Introduction
The allergy to peanuts (
Arachis hypogea) is one of the most common in industrialized societies. Research on the prevalence of peanut allergy carried out under the integrated project on food allergy EuroPrevall showed the occurrence of sensitization to peanut in adult population to be 9.3 % in USA and between 4.2 and 0.8 % in European countries [
1]. However, the level of sensitization reaches approximately 10 % in the population of 8-year-old children in the UK [
2]. Clinical peanut allergy is detectable in 1–2 % of the total population [
2] with an increasing prevalence in US [
3] and stable tendency in the UK [
4].
Peanut 7S globulin (Ara h 1) is one of the most potent peanut allergens. It was reported that 75 % of peanut allergic individuals have serum-specific IgE against this protein [
5]. The 63.5 kDa monomers of Ara h 1 form a highly stable homotrimer held together primarily through hydrophobic interactions between residues on α-helical bundles located on the ends of each monomer [
6,
7]. Koppelman et al. [
8] showed rapid hydrolysis of Ara h 1 by pepsin, which has been confirmed by other authors [
9,
10]. However, a trimeric complex of Ara h 1 may protect from digestion of a linear IgE epitopes which are located mainly in the areas of the subunit–subunit contacts [
11]. Thus, fragments of Ara h 1 containing several intact IgE-binding epitopes are able to reach the gut mucosa and influence the intestinal homeostasis [
11].
The Maillard reaction (MR, glycation), a non-enzymatic reaction between free amino groups of protein (usually the ε-amino group of lysine residues) and carbonyl groups of reducing sugars is one of the most widespread modifications of proteins that occurs during food processing. The mechanism of glycation is complex and involves a cascade of chemical rearrangements which finally leads to formation of numerous Maillard reaction products (MRPs) including components that may impact on human health (acrylamide, heterocyclic amines (HCAs), glycation/lipoxidation end products) [
12,
13]. Recent studies showing MR as a crucial factor changing the biological properties of food proteins [
14‐
17] highlighted the importance of this reaction not only in an areas of food chemistry [
18] and flavour chemistry [
19] but also human pathology [
17,
20‐
23]. In our previous studies, we demonstrated the influence of different temperatures of MR (37, 60 and 145 °C) on biological activity and biochemical characteristic of purified hazelnut 7S globulin, Cor a 11. We showed that the MR influences such parameters as sensitivity of Cor a 11 to proteolysis, binding capacity for human IgE or rabbit IgG and degranulation capacity of basophiles [
15]. We also demonstrated that roasting of Ara h 1 at 145 °C modulates its allergenicity by increasing the basophil degranulation capacity [
16]. However, the effect of roasting on the biological properties appears to be specific for different proteins. Thus, there is a need for answering the question how the processing influence the biological activity of different proteins what is especially relevant in terms of proteins with allergenic potential [
16]. In this approach, extremely important is the role of MR occurring in food systems on the biological properties of food allergens especially with respect to their interaction with gut epithelia. Careful planning and management of the conditions under which MR takes place may provide safe food, free from Maillard reaction products (MRPs) that have been described as harmful to health [
13,
20].
Caco-2 cells are widely used as model for human epithelium cells [
14,
17,
21,
22,
24,
25] since Hidalgo reported that they resemble the morphology and function of human intestine epithelial cells [
26]. The Caco-2 model allows to analyse biological activity of food components (e.g. MRPs) towards human intestine epithelial cells, reflected in cell proliferation, enzymatic activity, apoptosis and cytokine secretion. Characterization of food compounds in terms of promotion or inhibition of cell proliferation provides information about physiological conditions and growth rate of the cells, forming the basis for numerous in vitro assays of a specific cell response, for example interleukin secretion [
21,
22,
27‐
29]. Intestinal epithelial cells may affect the local immune system by their cytokine secretion and thus promote inflammatory response. Interleukin-8 (IL-8) has been identified as a key mediator of epithelial immune responses which influences several factors including activation, growth, differentiation, and migration of neutrophils, basophils and T cells [
30,
31]. Therefore, the modulation of expression of IL-8 by intestinal epithelial cells via MRPs may provide new information in the field of immunomodulation by food especially in terms of conditions such as food allergy.
