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IgE-binding and in vitro gastrointestinal digestibility of egg allergens in the presence of polysaccharides
Food Hydrocolloids 30 (2013) 597e605
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IgE-binding and in vitro gastrointestinal digestibility of egg allergens in the presence of polysaccharides Rodrigo Jiménez-Saiz, Iván López-Expósito, Elena Molina, Rosina López-Fandiño* Instituto de Investigación en Ciencias de la Alimentación (CIAL) CSIC-UAM, Nicolás Cabrera 9, 28049 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 March 2012 Accepted 25 July 2012
This work studies the IgE-binding and in vitro gastrointestinal digestibility of the main egg allergens, ovalbumin (OVA) and ovomucoid (OM), in the presence of pectin (P), gum arabic (G) and xylan (X), functional biopolymers commonly used in the food industry. To this aim, solutions of OVA or OM and P, G or X were digested by using a model that mimics physiological conditions. Gastric and duodenal digests were analysed by SDS-PAGE, RP-HPLC and SEC and the specific human-IgE binding capacity was assessed by immunoblotting and ELISA using sera from egg-sensitized patients. The reactivity towards human IgE of OVA and OM was considerably increased in the presence of the polysaccharides and their susceptibility to digestion was diminished when compared with the isolated proteins. As a result, the duodenal digests obtained in the presence of polysaccharides retained more IgE-binding than the isolated protein digests. Overall, the present results underline the importance of the food matrix in the digestibility of food allergens and in their potential to trigger an immune response. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Food allergy Matrix effect Egg allergens Polysaccharides In vitro digestion IgE-binding
1. Introduction Egg proteins are broadly used in the food industry due to their unique functional properties, such as emulsifying, foaming and gelling. In addition, the high biological quality of egg proteins adds more value to the food products that include egg or its constituents as ingredients (Kovacs-Nolan, Phillips, & Mine, 2005; Mine, 2002). However, egg is an important source of allergens, which are mainly contained in the egg white, with ovalbumin (OVA or Gal d 2) and ovomucoid (OM or Gal d 1) being the major ones (Mine & Yang, 2008). Among food allergies, allergy to egg is, together with peanut and milk, the most common in children and infants with a prevalence that varies between 7.9 and 10% (Osborne et al., 2011; Sicherer, 2011). Among the conditions required for food proteins to trigger an allergic reaction is their ability to keep the integrity of their allergenic determinants through the gastrointestinal tract. To date, most of the studies dealing with the influence of the gastro duodenal digestion on the potential allergenicity of foods have been carried out on isolated proteins (Moreno, 2007). However, exposure of allergic individuals to pure allergens is rare and, in fact, the stability
Abbreviations: OVA, ovalbumin; OM, ovomucoid; P, pectin; G, gum arabic; X, xylan; GD, gastric digest; DD, duodenal digest; CCD, carbohydrate determinants. * Corresponding author. Tel.: þ34 91 0017941; fax: þ34 91 0017905. E-mail addresses: [email protected], rosina@ifi.csic.es (R. López-Fandiño). 0268-005X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodhyd.2012.07.014
of proteins to digestion can be altered in the presence of various components that form part of the food matrix (Teuber, 2002). Thus, it has been shown that the presence of soluble polysaccharides commonly used in the preparation of a wide range of foods, as stabilizers, thickeners and emulsifiers, reduces protein digestibility. The increase of mixture viscosity, the interactions between the two types of macromolecules and the inhibition of enzymatic activity have been pointed out to explain this observation (Mouecoucou, Fremont, Sanchez, Villaume, & Mejean, 2004; Mouecoucou et al., 2003). Polovic et al. (2009) reported the existence of a protective matrix effect on the digestion of pectin-rich crude extracts of various fruits. Pectin can form gels in the conditions present during in vivo and in vitro gastric digestion, providing a physical obstacle to the mobility of both pepsin and the protein substrate in the reaction mixture (Polovic et al., 2007). In addition, protective effects of structural polysaccharides of plant origin on the in vitro digestion of peanut allergens and b-lactoglobulin by pepsin and trypsin have been reported (Mouecoucou, Fremont, et al., 2004; Mouecoucou, Villaume, Sanchez, & Mejean, 2004). Although some studies did not go further than the digestibility assessment (Mouecoucou et al., 2003; Polovic et al., 2009), others have shown that, despite milk and peanut proteins are more stable towards hydrolysis, their digests show less IgG/IgE binding capacity, which was attributed to the masking of epitopes because of the interaction of the polysaccharides with the digestion products (Mouecoucou, Fremont, et al., 2004; Mouecoucou, Fremont, Villaume, Sanchez, & Mejean, 2007). Regarding egg allergens,
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there are very few studies on the effect of interactions with carbohydrates on their digestibility and immunogenicity (JiménezSaiz, Belloque, Molina, & López-Fandiño, 2011). Considering that foods are complex multicomponent mixtures that can contain proteins and polysaccharides, in many cases interacting as mixed biopolymers (Dickinson, 1998), and that such interactions can also form in the stomach after ingestion, the aim of the present work was to investigate the in vitro gastrointestinal behaviour and the resulting human IgE-binding of the main egg allergens, OVA and OM, in the presence of the polysaccharides: pectin (P), gum arabic (G) and xylan (X). 2. Materials and methods 2.1. Proteinepolysaccharide mixtures Hen egg white OVA grade VI, ovoinhibitor-depleted OM, high methylated P, G from acacia tree and X from beechwood were from SigmaeAldrich (St. Louis, MO, USA). Proteins and polysaccharides were dissolved separately (20 mg/mL) in 0.1 M NaCl and the pH was adjusted to 3 with 0.1 N HCl. The polysaccharides were heated at 60 C for 15 min to improve their solubility. Then, OVA and OM solutions were mixed with the solutions of each polysaccharide (1:1, v/v) and shaken gently overnight at room temperature (RT). 2.2. Gastric and duodenal digestions The digestibility of OVA, OM and their mixtures with each polysaccharide (P, G, X) was studied using an in vitro model system in two steps, which imitates gastric and duodenal digestion in vivo (Martos, Contreras, Molina, & López-Fandiño, 2010). Gastric digestions were performed in simulated gastric fluid (SGF, 35 mM NaCl) at pH 2.0, for 60 min at 37 C, with 172 U/mg of porcine pepsin (EC 3.4.23.1, 3210 U/mg protein, SigmaeAldrich). The reactions were stopped by rising pH to 7.0 with 1 M NaHCO3. Duodenal digestions were performed at 37 C by using the 60 min gastric digests adjusted to pH 7.0, as described above, with the addition of: 1 M CaCl2, 0.25 M Bis-Tris pH 6.5 and 0.125 M bile salts. Porcine pancreatic lipase (EC 232-619-9), colipase (EC 259490-1), trypsin (EC 232-650-8. 10100 BAEE units/mg protein) and a-chymotrypsin (EC 232-671-2: 55 units/mg protein) from SigmaeAldrich were used. Aliquots were taken after 60 min of gastric digestion and 30 min of duodenal digestion. Duplicate digestions were conducted for each condition. 2.3. SDS-PAGE Samples, diluted in a buffer containing 62.5 mM TriseHCl pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) b-mercaptoethanol and 0.0025% (w/v) bromophenol blue, were heated for 5 min at 95 C and loaded on 12% Bis-tris polyacrylamide gels (CriterionÔ XT). Electrophoretic separations were carried out at 150 V in a criterion cell using XT-MES running buffer. Gels were stained for protein with Coomassie Blue G-250 and for polysaccharide with Schiff reactive, following Zacharius, Zell, Morrison, and Woodlock (1969). 2.4. Reverse phase high-performance liquid chromatography (RPHPLC) The RP-HPLC analyses were performed using a Waters 600 HPLC instrument (Waters, Milford, MA, USA) and a 250 mm 4.6 mm Widepore C18 column (Bio-Rad, Richmond, CA, USA). Operating conditions were as follows: column at RT; flow rate, 1 mL/min; injection volume, 50 mL; solvent A, 0.37 mL/L TFA in Milli-Q water,
