesta collected 3 h after the meal, vicilin was probably quite insoluble due to the greater294

pH (5.0-6.0; Rangel et al., 2003). Then, it may have become totally soluble during the 295

gastric phase at pH < 3.0. Consequently, vicilin may have been more easily emptied from 296

the stomach with the liquid phase and would no longer be detected in the stomach. 297

Pea protein digestion in the SI seemed to be more efficient in pigs than in other 298

animal species. Indeed, in rats and chicken, only the α-polypeptides of legumin were 299

degraded, the β-polypeptides being totally resistant (Aubry et al., 1986; Crevieu et al., 300

1997). The albumin PA2 was shown to resist trypsin hydrolysis in vitro (Gruen et al., 301

1987; Perrot et al., 1999) and SI digestion in chickens (Crevieu et al., 1997). In young 302

piglets used in the present study and growing pigs used in an earlier study (Le Gall et al. , 303

2005a), we observed a cleaved peptide of MW 15 kDa in digesta of the mid and distal SI. 304

These data indicate that a part of PA2 is susceptible to digestion in the gut of mammals, as 305

opposed to birds. 306

Endogenous Protein307

Many proteins present in GIT digesta are of endogenous origin and mainly consist 308

of enzymes and antibodies according to the literature. These proteins themselves could be 309

partly or totally degraded and reabsorbed during the digestion process. Proteins of greater310

MW may correspond to glucoamylase or pancreatic alpha-amylase, or both, as previously 311

identified at the terminal ileum of growing pigs (Le Gall et al. , 2005a). Proteins of 312




14

intermediary MW may be fragments of antibodies (Le Gall et al., 2005a) and proteins of 313

the pancreatic serine protease family (trypsin and chymotrypsin; Salgado, et al., 2002).314

Surprisingly, many of these endogenous proteins were present less in digesta after pea 315

consumption, but this may be explained by our analytical method. Indeed, the same 316

amount of indigestible soluble protein was deposited in each track of gels. Therefore, a 317

lower proportion of endogenous protein in the digesta did not indicate that the ileal flow of 318

endogenous protein was lower. A greater proportion of dietary undigested protein, like 319

lectins in the pea digesta, will result in a lower proportion of endogenous protein. Indeed, 320

based on the published tables (INRA-AFZ, 2004), the ileal digestibility of pea protein 321

seems to be lower than of milk and fish protein. These differences are due to the presence 322

of PI and lectin, which are known to increase the ileal flow of dietary protein, while the 323

same components and fiber increase the endogenous losses. At the ileum, only 1 band (i5) 324

was present in digesta after pea consumption. This band was previously identified as 325

corresponding to proteins of the pancreatic serine protease family (Salgado et al., 2002). 326

Greater endogenous losses with pea-based diets have been ascribed to cotyledon cell-wall 327

fiber displaying a high water holding capacity (Leterme et al., 1998).328

In this study, the major storage pea proteins, vicilin and legumin, were rapidly 329

broken down in the stomach and were subsequently digested in the SI of piglets. Lectin 330

remained virtually intact throughout the stomach and SI. The pea albumin PA2 transiently 331

survived as a 15 kDa peptide located at the N-terminus of the molecule. This may 332

contribute to the lower digestibility and availability of sulfur AA in pigs fed diets based on 333

field peas because pea albumins are rich in these AA. To improve the use of field peas in 334

pig diets, cultivars with a high globulin to albumin ratio should be used.335




15

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182.339

Beynon, R. J., and J. S. Bond. 1989. Proteolytic Enzymes. A Practical Approach. IRL 340

Press, Oxford, UK.341

Bush, R. S., R. Toullec, I. Caugant, and P. Guilloteau. 1992. Effects of raw pea flour on 342

nutrient digestibility and immune response in the preruminant calf. J. Dairy Sci. 343

75:3539-3552.344

Carbonaro, M., G. Grant, M. Cappelloni, and A. Pusztai. 2000. Perspectives into factors 345

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capacity in the pig; the effects of age and weaning. 1. Studies in anaesthetized pigs. 349

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Crevieu, I., B. Carre, A. M. Chagneau, L. Quillien, J. Guéguen, and S. Berot. 1997. 351

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chickens. J. Agric. Food Chem. 45:1295-13.353

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trypsin inhibitor activity in pigs. Can. J. Anim. Sci. 80:643-652.363

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albumin: implication of a sulphydryl group. J. Sci. Food Agric. 41:167-178.365

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of pea legumin induced by pH and ionic strength. J. Sci. Food Agric. 44:167-182.367

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destinées aux animaux d’élevage. 2nd ed. INRA Editions, Paris, France.369

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mode of action and effects of antinutritional factors from legumes seeds in non-371

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Legume Seeds and Rapeseed. Wageningen, Netherland.373

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terminal ileum in growing pigs. J. Nutr. 135:1215-1222.376

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treatments on hydrolysis susceptibility of pea protein (Pisum sativum L.). J. Agric. 378

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capacity of pea inner fibers affects the ileal flow of endogenous amino acids in 381

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Makkink, C. A., P. J. Berntsen, B. M. op den Kamp, B. Kemp, and M. W. Verstegen. 383

1994. Gastric protein breakdown and pancreatic enzyme activities in response to 2384

different dietary protein sources in newly weaned pigs. J. Anim. Sci. 72:2843-2850.385




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Mariscal-Landin, G., Y. Lebreton, and B. Sève. 2002. Apparent and standardised true ileal 386

digestibility of protein and amino acids from faba bean, lupin and pea, provided as 387

whole seeds, dehulled or extruded in pig diets. Anim. Feed Sci. Technol. 97:183-388

198.389

Perrot, C., L. Quillien, and J. Guéguen. 1999. Identification by immunoblotting of pea 390

(Pisum sativum L.) proteins resistant to in vitro enzymatic hydrolysis. Sci. Alim. 391

19:377-390.392

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cowpea protein isolate. J. Agric. Food Chem. 51:5792-5797.395

Rubio, L. A., G. Grant, S. Bardocz, P. Dewey, and A. Pusztai. 1991. Nutritional response 396

of growing rats to faba beans (Vicia faba L., minor) and faba bean fractions. Br. J.397

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R. Toullec, and J. P. Lallès. 2002. Legume grains enhance ileal losses of specific 400

endogenous serine-protease proteins in weaned pigs. J. Nutr. 132:1913-1920.401

Sève, B. 1994. Alimentation du porc en croissance: intégration des concepts de protéine 402

idéale, de disponibilité digestive des acides aminés et d'énergie nette. INRA Prod.403

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Subirade, M., J. Guéguen, and M. Pezolet. 1994. Conformational changes upon dissociation 405

of a globular protein from pea: a Fourier transform infrared spectroscopy study. 406

Biochim. Biophys. Acta. 1205:239-247.407




18

Table 1. Composition of diets, as-fed basis408

Diet

_____________________

Control1 Pea2

Ingredient, g/kg

Field peas 0.00 350.00

Maltodextrin3 561.90 324.50

Fish soluble protein concentrate4 117.90 77.60

Skim milk powder 280.00 184.60

Sunflower oil 0.00 18.00

CaCO3 9.20 12.20

CaHPO4, H2O 22.90 23.20

Vitamin/microelements mixture5 5.00 5.00

DL-Met 1.55 2.60

L-Thr 1.03 1.55

L-Trp 0.52 0.75

Analyzed chemical composition

DM, % 91.23 88.77

CP, % (N x 6.25), DM basis 21.5 21.9

Lys, DM basis 1.39 1.33

Minerals, DM basis 6.53 6.77

Calculated NE, MJ/kg 10.54 10.531 Contained (g per kilogram as-fed basis, analyzed): Met, 6.46; Cys, 1.42; Lys, 13.95; Thr, 8.12; Trp, 2.37; Arg,409

9.17; Ile, 7.90; Leu, 14.46; Val, 9.34; His, 4.33; and Phe, 7.66.4102 Contained (g per kilogram as-fed basis, analyzed): Met, 6.31; Cys, 1.41; Lys, 13.31; Thr, 8.13; Trp, 2.40; Arg,411