and solvent B, 0.27 mL/L TFA in HPLC grade acetonitrile. A linear gradient of solvent B in A, from 0 to 60% in 60 min, followed by 60% B for 30 min, was used. Absorbance was recorded at 220 nm with a Waters 2487 l dual detector. The software Empower 2000 system data (Waters) was used. 2.5. Size exclusion chromatography (SEC) The SEC analyses were performed using a Waters 600 HPLC instrument (Waters) and a 7.8 mm 300 mm TSK-gel G2000SWxl column (Tosoh Bioscience, Tokyo, Japan). Operation conditions were as follows: column at RT, flow rate, 1 mL/min; injection volume, 30 mL. Samples were eluted in isocratic mode by using 0.1 M NaCl, pH 7. Absorbance was recorded at 220 nm with a Waters 2487 l dual detector. The software Empower 2000 system data (Waters) was used. Fractions of interest were manually collected and frozen at 30 C for further use. 2.6. Human IgE-binding A total of 15 sera from children with clinically proved allergy to egg were mixed in five different pools of three sera each, as shown in Table 1. The sera were collected from the Maternal and Child Gregorio Marañón Hospital (Madrid, Spain). The patients’ specific seric IgE levels were determined by CAP (GE HealthCare, Waukesha, MI, USA). Unstained SDS-PAGE gels were soaked in transfer buffer (48 mM Tris, 39 mM glycine, 20% methanol, pH 9.2) for 20 min. Proteins were electroblotted into nitrocellulose membranes using a TransBlotÒ SD apparatus for 30 min at 18 V. The membranes were incubated in Tris buffered saline containing 0.05% (v/v) Tween 20 and 1% (w/v) bovine serum albumin (TBST-1% BSA) for 3 h and dipped into a pool of human sera (1/40 diluted in TBST-1% BSA) for 24 h at 4 C. Then, the membrane was incubated with biotinconjugated anti-human-IgE antibody (Southern Biotech, Birmingham, AL, USA), 1/500 diluted in TBST-1% BSA, overnight at 4 C. Finally, horseradish peroxidase (HRP)-streptavidin (DakoCytomation, Glostrup, Denmark) diluted 1:3000 in TBST-1% BSA was added and incubated for 1 h. All incubations were followed by 5 washing steps with TBST of 5 min each. The chemiluminescent substrate AmershamÔ ECLÔ Prime (GE HealthCare, Uppsala, Sweden) was used and light emission detected for 5 min in the VersaDocÔ imaging system. Human IgE-binding of the proteinepolysaccharide mixtures and their duodenal digests was also assessed by inhibition ELISA as Table 1 Specific IgE levels (kU/L) towards whole egg, egg white, ovalbumin and ovomucoid of the human sera and their distribution in the pools used throughout the study. Serum number IgE (kU/L)
Pool number
Whole egg Egg white Ovalbumin Ovomucoid 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
e e e e >100 >100 e >100 e e e e e e e
10.2 >100 e 7 e e 7.44 e e >100 7.7 11.6 >100 9.17 20.1
11.1 78.9 15 7.84 e e 3.03 e 62 e e e e e e
2.94 69.2 16 1.4 e e 7.51 e 80 e e e e e e
1 1 1 2 2 2 3 3 3 4 4 4 5 5 5
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previously reported (Jiménez-Saiz, Martos, Carrillo, López-Fandiño, & Molina, 2011) with slight variations: commercial OVA or OM, diluted in 0.01 M phosphate buffer, pH 7.4 (PBS), to 80 mg/mL, were used as coating antigens and polyclonal rabbit anti-human IgE (A0094, Dako, Glostrup, Denmark) and polyclonal swine anti-rabbit immunoglobulin labelled with HRP (P0399, Dako), diluted 1:1000 and 1:2000 (v/v), respectively, in PBS containing 0.05% Tween 20 (PBST), were used for detection. The IgE-binding results were expressed as the effective sample concentration for 50% of the maximum binding (EC50, mg/mL) following Jiménez-Saiz, Belloque, et al. (2011). The ability to bind human-IgE of the fractions collected by SEC was assessed by indirect ELISA. Plates were coated with the nondiluted samples and incubated overnight at 4 C. After overnight incubation, the plates were washed with PBST and incubated with sera from allergic patients. Following 2 h of incubation, plates were washed and incubated with rabbit anti-human IgE. After
599
a washing step, polyclonal swine anti-rabbit immunoglobulin labelled with HRP was added and incubated for 1 h. Then, the plate was washed and the tyramide-biotin and HRP-streptavidin amplification system was used following the instructions of the manufacturer (ELAST ELISA amplification system, PerkineElmer Life Sciences, Waltham, MA). 3,30 ,5,50 -tetramethylbenzidine (TMB, ready-to-use solution; SigmaeAldrich) was used as substrate and the reaction was stopped with sulphuric acid. The absorbance was measured at 450 nm in plate reader (Multiskan FC, Thermo Scientific, Finland). 2.7. Statistical analyses The ELISA results, either as EC50 or as AU at 450 nm, were expressed as means for n ¼ 2. Significant differences (P < 0.05) were evaluated by one-way analysis of variance followed by post hoc multiple-comparison using Tukey’s test.
Fig. 1. SDS-PAGE analysis with Coomassie staining (a) and immunoblot using a pool of human sera from allergic patients (pool 4) (b) of ovalbumin digested in the absence and presence of polysaccharides. MW: molecular mass marker. The patterns of undigested (N) ovalbumin (OVA), its mixtures with pectin (P), gum arabic (G) or xylan (X) , and their gastric (GD) and duodenal digests (DD) are shown. The IgE levels of the individual serum used in the pool are shown in Table 1.
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3. Results 3.1. Digestibility of the mixtures of OVA and OM with polysaccharides The mixtures of OVA and OM with P, G and X, as well as their gastric and duodenal digests, were analysed by SDS-PAGE (Figs. 1a and 2a) and RP-HPLC (Figs. 3 and 4). Before in vitro hydrolysis, the mixtures of the proteins with the polysaccharides showed a smeared SDS-PAGE pattern stainable with Schiff reagent in the area of the gel corresponding to molecular masses higher than 50 kDa (results not shown). Coomassie staining did not show changes in the characteristic band patterns of OVA and OM in the presence of P, G and X; however, the existence of higher molecular mass protein material suggested that a portion of the proteins interacted with the polysaccharides in the mixed samples to form new polydispersed bands (Figs. 1a and 2a, lanes 5, 8 and 11).
Gastric digestion of OVA was slightly hampered in the presence of the polysaccharides (Fig. 1a). Furthermore, the 40.1 kDa fragment, Ala23ePro385, released by pepsin action (Martos et al., 2010), which corresponds to the peak eluting at 51 min in RP-HPLC (not shown), and, in general terms, the peptide fragments released during the simulated gastric digestion showed higher intensity in the presence of the polysaccharides, what suggested that they protected, to some extent, OVA and their digestion products from further hydrolysis by pepsin. In Fig. 3, which shows the RP-HPLC profiles of the duodenal digests, the higher resistance to digestion of OVA and of the Ala23ePro385 fragment, in the presence of P, G and X is still observed, although the protector effect on the duodenal peptides was reduced. In the case of OM digested under simulated gastric conditions, there were no differences among the SDS-PAGE (Fig. 2a) or RP-HPLC profiles (data not shown) in the absence or presence of polysaccharides. However, the digestibility of OM mixed with the polysaccharides under in vitro duodenal conditions
Fig. 2. SDS-PAGE analysis with Coomassie staining (a) and immunoblot using a pool of human sera from allergic patients (pool 5) (b) of ovomucoid digested in the absence and presence of polysaccharides. MW: molecular mass marker. The patterns of undigested (N) ovomucoid (OM), its mixtures with pectin (P), gum arabic (G) or xylan (X) and their gastric (GD) and duodenal digests (DD) are shown. The IgE levels of the individual serum used in the pool are shown in Table 1.
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601
Duodenal digests 0.12 0.10 0.08 0.06
DD OVA
0.04
DD OVA-P
0.02 AU 220 nm
0.00 0.10 0.08 0.06 0.04
DD OVA
0.02
DD OVA-G
0.00 0.10 0.08 0.06 0.04
DD OVA
0.02 0.00 10.0
DD OVA-X
15.0
20.0
25.0
30.0
35.0
40.0
45.0
50.0
55.0
60.0
65.0
70.0
Time (minutes)
Fig. 3. RP-HPLC analyses of in vitro duodenal digests (DD) of ovalbumin (OVA) and of mixtures of OVA with pectin (P), gum arabic (G), and xylan (X). Fig. 4. RP-HPLC analyses of in vitro duodenal digests (DD) of ovomucoid (OM) and of mixtures of OM with pectin (P), gum arabic (G), and xylan (X).
AU 220 nm
a 0.10
OVA-P
0.08
GD OVA-P DD OVA-P
0.06
*
0.04
**
0.02 0.00 2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
Time (minutes)
AU 220 nm
b 0.10
OVA-G
0.08
GD OVA-G DD OVA-G
0.06
*
0.04
**
0.02 0.00 2.0
3.0
4.0
5.0
6.0
7.0
8.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
Time (minutes)
c AU 220 nm
9.0
0.10
OVA-X
0.08
GD OVA-X
0.06
DD OVA-X
0.04
** **
0.02 0.00 2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
Time (minutes) Fig. 5. SEC analyses of mixtures of ovalbumin (OVA) with pectin (P) (a), gum arabic (G) (b), and xylan (X) (c) and their respective gastric (GD) and duodenal digests (DD).
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was slightly reduced, especially in the presence of P, as it can be seen in the RP-HPLC analyses shown in Fig. 4. SEC analyses were conducted on OVA and OM mixed with the polysaccharides and on their gastric and duodenal digests (Figs. 5 and 6). These showed peaks corresponding to high molecular mass forms in the presence of P and G (marked with * in Figs. 5 and 6), which were not found in the OVA or OM samples used as controls (data not shown), nor in the mixtures of OVA and OM with the neutral polysaccharide X (Figs. 5c and 6c). With the advance of digestion, new peaks corresponding to hydrolysis products with lower size were detected in the chromatograms. In addition, the high molecular mass forms increased in intensity during gastric digestion, and new peaks (marked with ** in Figs. 5 and 6) appeared, suggesting the binding of polysaccharides to peptide fragments released by pepsin. Upon simulated duodenal digestion, the first peak (marked with ** in Fig. 5) was not longer detectable in any of the OVA-polysaccharide mixtures, while the area of the second peak decreased in OVA and OM mixed with P, G and X.
3.2. IgE-binding of the mixtures of OVA and OM with polysaccharides and their digests The IgE-binding of OVA, OM, their mixtures with P, G and X and their in vitro digests was assessed by SDS-PAGE followed by immunoblotting (Figs. 1b and 2b) and by inhibition ELISA (Table 2). Inhibition ELISA could not detect any response from G and provided very high EC50 values for X and P (3.7 g/mL and 63.75 mg/mL, respectively), indicating that the polysaccharides did not bind IgE from egg allergic patients. The immunoblotting showed that the reactivity towards IgE of OVA and OM was considerably increased in the presence of the polysaccharides and the diffuse bands of high molecular mass, which presumably contained both proteins and polysaccharides, as judged by Shift and Coomassie staining, were strongly recognized by IgE (Figs. 1 and 2). The IgE binding was particularly enhanced in the mixtures with P, in the case of OVA, and with X, in the case of OM. Inhibition ELISA also indicated that the mixtures of OVA and
a 0.12
OM-P
0.10
GD OM-P
0.08
DD OM-P
0.06 0.04
*
0.02 0.00 2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
17.0
18.0
Time (minutes)
b 0.12
OM-G
0.10
GD OM-G
0.08
DD OM-G
0.06
*
0.04 0.02 0.00 2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
Time (minutes)
c 0.12
OM-X
0.10
GD OM-X
0.08
DD OM-X
0.06 0.04
**
0.02 0.00 2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.028 16.0
17.0
18.0
Time (minutes) Fig. 6. SEC analyses of mixtures of ovomucoid (OM) with pectin (P) (a), gum arabic (G) (b), and xylan (X) (c) and their respective gastric (GD) and duodenal digests (DD).
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4. Discussion The present study shows that the reactivity towards human IgE of the major egg allergens, OVA and OM, was considerably increased in the presence of three different polysaccharides commonly used in the food industry. This could be attributed to the interaction, at acidic pH, of OVA and OM with P, G and X, producing complexes which were strongly recognized by IgE (Figs. 1 and 2; Table 2). In addition, the mixture with the polysaccharides slightly reduced the in vitro gastrointestinal stability of the allergens. The lower digestibility of
M -X O D D
D
a
a
2
1
b
b
0
M -X D D
D
D
D
D
O
O
O
M -P
+ C
Pool 3 a
a 2
1
b
b M -X O
O D
D D
M -P
0
D
OM with the polysaccharides exhibited a significantly (P < 0.05) increased reactivity, although the response depended on the pool of human sera considered and there were not significant differences among P, G and X (Table 2). The gastric and duodenal digests of the mixtures of OVA and polysaccharides also showed an increased IgE binding when compared with those of isolated OVA, as estimated by the higher intensity of the intact OVA bands and OVA degradation products in the immunoblot (Fig. 1b). The degradation products of OM, either in the absence or presence of polysaccharides, could not be transferred to the nitrocellulose membrane or were not retained in the membrane due to their small size (Fig. 2b). Inhibition ELISA confirmed that the duodenal digests of OVA and OM in the presence of P, G and X had lower EC50 than the duodenal digests of the isolated proteins (P < 0.05, Table 2). In general terms, the duodenal digests presented a reduced but still relevant IgE binding, compared with the undigested proteins or protein mixtures, especially in the case of OVA, although it greatly varied depending on the sera pool used. Thus, pool 2 exhibited a higher reactivity towards the duodenal digests than towards the intact proteins. Finally, the potential immunoreactivity of the compounds attributed to the interaction of OVA and OM degradation products with the polysaccharides was estimated. For this purpose, and as an example, the peaks eluting between 5 and 6 min in the SEC analyses of the duodenal digests of OM in the presence of P, G or X (marked with * or ** in Fig. 6) were collected, and their IgE-binding activity was checked by indirect ELISA (Fig. 7). The three collected fractions showed the ability to recognize IgE from egg allergic patients, with those formed in the presence of X being the most reactive in terms of IgE-binding.
M -G O
219a 103b 63.8b 120a,b
D
21.8a 5.15b 9.20b 10.9b
+
559a 255b 200b 254b
C
OM OM-P OM-G OM-X
Pool 2
M -G
66.8a 75.0a 62.3a 65.0a
AU 450 nm
DD DD DD DD
19.6a 13.4b 12.3a,b 13.3a,b
O M -G
23.4a 11.0b 13.0b 8.56b
0
AU 450 nm
OM OM-P OM-G OM-X
b
D D
110a 86.5a,b 72.9b 71.5b
b
M -P
13.9a 9.35b 8.55b 10.5a,b
1
O
151a 93.2b 102b,c 114c
52.3a 33.0b 36.4b 42.8a,b
2
D
OVA OVA-P OVAG OVA-X
41.9a 18.6b 27.8b 23.4b
Pool 3
D
DD DD DD DD
21.0a 16.6b 19.2a,b 19.2a,b
Pool 2
a
+
OVA OVA-P OVA-G OVA-X
Pool 1
a
C
Sample
Pool 1
AU 450 nm
Table 2 Binding to human IgE, expressed as EC50 (mg/ml), of ovalbumin (OVA) and ovomucoid (OM) in the absence or presence of pectin (P), gum arabic (G) and xylan (X) and their respective duodenal digests (DD) tested with 3 different pools of sera of egg allergic patient by inhibition ELISA. Results are means for n ¼ 2. Different letters indicate significant differences (P < 0.05) within each group. The IgE levels of the individual serum used in the pools are shown in Table 1.
603
Fig. 7. Binding to human IgE of the fractions collected after SEC analyses of the duodenal digests (DD) of the mixtures of ovomucoid (OM) with pectin (P), gum arabic (G), and xylan (X) marked with * or ** in Fig. 6. Cþ: positive control (80 mg/mL of OM in PBS). The bars represent the standard error and different letters indicate significant differences (P < 0.05) within each pool. The IgE levels of the individual sera used in the pool are shown in Table 1.
OVA in the presence of P, G and X supports that it could have interacted with the polysaccharides, particularly under the acidic conditions of gastric digestion (Figs. 1 and 3). In the case of OM, which is normally hydrolysed rapidly by pepsin (Kovacs-Nolan, Zhang, Hayakawa, & Mine, 2000), the protective effect was only detected upon duodenal digestion (Figs. 2 and 4). This agrees with previous works that reported that the establishment of interactions between food proteins and polysaccharides exerts a protective effect towards proteolysis (Mouecoucou, Fremont, et al., 2004; Mouecoucou et al., 2007, 2003; Mouecoucou, Villaume, et al., 2004; Polovic et al., 2007, 2009). However, to the best of our knowledge, the IgE binding of the complexes formed had not been assessed.