10.89; Ile, 7.46; Leu, 13.37; Val, 8.60; His, 4.18; and Phe, 7.71.4123 Provided by Cargill, France. Reference: C*Dry MD 01904; D. E.: 3.5 to 6.5 analyzed by Luff-Schoorl. 4134 Contained (g per kilogram as-fed basis, analyzed): CP, 800; ether extract, 100; ash, 60; and water solubility, 80%. 414

Provided by the Coopérative de traitement des produits de la pêche, Concarneau, France.4155 Providing the following amount of vitamins and minerals per kilogram as fed basis: calcium carbonate 416

(excipients) 550.69; Zn (ZnO), 100 mg; Cu (CuSO4), 20 mg; Mn (MnO), 40 mg; Fe (FeSO4), 100 mg; I (KI), 1 mg; Co417(CoSO4), 2 mg; Se (Na 2SeO3), 0.3 mg; vitamin A, 15,000 IU; vitamin D3, 3,000 IU; vitamin E, 40 IU; vitamin B1, 2 mg; 418vitamin B2, 10 mg; pantothenic acid, 15 mg; vitamin B6, 10 mg; vitamin B12, 0.05 mg; vitamin PP, 30 mg; folic acid, 2 mg; 419vitamin K3, 2 mg; biotin, 0.2 mg; choline chloride, 800 mg; and vitamin C, 100 mg. Provided by Centralys, Trappes, France.420

421422423424




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Table 2. Performance data for piglets fed the control and the pea diet425426

Diet1

__________________________________

Control Pea

SEMP-value

BW at weaning, kg 8.0 8.1 0.2 0.81

BW gain, g/d 158 162 15 0.82

G:F 0.59 0.60 0.05 0.59

ADFI, g/kg BW0.75 53.4 54.7 0.8 0.541Twelve pigs per treatment.427

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Table 3. Relative weight (g/kg BW) of the entire empty gastrointestinal tract and its segments in piglets fed the control and the pea diet430431

Diet1

___________________________

Control Pea

SEMP-value

Gastrointestinal tract 63.7 70.3 1.7 0.015

Stomach 6.6 7.0 0.2 0.11

Proximal SI2 16.2 16.7 0.5 0.52

Mid SI 14.7 15.4 0.6 0.43

Distal SI 9.1 8.7 0.3 0.42

Cecum 5.1a 8.1b 0.3 < 0.001

Proximal colon 3.8a 5.9b 0.3 < 0.001

Distal colon 1.7 2.0 0.1 0.11

Mesentery + pancreas 6.6 6.6 0.3 0.89

a,b Within a row, values not sharing a common superscript letter differ (P < 0.05). 4321Twelve pigs per treatment.4332Small intestine.434

435



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Table 4. Residual DM in the stomach and pH of digesta contents in piglets fed the control or the pea diet436

Time after the meal, h

Diet1

__________________________________________________________

Control Pea

3 6 9 3 6 9

SEM

P-value

________________________________________________________

T2 T linear T quadratic D3 T × D4 T linear × D T quadractic × D

Residual DM in the

stomach, %551 25 15 56 17 12 7 < 0.001 < 0.001 0.05 0.69 0.61 0.54 0.45

Digesta pH

Stomach 4.71 3.88 2.87 4.58 2.92 2.18 0.27 < 0.001 < 0.001 0.44 0.02 0.31 0.31 0.26

Proximal SI6 7.62 7.61 7.23 7.46 7.56 7.39 0.06 0.001 0.002 0.009 0.76 0.06 0.02 0.71

Mid SI 6.66 6.76 6.86 6.80 6.64 6.74 0.15 0.79 0.65 0.62 0.79 0.62 0.41 0.61

Distal SI 7.14 7.25 7.35 6.97 6.69 7.10 0.16 0.28 0.29 0.23 0.02 0.45 0.79 0.23

1Twelve pigs per treatment.4372Time.4383Diet.4394Time by diet interaction.4405Percentage of DM intake at the last meal before slaughter.4416Small intestine.442