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The non-covalent interaction between proteins and polysaccharides in solution depends on the conditions of pH and ionic strength and on the distribution of charged, hydrophobic, hydrogen bonding groups, etc. This implies that such associations may reverse, although not necessarily, on changing the temperature or the pH (Dickinson, 1998). G and P, contain uronic acid, which is responsible for their anionic character, while X, composed of xylose, is neutral. At acidic pH, typical of the stomach (between 1.5 and 3; Mackie & Macierzanka, 2010), proteins below their isoelectric point, as it is the case of OVA and OM, can establish electrostatic associations with anionic polysaccharides. Such interactions, reinforced with non-ionic attractions, like hydrogen bonds, can lead to complex formation; while non-ionic interactions themselves are enough to establish associations between positively charged proteins and neutral polysaccharides such as X (Dickinson, 1998). In addition, at low pH, the unfolding of the native structure of the proteins could increase the chance of interaction (Rodriguez Patino & Pilosof, 2011). At neutral pH, typical of the duodenum (between 6 and 7; Kaukonen, Boyd, Porter, & Charman, 2004), proteins and anionic polysaccharides usually carry a negative net charge, but electrostatic interaction may still take place between the negative charges on polysaccharides and positively charged regions on proteins, that could also be reinforced with non-ionic attractions (Dickinson, 1998; Mouecoucou, Fremont, et al., 2004; Mouecoucou, Villaume, et al., 2004). Overall, the degree of protection varied depending on the polysaccharide. The highest resistance to digestion was found in OVA or OM and P mixtures. It is well known that P is prone to form gels at low pH, which could hinder digestion by increasing the viscosity of the gastric environment, although this capacity is reduced at higher pHs (Polovic et al., 2007). Nevertheless, P gels can resist enough at the duodenal level to exert a protector effect on the allergen Act c 1 from kiwi (Polovic et al., 2009). Previous studies claimed that it is the association of the polysaccharides with the degradation products, rather than the interaction with the undigested protein, what slows down digestion (Mouecoucou, Fremont, et al., 2004; Mouecoucou et al., 2007; Mouecoucou, Villaume, et al., 2004). In our work, SDS-PAGE analyses did not provide evidence for the interaction of the polysaccharides with the hydrolysis products of OVA or OM. However, the results of SEC analyses suggested that P and G could have interacted electrostatically with OVA and OM fragments released by pepsin, resisting, at least partially, duodenal digestion (Figs. 5 and 6). It is also likely that, under duodenal conditions, new associations were established between anionic polysaccharides and protein fragments containing basic amino acids released by trypsin (Arg and Lys). On the other hand, X probably established mainly non-ionic interactions with peptides arising from gastric and/or duodenal digestion. The in vitro duodenal digests of OVA and OM mixed with P, G and X showed a higher binding activity to IgE (Table 2) that could be, in principle, attributed to the lower digestion degree of the proteins. However, it was noteworthy that, in the presence of the polysaccharides, the reactivity towards IgE of both proteins significantly increased before hydrolysis, while their digestibility, and in particular that of OM, was only slightly affected. This suggested that there could be another explanation for the increased immunoreactivity observed. In fact, we found that the compounds attributed to the interaction of OM degradation products with the polysaccharides were able to bind IgE and those formed with X were very reactive (Fig. 7). It has been reported that the interaction of polysaccharides with gastric and duodenal digests of milk or peanut proteins reduces the IgE-binding, what was attributed to a masking effect on the reactive epitopes (Mouecoucou, Fremont, et al., 2004; Mouecoucou et al., 2007). However, our study
indicates that, depending on the nature of the allergens and their digests, their interactions with polysaccharides could also enhance their contact with IgE. Different glycosylation motifs on proteins are known as cross-reactive carbohydrate determinants (CCD) because of their ability to bind specific IgE antibodies (Commins & PlattsMills, 2010). In particular, it has been reported that residues of b 1,2-xylose exhibit in vitro CCD reactivity in pollen and food allergy, although its clinical relevance might be low (KaulfürstSoboll, Mertens, Brehler, & von Schaewen, 2011). 5. Conclusions The present study shows that the interaction of the main egg allergens, OVA and OM, with P, G and X increased their IgE binding and hampered their digestibility. The in vitro duodenal digests of OVA and OM in the presence of the polysaccharides retained a higher IgE-binding, probably as a result of the interaction between the polysaccharides and the peptides derived from protein digestion. Overall, the present results underline the importance of the food matrix in the digestibility of food allergens and in their potential ability to trigger an immune response. Acknowledgements This work was supported by the projects AGL2008-01740, AGL2011-24740 and CONSOLIDER CSD 2007-00063-FUN-C-FOOD. Jiménez-Saiz acknowledges the financial support of MICINN through a FPU grant. References Commins, S. P., & Platts-Mills, T. A. E. (2010). Allergenicity of carbohydrates and their role in anaphylactic events. Current Allergy and Asthma Reports, 10, 29e33. Dickinson, E. (1998). Stability and rheological implications of electrostatic milk proteinepolysaccharide interactions. Trends in Food Science & Technology, 9(10), 347e354. Jiménez-Saiz, R., Belloque, J., Molina, E., & López-Fandiño, R. (2011). Human immunoglobulin E (IgE) binding to heated and glycated ovalbumin and ovomucoid before and after in vitro digestion. Journal of Agricultural and Food Chemistry, 59(18), 10044e10051. Jiménez-Saiz, R., Martos, G., Carrillo, W., López-Fandiño, R., & Molina, E. (2011). Susceptibility of lysozyme to in-vitro digestion and immunoreactivity of its digests. Food Chemistry, 127(4), 1719e1726. Kaukonen, A. M., Boyd, B. J., Porter, C. J. H., & Charman, W. N. (2004). Drug solubilization behaviour during in vitro digestion of simple triglyceride lipid solution formulations. Pharmaceutical Research, 21, 245e253. Kaulfürst-Soboll, H., Mertens, M., Brehler, R., & von Schaewen, A. (2011). Reduction of cross-reactive carbohydrate determinants in plant foodstuff: elucidation of clinical relevance and implications for allergy diagnosis. PloS ONE, 6(3), e17800. Kovacs-Nolan, J., Phillips, M., & Mine, Y. (2005). Advances in the value of eggs and egg components for human health. Journal of Agricultural and Food Chemistry, 53(22), 8421e8431. Kovacs-Nolan, J., Zhang, J. W., Hayakawa, S., & Mine, Y. (2000). Immunochemical and structural analysis of pepsin-digested egg white ovomucoid. Journal of Agricultural and Food Chemistry, 48(12), 6261e6266. Mackie, A., & Macierzanka, A. (2010). Colloidal aspects of protein digestion. Current Opinion in Colloid & Interface Science, 15(1e2), 102e108. Martos, G., Contreras, P., Molina, E., & López-Fandiño, R. (2010). Egg white ovalbumin digestion mimicking physiological conditions. Journal of Agricultural and Food Chemistry, 58(9), 5640e5648. Mine, Y. (2002). Recent advances in egg protein functionality in the food system. World’s Poultry Science Journal, 58(1), 31e39. Mine, Y., & Yang, M. (2008). Recent advances in the understanding of egg allergens: basic, industrial, and clinical perspectives. Journal of Agricultural and Food Chemistry, 56(13), 4874e4900. Moreno, F. J. (2007). Gastrointestinal digestion of food allergens: effect on their allergenicity. Biomedicine & Pharmacotherapy, 61(1), 50e60. Mouecoucou, J., Fremont, S., Sanchez, C., Villaume, C., & Mejean, L. (2004). In vitro allergenicity of peanut after hydrolysis in the presence of polysaccharides. Clinical and Experimental Allergy, 34(9), 1429e1437. Mouecoucou, J., Fremont, S., Villaume, C., Sanchez, C., & Mejean, L. (2007). Polysaccharides reduce in vitro IgG/IgE-binding of beta-lactoglobulin after hydrolysis. Food Chemistry, 104(3), 1242e1249. Mouecoucou, J., Sanchez, C., Villaume, C., Marrion, O., Fremont, S., Laurent, F., et al. (2003). Effects of different levels of gum arabic, low methylated pectin and
R. Jiménez-Saiz et al. / Food Hydrocolloids 30 (2013) 597e605 xylan on in vitro digestibility of beta-lactoglobulin. Journal of Dairy Science, 86(12), 3857e3865. Mouecoucou, J., Villaume, C., Sanchez, C., & Mejean, L. (2004). Beta-lactoglobulin/ polysaccharide interactions during in vitro gastric and pancreatic hydrolysis assessed in dialysis bags of different molecular weight cut-offs. Biochimica et Biophysica Acta-General Subjects, 1670(2), 105e112. Osborne, N. J., Koplin, J. J., Martin, P. E., Gurrin, L. C., Lowe, A. J., Matheson, M. C., et al. (2011). Prevalence of challenge-proven IgE-mediated food allergy using population-based sampling and predetermined challenge criteria in infants. Journal of Allergy and Clinical Immunology, 127(3), 668e676.e2. Polovic, N., Blanusa, M., Gavrovic-Jankulovic, M., Atanaskovic-Markovic, M., Burazer, L., Jankov, R., et al. (2007). A matrix effect in pectin-rich fruits hampers digestion of allergen by pepsin in vivo and in vitro. Clinical and Experimental Allergy, 37(5), 764e771.