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Table 5. Densitometry of protein bands in digesta of the first, second, and third segment of the small intestine (DU1 · g-1 protein)443

Time after the meal, h

Diet2

______________________________________________________________________

Control Pea 3 6 9 3 6 9

SEM

P-value

T3 T linear T quadratic D4 T × D5 T linear × D T quadractic × D

Proximal SI6,7

d1, 45 kDa 0.255 0.317 0.347 0.059 0.096 0.148 0.067 0.459 0.223 0.943 0.003 0.980 0.977 0.845d2, 28 kDa 0.138 0.221 0.040 0.474 0.620 0.515 0.145 0.612 0.854 0.334 0.006 0.901 0.654 0.983d3, 25 kDa 0.000 0.000 0.008 0.806 0.069 0.088 0.092 0.002 0.002 0.033 0.001 0.002 0.002 0.036d4, 20 kDa, lectin 0.000 0.000 0.018 0.566 0.753 0.233 0.143 0.230 0.262 0.171 0.001 0.275 0.311 0.192

Mid SI8

j1, 90 kDa 0.302 0.534 0.333 0.110 0.000 0.000 0.104 0.631 0.711 0.383 0.001 0.285 0.508 0.153j2, 68 kDa 0.320 0.318 0.607 0.067 0.092 0.043 0.101 0.756 0.505 0.750 0.043 0.63 0.432 0.591j3, 55 kDa 3.470 2.250 1.710 0.650 2.380 0.750 0.594 0.191 0.179 0.210 0.024 0.072 0.139 0.068j4, 28 kDa 0.530 0.540 0.240 0.310 0.370 0.150 0.090 0.023 0.024 0.810 0.045 0.788 0.497 0.987j5, 26 kDa 0.094 0.374 0.175 0.044 0.073 0.067 0.059 0.054 0.886 0.070 0.006 0.117 0.812 0.479j6, 20 kDa, lectin 0.000 0.000 0.000 1.510 1.700 0.600 0.157 0.007 0.011 0.030 < 0.001 0.007 0.011 0.031

Distal SI9

i1, 90 kDa 0.714 0.748 0.794 0.317 0.730 0.366 0.154 0.356 0.681 0.174 0.041 0.36 0.922 0.161i2, 68 kDa 0.727 0.630 0.574 0.194 0.619 0.421 0.104 0.283 0.727 0.126 0.015 0.062 0.088 0.085i3, 55 kDa 4.37 3.92 1.03 0.108 0.424 0.658 0.625 0.071 0.041 0.262 < 0.001 0.017 0.007 0.293i4, 35 kDa 0.687 0.451 0.263 0.000 0.000 0.000 0.091 0.098 0.034 0.881 < 0.001 0.099 0.034 0.881i5, 28 kDa 0.371 0.501 0.449 2.130 2.860 3.070 0.389 0.394 0.211 0.609 < 0.001 0.539 0.286 0.804i6, 20 kDa, lectin 0.000 0.000 0.000 1.780 3.480 2.770 0.270 0.022 0.087 0.021 < 0.001 0.022 0.087 0.021

1Densitometric unit.4442Twelve pigs per treatment.4453Time.4464Diet.4475Time by diet interaction.4486Small intestine.4497Duodenal protein band number 1 to 4, molecular weight in kDa.4508Jejunal protein band number 1 to 6, molecular weight in kDa.4519Ileal protein band number 1 to 6, molecular weight in kDa.452



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FIGURE CAPTION453454

Figure 1. Protein digestion in the stomach of piglets fed a control diet (C) or a diet 455

containing pea (P) as revealed by (A) SDS-PAGE in reducing conditions and Coomassie 456

blue staining, and (B) immunoblotting with antibodies against pea vicilin (a), legumin (b), 457

lectin β subunit (c), and albumin PA2 (d) for the diet extracts and the digesta extracts from 458

the piglets fed the pea diet (no antibody reacted with proteins in the controls). The digesta 459

were collected by the slaughter technique at 3, 6, and 9 h after the meal and extracted for 460

proteins before analysis. Individual tracks represent individual samples. M: molecular 461

mass markers in kDa on the left.462

463

Figure 2. Protein digestion in the proximal small intestine (SI1) of piglets fed a control diet 464

(C) or a diet containing pea (P) as revealed by (A) SDS-PAGE in reducing conditions and 465

Coomassie blue staining, and (B) immunoblotting with antibodies against (a) pea legumin, 466

(b) lectin β subunit, and (c) albumin PA2 for the diet extracts and the digesta extracts from 467

the piglets fed the pea diet (no antibody reacted with proteins in the controls). The digesta 468

were collected by the slaughter technique at 3, 6, and 9 h after the meal and extracted for 469


mass markers in kDa on the left. The numbered bands in A refer to those further analyzed 471

by densitometry.472

473

Figure 3. Protein digestion in the mid small intestine (SI2) of piglets fed a control diet (C) 474

or a diet containing pea (P) as revealed by (A) SDS-PAGE in reducing conditions and 475

Coomassie blue staining, and (B) immunoblotting with antibodies against (a) lectin β476

subunit and (b) albumin PA2 for the diet extracts and the digesta extracts from the piglets 477

fed the pea diet (no antibody reacted with proteins in the controls). The digesta were 478




24

collected by the slaughter technique at 3, 6, and 9 h after the meal and extracted for 479



by densitometry.482

483

Figure 4. Protein digestion in the distal small intestine (SI3) of piglets fed a control diet (C) 484

or a diet containing pea (P) as revealed by (A) SDS-PAGE in reducing conditions and 485

Coomassie blue staining, and (B) immunoblotting with antibodies against (a) lectin β486

subunit and (b) albumin PA2 for the diet extracts and the digesta extracts from the piglets 487

fed the pea diet (no antibody reacted with proteins in the controls). The digesta were 488

collected by the slaughter technique at 3, 6, and 9 h after the meal and extracted for 489



by densitometry.492

493

Figure 5. Separation by SDS-PAGE (A) and identification by immunoblotting (B) of pea 494

albumin PA2 in the pea flour (P) and of PA2 polypeptides present in ileal (SI3) digesta of 495

piglets fed the pea diet. These undigested polypeptides were first immuno-precipitated 496

from digesta using protein A technology. Track 1: molecular weight markers in kDa. Track 497

2: pea proteins (A) and PA2 (B) in the pea flour extract. Track 3: Rabbit IgG antibodies 498

(Ac) specific for PA2. Track 4: immuno-precipitated (Immunoprec.) proteins including the 499

anti-PA2 rabbit antibodies and the undigested PA2 polypeptides (A) and immunoblotting 500

revelation with the anti-PA2 antibodies. Track 5: similar to track 4 for (A) and revelation 501

with an anti-rabbit IgG antibody alone (5). The undigested PA2 polypeptides specifically 502

detected and analyzed by mass spectrometry were obtained by difference between the band 503




25

patterns present on track 4 and those present on track 5 of the immunoblot (B). They are 504

identified with black arrowheads on panel A.505




26

A: SDS-PAGE stained gels for proteins506

Control Pea507 M C 3 6 9 M P 3 6 9508

509510

B: Pea protein detection by immunoblotting511a) Vicilin b) Legumin 512M C P 3 C 3 6 M C P 3 6 9513

---- ---------- --------------- -------------- ----------- -------514

515c) Lectin d) PA2 516M C P 3 6 9 M C P 3 6 9517

------------- ----------- ----- --------------- ---------- -----518

519520

Figure 1521

9766

45

30

20.1

14.4

1158051

30

25

17

1158051

3025

17

- Convicilin- Vicilin 50- Vicilin 33 - α major

- α- cleaved

-β

- PA2

- Lectin (β)




27

A: SDS-PAGE stained gels for proteins522Control Pea523

M C 3 6 9 M P 3 6 9524

525526

B: Pea protein detection by immunoblotting527a) Legumin b) Lectin c) PA2 528 M P C 3 6 9 3 6 9 3 6 3 6 M P 3 6 9 M C C P 3 6 9 529

---- --- --- --- --- --- --- --- --- --- ------------- ----------- --------- ------------- ----------- ------530