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Polovic, N. D., Pjanovic, R. V., Burazer, L. M., Velickovic, S. J., Jankov, R. M., & Velickovic, T. D. C. (2009). Acid-formed pectin gel delays major incomplete kiwi fruit allergen Act c 1 proteolysis in in vitro gastrointestinal digestion. Journal of the Science of Food and Agriculture, 89(1), 8e14. Rodriguez Patino, J. M., & Pilosof, A. M. R. (2011). Proteinepolysaccharide interactions at fluid interfaces. Food Hydrocolloids, 25(8), 1925e1937. Sicherer, S. H. (2011). Epidemiology of food allergy. Journal of Allergy and Clinical Immunology, 127(3), 594e602. Teuber, S. S. (2002). Hypothesis: the protein body effect and other aspects of food matrix effects. In T. J. G. S. M. Fu (Ed.), Genetically engineered foods assessing potential allergenicity, Vol. 964 (pp. 111e116). Zacharius, R. M., Zell, T. E., Morrison, J. H., & Woodlock, J. J. (1969). Glycoprotein staining following electrophoresis on acrylamide gels. Analytical Biochemistry, 30(1), 148e152.
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
IgE-binding and in vitro gastrointestinal digestibility of egg allergens in the presence of polysaccharides Rodrigo Jiménez-Saiz, Iván López-Expósito, Elena Molina, Rosina López-Fandiño* Instituto de Investigación en Ciencias de la Alimentación (CIAL) CSIC-UAM, Nicolás Cabrera 9, 28049 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 March 2012 Accepted 25 July 2012
This work studies the IgE-binding and in vitro gastrointestinal digestibility of the main egg allergens, ovalbumin (OVA) and ovomucoid (OM), in the presence of pectin (P), gum arabic (G) and xylan (X), functional biopolymers commonly used in the food industry. To this aim, solutions of OVA or OM and P, G or X were digested by using a model that mimics physiological conditions. Gastric and duodenal digests were analysed by SDS-PAGE, RP-HPLC and SEC and the specific human-IgE binding capacity was assessed by immunoblotting and ELISA using sera from egg-sensitized patients. The reactivity towards human IgE of OVA and OM was considerably increased in the presence of the polysaccharides and their susceptibility to digestion was diminished when compared with the isolated proteins. As a result, the duodenal digests obtained in the presence of polysaccharides retained more IgE-binding than the isolated protein digests. Overall, the present results underline the importance of the food matrix in the digestibility of food allergens and in their potential to trigger an immune response. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Food allergy Matrix effect Egg allergens Polysaccharides In vitro digestion IgE-binding
1. Introduction Egg proteins are broadly used in the food industry due to their unique functional properties, such as emulsifying, foaming and gelling. In addition, the high biological quality of egg proteins adds more value to the food products that include egg or its constituents as ingredients (Kovacs-Nolan, Phillips, & Mine, 2005; Mine, 2002). However, egg is an important source of allergens, which are mainly contained in the egg white, with ovalbumin (OVA or Gal d 2) and ovomucoid (OM or Gal d 1) being the major ones (Mine & Yang, 2008). Among food allergies, allergy to egg is, together with peanut and milk, the most common in children and infants with a prevalence that varies between 7.9 and 10% (Osborne et al., 2011; Sicherer, 2011). Among the conditions required for food proteins to trigger an allergic reaction is their ability to keep the integrity of their allergenic determinants through the gastrointestinal tract. To date, most of the studies dealing with the influence of the gastro duodenal digestion on the potential allergenicity of foods have been carried out on isolated proteins (Moreno, 2007). However, exposure of allergic individuals to pure allergens is rare and, in fact, the stability
Abbreviations: OVA, ovalbumin; OM, ovomucoid; P, pectin; G, gum arabic; X, xylan; GD, gastric digest; DD, duodenal digest; CCD, carbohydrate determinants. * Corresponding author. Tel.: þ34 91 0017941; fax: þ34 91 0017905. E-mail addresses: [email protected], rosina@ifi.csic.es (R. López-Fandiño). 0268-005X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodhyd.2012.07.014
of proteins to digestion can be altered in the presence of various components that form part of the food matrix (Teuber, 2002). Thus, it has been shown that the presence of soluble polysaccharides commonly used in the preparation of a wide range of foods, as stabilizers, thickeners and emulsifiers, reduces protein digestibility. The increase of mixture viscosity, the interactions between the two types of macromolecules and the inhibition of enzymatic activity have been pointed out to explain this observation (Mouecoucou, Fremont, Sanchez, Villaume, & Mejean, 2004; Mouecoucou et al., 2003). Polovic et al. (2009) reported the existence of a protective matrix effect on the digestion of pectin-rich crude extracts of various fruits. Pectin can form gels in the conditions present during in vivo and in vitro gastric digestion, providing a physical obstacle to the mobility of both pepsin and the protein substrate in the reaction mixture (Polovic et al., 2007). In addition, protective effects of structural polysaccharides of plant origin on the in vitro digestion of peanut allergens and b-lactoglobulin by pepsin and trypsin have been reported (Mouecoucou, Fremont, et al., 2004; Mouecoucou, Villaume, Sanchez, & Mejean, 2004). Although some studies did not go further than the digestibility assessment (Mouecoucou et al., 2003; Polovic et al., 2009), others have shown that, despite milk and peanut proteins are more stable towards hydrolysis, their digests show less IgG/IgE binding capacity, which was attributed to the masking of epitopes because of the interaction of the polysaccharides with the digestion products (Mouecoucou, Fremont, et al., 2004; Mouecoucou, Fremont, Villaume, Sanchez, & Mejean, 2007). Regarding egg allergens,
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there are very few studies on the effect of interactions with carbohydrates on their digestibility and immunogenicity (JiménezSaiz, Belloque, Molina, & López-Fandiño, 2011). Considering that foods are complex multicomponent mixtures that can contain proteins and polysaccharides, in many cases interacting as mixed biopolymers (Dickinson, 1998), and that such interactions can also form in the stomach after ingestion, the aim of the present work was to investigate the in vitro gastrointestinal behaviour and the resulting human IgE-binding of the main egg allergens, OVA and OM, in the presence of the polysaccharides: pectin (P), gum arabic (G) and xylan (X). 2. Materials and methods 2.1. Proteinepolysaccharide mixtures Hen egg white OVA grade VI, ovoinhibitor-depleted OM, high methylated P, G from acacia tree and X from beechwood were from SigmaeAldrich (St. Louis, MO, USA). Proteins and polysaccharides were dissolved separately (20 mg/mL) in 0.1 M NaCl and the pH was adjusted to 3 with 0.1 N HCl. The polysaccharides were heated at 60 C for 15 min to improve their solubility. Then, OVA and OM solutions were mixed with the solutions of each polysaccharide (1:1, v/v) and shaken gently overnight at room temperature (RT). 2.2. Gastric and duodenal digestions The digestibility of OVA, OM and their mixtures with each polysaccharide (P, G, X) was studied using an in vitro model system in two steps, which imitates gastric and duodenal digestion in vivo (Martos, Contreras, Molina, & López-Fandiño, 2010). Gastric digestions were performed in simulated gastric fluid (SGF, 35 mM NaCl) at pH 2.0, for 60 min at 37 C, with 172 U/mg of porcine pepsin (EC 3.4.23.1, 3210 U/mg protein, SigmaeAldrich). The reactions were stopped by rising pH to 7.0 with 1 M NaHCO3. Duodenal digestions were performed at 37 C by using the 60 min gastric digests adjusted to pH 7.0, as described above, with the addition of: 1 M CaCl2, 0.25 M Bis-Tris pH 6.5 and 0.125 M bile salts. Porcine pancreatic lipase (EC 232-619-9), colipase (EC 259490-1), trypsin (EC 232-650-8. 10100 BAEE units/mg protein) and a-chymotrypsin (EC 232-671-2: 55 units/mg protein) from SigmaeAldrich were used. Aliquots were taken after 60 min of gastric digestion and 30 min of duodenal digestion. Duplicate digestions were conducted for each condition. 2.3. SDS-PAGE Samples, diluted in a buffer containing 62.5 mM TriseHCl pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) b-mercaptoethanol and 0.0025% (w/v) bromophenol blue, were heated for 5 min at 95 C and loaded on 12% Bis-tris polyacrylamide gels (CriterionÔ XT). Electrophoretic separations were carried out at 150 V in a criterion cell using XT-MES running buffer. Gels were stained for protein with Coomassie Blue G-250 and for polysaccharide with Schiff reactive, following Zacharius, Zell, Morrison, and Woodlock (1969). 2.4. Reverse phase high-performance liquid chromatography (RPHPLC) The RP-HPLC analyses were performed using a Waters 600 HPLC instrument (Waters, Milford, MA, USA) and a 250 mm 4.6 mm Widepore C18 column (Bio-Rad, Richmond, CA, USA). Operating conditions were as follows: column at RT; flow rate, 1 mL/min; injection volume, 50 mL; solvent A, 0.37 mL/L TFA in Milli-Q water,
and solvent B, 0.27 mL/L TFA in HPLC grade acetonitrile. A linear gradient of solvent B in A, from 0 to 60% in 60 min, followed by 60% B for 30 min, was used. Absorbance was recorded at 220 nm with a Waters 2487 l dual detector. The software Empower 2000 system data (Waters) was used. 2.5. Size exclusion chromatography (SEC) The SEC analyses were performed using a Waters 600 HPLC instrument (Waters) and a 7.8 mm 300 mm TSK-gel G2000SWxl column (Tosoh Bioscience, Tokyo, Japan). Operation conditions were as follows: column at RT, flow rate, 1 mL/min; injection volume, 30 mL. Samples were eluted in isocratic mode by using 0.1 M NaCl, pH 7. Absorbance was recorded at 220 nm with a Waters 2487 l dual detector. The software Empower 2000 system data (Waters) was used. Fractions of interest were manually collected and frozen at 30 C for further use. 2.6. Human IgE-binding A total of 15 sera from children with clinically proved allergy to egg were mixed in five different pools of three sera each, as shown in Table 1. The sera were collected from the Maternal and Child Gregorio Marañón Hospital (Madrid, Spain). The patients’ specific seric IgE levels were determined by CAP (GE HealthCare, Waukesha, MI, USA). Unstained SDS-PAGE gels were soaked in transfer buffer (48 mM Tris, 39 mM glycine, 20% methanol, pH 9.2) for 20 min. Proteins were electroblotted into nitrocellulose membranes using a TransBlotÒ SD apparatus for 30 min at 18 V. The membranes were incubated in Tris buffered saline containing 0.05% (v/v) Tween 20 and 1% (w/v) bovine serum albumin (TBST-1% BSA) for 3 h and dipped into a pool of human sera (1/40 diluted in TBST-1% BSA) for 24 h at 4 C. Then, the membrane was incubated with biotinconjugated anti-human-IgE antibody (Southern Biotech, Birmingham, AL, USA), 1/500 diluted in TBST-1% BSA, overnight at 4 C. Finally, horseradish peroxidase (HRP)-streptavidin (DakoCytomation, Glostrup, Denmark) diluted 1:3000 in TBST-1% BSA was added and incubated for 1 h. All incubations were followed by 5 washing steps with TBST of 5 min each. The chemiluminescent substrate AmershamÔ ECLÔ Prime (GE HealthCare, Uppsala, Sweden) was used and light emission detected for 5 min in the VersaDocÔ imaging system. Human IgE-binding of the proteinepolysaccharide mixtures and their duodenal digests was also assessed by inhibition ELISA as Table 1 Specific IgE levels (kU/L) towards whole egg, egg white, ovalbumin and ovomucoid of the human sera and their distribution in the pools used throughout the study. Serum number IgE (kU/L)
Pool number
Whole egg Egg white Ovalbumin Ovomucoid 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
e e e e >100 >100 e >100 e e e e e e e
10.2 >100 e 7 e e 7.44 e e >100 7.7 11.6 >100 9.17 20.1
11.1 78.9 15 7.84 e e 3.03 e 62 e e e e e e
2.94 69.2 16 1.4 e e 7.51 e 80 e e e e e e
1 1 1 2 2 2 3 3 3 4 4 4 5 5 5
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previously reported (Jiménez-Saiz, Martos, Carrillo, López-Fandiño, & Molina, 2011) with slight variations: commercial OVA or OM, diluted in 0.01 M phosphate buffer, pH 7.4 (PBS), to 80 mg/mL, were used as coating antigens and polyclonal rabbit anti-human IgE (A0094, Dako, Glostrup, Denmark) and polyclonal swine anti-rabbit immunoglobulin labelled with HRP (P0399, Dako), diluted 1:1000 and 1:2000 (v/v), respectively, in PBS containing 0.05% Tween 20 (PBST), were used for detection. The IgE-binding results were expressed as the effective sample concentration for 50% of the maximum binding (EC50, mg/mL) following Jiménez-Saiz, Belloque, et al. (2011). The ability to bind human-IgE of the fractions collected by SEC was assessed by indirect ELISA. Plates were coated with the nondiluted samples and incubated overnight at 4 C. After overnight incubation, the plates were washed with PBST and incubated with sera from allergic patients. Following 2 h of incubation, plates were washed and incubated with rabbit anti-human IgE. After
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a washing step, polyclonal swine anti-rabbit immunoglobulin labelled with HRP was added and incubated for 1 h. Then, the plate was washed and the tyramide-biotin and HRP-streptavidin amplification system was used following the instructions of the manufacturer (ELAST ELISA amplification system, PerkineElmer Life Sciences, Waltham, MA). 3,30 ,5,50 -tetramethylbenzidine (TMB, ready-to-use solution; SigmaeAldrich) was used as substrate and the reaction was stopped with sulphuric acid. The absorbance was measured at 450 nm in plate reader (Multiskan FC, Thermo Scientific, Finland). 2.7. Statistical analyses The ELISA results, either as EC50 or as AU at 450 nm, were expressed as means for n ¼ 2. Significant differences (P < 0.05) were evaluated by one-way analysis of variance followed by post hoc multiple-comparison using Tukey’s test.
Fig. 1. SDS-PAGE analysis with Coomassie staining (a) and immunoblot using a pool of human sera from allergic patients (pool 4) (b) of ovalbumin digested in the absence and presence of polysaccharides. MW: molecular mass marker. The patterns of undigested (N) ovalbumin (OVA), its mixtures with pectin (P), gum arabic (G) or xylan (X) , and their gastric (GD) and duodenal digests (DD) are shown. The IgE levels of the individual serum used in the pool are shown in Table 1.
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3. Results 3.1. Digestibility of the mixtures of OVA and OM with polysaccharides The mixtures of OVA and OM with P, G and X, as well as their gastric and duodenal digests, were analysed by SDS-PAGE (Figs. 1a and 2a) and RP-HPLC (Figs. 3 and 4). Before in vitro hydrolysis, the mixtures of the proteins with the polysaccharides showed a smeared SDS-PAGE pattern stainable with Schiff reagent in the area of the gel corresponding to molecular masses higher than 50 kDa (results not shown). Coomassie staining did not show changes in the characteristic band patterns of OVA and OM in the presence of P, G and X; however, the existence of higher molecular mass protein material suggested that a portion of the proteins interacted with the polysaccharides in the mixed samples to form new polydispersed bands (Figs. 1a and 2a, lanes 5, 8 and 11).
Gastric digestion of OVA was slightly hampered in the presence of the polysaccharides (Fig. 1a). Furthermore, the 40.1 kDa fragment, Ala23ePro385, released by pepsin action (Martos et al., 2010), which corresponds to the peak eluting at 51 min in RP-HPLC (not shown), and, in general terms, the peptide fragments released during the simulated gastric digestion showed higher intensity in the presence of the polysaccharides, what suggested that they protected, to some extent, OVA and their digestion products from further hydrolysis by pepsin. In Fig. 3, which shows the RP-HPLC profiles of the duodenal digests, the higher resistance to digestion of OVA and of the Ala23ePro385 fragment, in the presence of P, G and X is still observed, although the protector effect on the duodenal peptides was reduced. In the case of OM digested under simulated gastric conditions, there were no differences among the SDS-PAGE (Fig. 2a) or RP-HPLC profiles (data not shown) in the absence or presence of polysaccharides. However, the digestibility of OM mixed with the polysaccharides under in vitro duodenal conditions
Fig. 2. SDS-PAGE analysis with Coomassie staining (a) and immunoblot using a pool of human sera from allergic patients (pool 5) (b) of ovomucoid digested in the absence and presence of polysaccharides. MW: molecular mass marker. The patterns of undigested (N) ovomucoid (OM), its mixtures with pectin (P), gum arabic (G) or xylan (X) and their gastric (GD) and duodenal digests (DD) are shown. The IgE levels of the individual serum used in the pool are shown in Table 1.