531Figure 2532

1158051

3025

17

976645

30

20.1

14.4

---d2

----------------------- d3- - ------ d4

-----------d1

---d2

- Lectin (β)

- PA2- β legumin




28

A: SDS-PAGE stained gels for proteins533Control Pea534

M C 3 6 9 M P 3 6 9 535

536537

B: Pea protein detection by immunoblotting538a) Lectin b) PA2 539M P 3 6 9 M P 3 6 9540

----------- ----------- -------- --------- ------------ -------------541

542543

Figure 3544

-j1-j2-j3

-j4-j5

-j6

976645

30

20.1

14.4

-j1-j2-j3

-j4

1158051

3025

17 - Lectin

- PA2

- Cleaved PA2




29

A: SDS-PAGE stained gels for proteins545

Control Pea546 M C 3 6 9 M P 3 6 9 547

548549

B: Pea protein detection by immunoblotting550a) Lectin b) PA2551M P 3 6 9 M P 3 6 9552

----------- ----------- --------- --------------- ------------ --------553

554555

Figure 4556

-i1

-i2-i3

-i4

-i5

-i6

-i1

-i2-i3

-i4

-i5

976645

30

20.1

14.4

1158051

3025

17 - Lectin (β) - Cleaved PA2




30

557

558

559560

Figure 5.561

1 2 3 4 5 1 2 3 4 5M P Ac Immunoprec. M PA2 Ac PA2 Ac

+Ac

MW A: SDS-PAGE gel B: Immunoblotting

115 80

51

30

25

17

12

345

Antibodies

PA2

Degraded antibodies




A: SDS-PAGE stained gels for proteins Control Pea M C 3 6 9 M P 3 6 9

B: Pea protein detection by immunoblottinga) Vicilin b) Legumin M C P 3 C 3 6 M C P 3 6 9

---- ---------- --------------- -------------- ----------- -------

c) Lectin d) PA2 M C P 3 6 9 M C P 3 6 9

------------- ----------- ----- --------------- ---------- -----

Figure 1

9766

45

30

20.1

14.4

1158051

30

25

17

1158051

3025

17

- Convicilin- Vicilin 50- Vicilin 33 - α major

- α- cleaved

-β

- PA2

- Lectin (β)




A: SDS-PAGE stained gels for proteins Control Pea

M C 3 6 9 M P 3 6 9

B: Pea protein detection by immunoblotting a) Legumin b) Lectin c) PA2 M P C 3 6 9 3 6 9 3 6 3 6 M P 3 6 9 M C C P 3 6 9 ---- --- --- --- --- --- --- --- --- --- ------------- ----------- --------- ------------- ----------- ------

Figure 2

115 80 51

30

25

17

97

66 45 30 20.1 14.4

---d2 ----------------------- d3

- - ------ d4

-----------d1

---d2

- Lectin (β)

- PA2 - β legumin





M C 3 6 9 M P 3 6 9

B: Pea protein detection by immunoblotting a) Lectin b) PA2 M P 3 6 9 M P 3 6 9

----------- ----------- -------- --------- ------------ -------------

Figure 3

-j1

-j2 -j3

-j4 -j5

-j6

97 66 45 30 20.1 14.4

-j1

-j2 -j3

-j4

115 80 51

30

25

17 - Lectin

- PA2

- Cleaved PA2





M C 3 6 9 M P 3 6 9

B: Pea protein detection by immunoblotting a) Lectin b) PA2 M P 3 6 9 M P 3 6 9

----------- ----------- --------- --------------- ------------ --------

Figure 4

-i1

-i2 -i3

-i4

-i5

-i6

-i1

-i2 -i3 -i4

-i5

97 66 45 30 20.1 14.4

115 80 51

30

25

17 - Lectin (β) - Cleaved PA2




1

2

34

Figure 55

1 2 3 4 5 1 2 3 4 5M P Ac Immunoprec. M PA2 Ac PA2 Ac

+Ac

MW A: SDS-PAGE gel B: Immunoblotting

115 80

51

30

25

17

12

345

Antibodies

PA2

Degraded antibodies




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