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601
Duodenal digests 0.12 0.10 0.08 0.06
DD OVA
0.04
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0.02 AU 220 nm
0.00 0.10 0.08 0.06 0.04
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25.0
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Fig. 3. RP-HPLC analyses of in vitro duodenal digests (DD) of ovalbumin (OVA) and of mixtures of OVA with pectin (P), gum arabic (G), and xylan (X). Fig. 4. RP-HPLC analyses of in vitro duodenal digests (DD) of ovomucoid (OM) and of mixtures of OM with pectin (P), gum arabic (G), and xylan (X).
AU 220 nm
a 0.10
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Time (minutes) Fig. 5. SEC analyses of mixtures of ovalbumin (OVA) with pectin (P) (a), gum arabic (G) (b), and xylan (X) (c) and their respective gastric (GD) and duodenal digests (DD).
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was slightly reduced, especially in the presence of P, as it can be seen in the RP-HPLC analyses shown in Fig. 4. SEC analyses were conducted on OVA and OM mixed with the polysaccharides and on their gastric and duodenal digests (Figs. 5 and 6). These showed peaks corresponding to high molecular mass forms in the presence of P and G (marked with * in Figs. 5 and 6), which were not found in the OVA or OM samples used as controls (data not shown), nor in the mixtures of OVA and OM with the neutral polysaccharide X (Figs. 5c and 6c). With the advance of digestion, new peaks corresponding to hydrolysis products with lower size were detected in the chromatograms. In addition, the high molecular mass forms increased in intensity during gastric digestion, and new peaks (marked with ** in Figs. 5 and 6) appeared, suggesting the binding of polysaccharides to peptide fragments released by pepsin. Upon simulated duodenal digestion, the first peak (marked with ** in Fig. 5) was not longer detectable in any of the OVA-polysaccharide mixtures, while the area of the second peak decreased in OVA and OM mixed with P, G and X.
3.2. IgE-binding of the mixtures of OVA and OM with polysaccharides and their digests The IgE-binding of OVA, OM, their mixtures with P, G and X and their in vitro digests was assessed by SDS-PAGE followed by immunoblotting (Figs. 1b and 2b) and by inhibition ELISA (Table 2). Inhibition ELISA could not detect any response from G and provided very high EC50 values for X and P (3.7 g/mL and 63.75 mg/mL, respectively), indicating that the polysaccharides did not bind IgE from egg allergic patients. The immunoblotting showed that the reactivity towards IgE of OVA and OM was considerably increased in the presence of the polysaccharides and the diffuse bands of high molecular mass, which presumably contained both proteins and polysaccharides, as judged by Shift and Coomassie staining, were strongly recognized by IgE (Figs. 1 and 2). The IgE binding was particularly enhanced in the mixtures with P, in the case of OVA, and with X, in the case of OM. Inhibition ELISA also indicated that the mixtures of OVA and
a 0.12
OM-P
0.10
GD OM-P
0.08
DD OM-P
0.06 0.04
*
0.02 0.00 2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
17.0
18.0
Time (minutes)
b 0.12
OM-G
0.10
GD OM-G
0.08
DD OM-G
0.06
*
0.04 0.02 0.00 2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
Time (minutes)
c 0.12
OM-X
0.10
GD OM-X
0.08
DD OM-X
0.06 0.04
**
0.02 0.00 2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.028 16.0
17.0
18.0
Time (minutes) Fig. 6. SEC analyses of mixtures of ovomucoid (OM) with pectin (P) (a), gum arabic (G) (b), and xylan (X) (c) and their respective gastric (GD) and duodenal digests (DD).
R. Jiménez-Saiz et al. / Food Hydrocolloids 30 (2013) 597e605
4. Discussion The present study shows that the reactivity towards human IgE of the major egg allergens, OVA and OM, was considerably increased in the presence of three different polysaccharides commonly used in the food industry. This could be attributed to the interaction, at acidic pH, of OVA and OM with P, G and X, producing complexes which were strongly recognized by IgE (Figs. 1 and 2; Table 2). In addition, the mixture with the polysaccharides slightly reduced the in vitro gastrointestinal stability of the allergens. The lower digestibility of
M -X O D D
D
a
a
2
1
b
b
0
M -X D D
D
D
D
D
O
O
O
M -P
+ C
Pool 3 a
a 2
1
b
b M -X O
O D
D D
M -P
0
D
OM with the polysaccharides exhibited a significantly (P < 0.05) increased reactivity, although the response depended on the pool of human sera considered and there were not significant differences among P, G and X (Table 2). The gastric and duodenal digests of the mixtures of OVA and polysaccharides also showed an increased IgE binding when compared with those of isolated OVA, as estimated by the higher intensity of the intact OVA bands and OVA degradation products in the immunoblot (Fig. 1b). The degradation products of OM, either in the absence or presence of polysaccharides, could not be transferred to the nitrocellulose membrane or were not retained in the membrane due to their small size (Fig. 2b). Inhibition ELISA confirmed that the duodenal digests of OVA and OM in the presence of P, G and X had lower EC50 than the duodenal digests of the isolated proteins (P < 0.05, Table 2). In general terms, the duodenal digests presented a reduced but still relevant IgE binding, compared with the undigested proteins or protein mixtures, especially in the case of OVA, although it greatly varied depending on the sera pool used. Thus, pool 2 exhibited a higher reactivity towards the duodenal digests than towards the intact proteins. Finally, the potential immunoreactivity of the compounds attributed to the interaction of OVA and OM degradation products with the polysaccharides was estimated. For this purpose, and as an example, the peaks eluting between 5 and 6 min in the SEC analyses of the duodenal digests of OM in the presence of P, G or X (marked with * or ** in Fig. 6) were collected, and their IgE-binding activity was checked by indirect ELISA (Fig. 7). The three collected fractions showed the ability to recognize IgE from egg allergic patients, with those formed in the presence of X being the most reactive in terms of IgE-binding.
M -G O
219a 103b 63.8b 120a,b
D
21.8a 5.15b 9.20b 10.9b
+
559a 255b 200b 254b
C
OM OM-P OM-G OM-X
Pool 2
M -G
66.8a 75.0a 62.3a 65.0a
AU 450 nm
DD DD DD DD
19.6a 13.4b 12.3a,b 13.3a,b
O M -G
23.4a 11.0b 13.0b 8.56b
0
AU 450 nm
OM OM-P OM-G OM-X
b
D D
110a 86.5a,b 72.9b 71.5b
b
M -P
13.9a 9.35b 8.55b 10.5a,b
1
O
151a 93.2b 102b,c 114c
52.3a 33.0b 36.4b 42.8a,b
2
D
OVA OVA-P OVAG OVA-X
41.9a 18.6b 27.8b 23.4b
Pool 3
D
DD DD DD DD
21.0a 16.6b 19.2a,b 19.2a,b
Pool 2
a
+
OVA OVA-P OVA-G OVA-X
Pool 1
a
C
Sample
Pool 1
AU 450 nm
Table 2 Binding to human IgE, expressed as EC50 (mg/ml), of ovalbumin (OVA) and ovomucoid (OM) in the absence or presence of pectin (P), gum arabic (G) and xylan (X) and their respective duodenal digests (DD) tested with 3 different pools of sera of egg allergic patient by inhibition ELISA. Results are means for n ¼ 2. Different letters indicate significant differences (P < 0.05) within each group. The IgE levels of the individual serum used in the pools are shown in Table 1.
603
Fig. 7. Binding to human IgE of the fractions collected after SEC analyses of the duodenal digests (DD) of the mixtures of ovomucoid (OM) with pectin (P), gum arabic (G), and xylan (X) marked with * or ** in Fig. 6. Cþ: positive control (80 mg/mL of OM in PBS). The bars represent the standard error and different letters indicate significant differences (P < 0.05) within each pool. The IgE levels of the individual sera used in the pool are shown in Table 1.
OVA in the presence of P, G and X supports that it could have interacted with the polysaccharides, particularly under the acidic conditions of gastric digestion (Figs. 1 and 3). In the case of OM, which is normally hydrolysed rapidly by pepsin (Kovacs-Nolan, Zhang, Hayakawa, & Mine, 2000), the protective effect was only detected upon duodenal digestion (Figs. 2 and 4). This agrees with previous works that reported that the establishment of interactions between food proteins and polysaccharides exerts a protective effect towards proteolysis (Mouecoucou, Fremont, et al., 2004; Mouecoucou et al., 2007, 2003; Mouecoucou, Villaume, et al., 2004; Polovic et al., 2007, 2009). However, to the best of our knowledge, the IgE binding of the complexes formed had not been assessed.
604
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The non-covalent interaction between proteins and polysaccharides in solution depends on the conditions of pH and ionic strength and on the distribution of charged, hydrophobic, hydrogen bonding groups, etc. This implies that such associations may reverse, although not necessarily, on changing the temperature or the pH (Dickinson, 1998). G and P, contain uronic acid, which is responsible for their anionic character, while X, composed of xylose, is neutral. At acidic pH, typical of the stomach (between 1.5 and 3; Mackie & Macierzanka, 2010), proteins below their isoelectric point, as it is the case of OVA and OM, can establish electrostatic associations with anionic polysaccharides. Such interactions, reinforced with non-ionic attractions, like hydrogen bonds, can lead to complex formation; while non-ionic interactions themselves are enough to establish associations between positively charged proteins and neutral polysaccharides such as X (Dickinson, 1998). In addition, at low pH, the unfolding of the native structure of the proteins could increase the chance of interaction (Rodriguez Patino & Pilosof, 2011). At neutral pH, typical of the duodenum (between 6 and 7; Kaukonen, Boyd, Porter, & Charman, 2004), proteins and anionic polysaccharides usually carry a negative net charge, but electrostatic interaction may still take place between the negative charges on polysaccharides and positively charged regions on proteins, that could also be reinforced with non-ionic attractions (Dickinson, 1998; Mouecoucou, Fremont, et al., 2004; Mouecoucou, Villaume, et al., 2004). Overall, the degree of protection varied depending on the polysaccharide. The highest resistance to digestion was found in OVA or OM and P mixtures. It is well known that P is prone to form gels at low pH, which could hinder digestion by increasing the viscosity of the gastric environment, although this capacity is reduced at higher pHs (Polovic et al., 2007). Nevertheless, P gels can resist enough at the duodenal level to exert a protector effect on the allergen Act c 1 from kiwi (Polovic et al., 2009). Previous studies claimed that it is the association of the polysaccharides with the degradation products, rather than the interaction with the undigested protein, what slows down digestion (Mouecoucou, Fremont, et al., 2004; Mouecoucou et al., 2007; Mouecoucou, Villaume, et al., 2004). In our work, SDS-PAGE analyses did not provide evidence for the interaction of the polysaccharides with the hydrolysis products of OVA or OM. However, the results of SEC analyses suggested that P and G could have interacted electrostatically with OVA and OM fragments released by pepsin, resisting, at least partially, duodenal digestion (Figs. 5 and 6). It is also likely that, under duodenal conditions, new associations were established between anionic polysaccharides and protein fragments containing basic amino acids released by trypsin (Arg and Lys). On the other hand, X probably established mainly non-ionic interactions with peptides arising from gastric and/or duodenal digestion. The in vitro duodenal digests of OVA and OM mixed with P, G and X showed a higher binding activity to IgE (Table 2) that could be, in principle, attributed to the lower digestion degree of the proteins. However, it was noteworthy that, in the presence of the polysaccharides, the reactivity towards IgE of both proteins significantly increased before hydrolysis, while their digestibility, and in particular that of OM, was only slightly affected. This suggested that there could be another explanation for the increased immunoreactivity observed. In fact, we found that the compounds attributed to the interaction of OM degradation products with the polysaccharides were able to bind IgE and those formed with X were very reactive (Fig. 7). It has been reported that the interaction of polysaccharides with gastric and duodenal digests of milk or peanut proteins reduces the IgE-binding, what was attributed to a masking effect on the reactive epitopes (Mouecoucou, Fremont, et al., 2004; Mouecoucou et al., 2007). However, our study
indicates that, depending on the nature of the allergens and their digests, their interactions with polysaccharides could also enhance their contact with IgE. Different glycosylation motifs on proteins are known as cross-reactive carbohydrate determinants (CCD) because of their ability to bind specific IgE antibodies (Commins & PlattsMills, 2010). In particular, it has been reported that residues of b 1,2-xylose exhibit in vitro CCD reactivity in pollen and food allergy, although its clinical relevance might be low (KaulfürstSoboll, Mertens, Brehler, & von Schaewen, 2011). 5. Conclusions The present study shows that the interaction of the main egg allergens, OVA and OM, with P, G and X increased their IgE binding and hampered their digestibility. The in vitro duodenal digests of OVA and OM in the presence of the polysaccharides retained a higher IgE-binding, probably as a result of the interaction between the polysaccharides and the peptides derived from protein digestion. Overall, the present results underline the importance of the food matrix in the digestibility of food allergens and in their potential ability to trigger an immune response. Acknowledgements This work was supported by the projects AGL2008-01740, AGL2011-24740 and CONSOLIDER CSD 2007-00063-FUN-C-FOOD. Jiménez-Saiz acknowledges the financial support of MICINN through a FPU grant. References Commins, S. P., & Platts-Mills, T. A. E. (2010). Allergenicity of carbohydrates and their role in anaphylactic events. Current Allergy and Asthma Reports, 10, 29e33. Dickinson, E. (1998). Stability and rheological implications of electrostatic milk proteinepolysaccharide interactions. Trends in Food Science & Technology, 9(10), 347e354. Jiménez-Saiz, R., Belloque, J., Molina, E., & López-Fandiño, R. (2011). Human immunoglobulin E (IgE) binding to heated and glycated ovalbumin and ovomucoid before and after in vitro digestion. Journal of Agricultural and Food Chemistry, 59(18), 10044e10051. Jiménez-Saiz, R., Martos, G., Carrillo, W., López-Fandiño, R., & Molina, E. (2011). Susceptibility of lysozyme to in-vitro digestion and immunoreactivity of its digests. Food Chemistry, 127(4), 1719e1726. Kaukonen, A. M., Boyd, B. J., Porter, C. J. H., & Charman, W. N. (2004). Drug solubilization behaviour during in vitro digestion of simple triglyceride lipid solution formulations. Pharmaceutical Research, 21, 245e253. Kaulfürst-Soboll, H., Mertens, M., Brehler, R., & von Schaewen, A. (2011). Reduction of cross-reactive carbohydrate determinants in plant foodstuff: elucidation of clinical relevance and implications for allergy diagnosis. PloS ONE, 6(3), e17800. Kovacs-Nolan, J., Phillips, M., & Mine, Y. (2005). Advances in the value of eggs and egg components for human health. Journal of Agricultural and Food Chemistry, 53(22), 8421e8431. Kovacs-Nolan, J., Zhang, J. W., Hayakawa, S., & Mine, Y. (2000). Immunochemical and structural analysis of pepsin-digested egg white ovomucoid. Journal of Agricultural and Food Chemistry, 48(12), 6261e6266. Mackie, A., & Macierzanka, A. (2010). Colloidal aspects of protein digestion. Current Opinion in Colloid & Interface Science, 15(1e2), 102e108. Martos, G., Contreras, P., Molina, E., & López-Fandiño, R. (2010). Egg white ovalbumin digestion mimicking physiological conditions. Journal of Agricultural and Food Chemistry, 58(9), 5640e5648. Mine, Y. (2002). Recent advances in egg protein functionality in the food system. World’s Poultry Science Journal, 58(1), 31e39. Mine, Y., & Yang, M. (2008). Recent advances in the understanding of egg allergens: basic, industrial, and clinical perspectives. Journal of Agricultural and Food Chemistry, 56(13), 4874e4900. Moreno, F. J. (2007). Gastrointestinal digestion of food allergens: effect on their allergenicity. Biomedicine & Pharmacotherapy, 61(1), 50e60. Mouecoucou, J., Fremont, S., Sanchez, C., Villaume, C., & Mejean, L. (2004). In vitro allergenicity of peanut after hydrolysis in the presence of polysaccharides. Clinical and Experimental Allergy, 34(9), 1429e1437. Mouecoucou, J., Fremont, S., Villaume, C., Sanchez, C., & Mejean, L. (2007). Polysaccharides reduce in vitro IgG/IgE-binding of beta-lactoglobulin after hydrolysis. Food Chemistry, 104(3), 1242e1249. Mouecoucou, J., Sanchez, C., Villaume, C., Marrion, O., Fremont, S., Laurent, F., et al. (2003). Effects of different levels of gum arabic, low methylated pectin and
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