In this study, we created a model system to investigate the biological changes of the major peanut allergen Ara h 1 treated with and without glucose under three different temperate/time conditions. As before [
15], we decided to use temperatures which are widely used in food industry as well as in experimental work to mimic the Maillard reaction in food. Heating at 37 °C, corresponding to human body temperature, has only a minor effect on protein structure and therefore contributes to gaining further knowledge on protein glycation in the body [
32]. Heating at 60 °C changes the secondary and tertiary structure of proteins [
33], creates an effective condition for MR [
34] and is also applied during food manufacturing [
35], while 145 °C is the temperature of peanut roasting [
36]. This approach allowed us to characterise the effect of these time/temperature conditions with respect to their effect on biochemical and molecular properties of Ara h 1 including:
1.
Physicochemical properties at different stages of MR,
2.
Sensitivity to pepsin proteolysis,
3.
Molecular interaction with gut epithelium in a model system using the Caco-2 cell line.
Discussion
In this study we examined three different conditions of MR on biochemical properties and biological activity of Ara h 1. We demonstrated temperature/time-dependent differences in biochemical characteristics of Ara h 1 glycated at different conditions, which is attributed to different stages of MR and diversity of obtained products. In our previous study on hazelnut 7S globulin, we applied identical temperature/time-treatment conditions as in this study to assess the effect of the MR [
15] in terms of its three main phases (early, advanced and final stage) [
40,
41]. An effective glycation of Cor a 11 occurred at all investigated conditions [
15], fully corroborating observations of this study on Ara h 1. MR at 37 °C did not influence the specific IgG or IgE binding to Cor a 11 [
15], suggesting minimal structural changes under these treatment conditions. Analysis of the physicochemical properties of Ara h 1 glycated at 37 °C, such as the lack of brown colour development (Table
1) and no differences between UV–vis spectra of Ara h 1 treated in and without glucose (data not shown), suggest that under these conditions MR did not proceed to the advanced stage. Pedrosa et al. [
32], studying the influence of glycation at 37 °C during 50 h on pea 7S globulin, showed that glycation at this mild condition does not significantly change the oligomeric structure of vicilin. However, the glycated vicilin derivatives appeared to be more stable most probably due to covalent attachment of carbohydrate moieties to the protein surface and the participation of carbohydrate hydroxyl groups in intersubunit hydrogen bonding [
32]. The results presented by Pedrosa et al. seem to be consistent with data obtained for Ara h 1 glycated at 37 °C; however, the stability of the protein was not directly examined. Fluorescence and browning development in MR are generally used as indicators of the reaction rate and formation of MRPs [
34,
42,
43]. We observed both an enhancement of fluorescence and brown colour development in the samples glycated at 60 and 145 °C indicating the advanced and final stage of MR. The low level of fructosamine compared to protein-bound fluorescent compounds (ratio about 3:1) indicate a formation of advanced Maillard reaction products (AGEs) at 60 °C. This is consistent with previous findings showing formation of AGEs during the first 24 h of protein incubation at 60 °C [
34]. An increased browning intensity of Ara h 1 glycated at 145 °C correlated with decreased intensity of protein-bound fluorescence, confirming the hypothesis of various authors that fluorescent compounds are precursors for browning products [
44,
45]. The kinetics and mechanism of the formation of both colour and fluorescent compounds seem to be highly material- and condition-dependent [
43,
46,
47], making speculations about the nature of formed fluorescent and brown compounds very difficult. It has been shown that above 100 °C, the degradation of the Amadori product is faster than its formation [
40] what most probably also took place during the glycation of Ara h 1 at 145 °C as suggested by the relations between fructosamine, protein-bound fluorescence and colour development. The diversity and heterogeneity of low-molecular products formed during glycation of Ara h 1 at 145 °C were corroborated by a smear observed on the SDS-PAGE. Similar effects of MR that occurred at temperatures above 100 °C were observed also by other authors [
11,
15]. The products obtained through the glycation of Ara h 1 at 60 °C as well as 37 °C seem to be more homogenous as shown on SDS-PAGE. The results obtained due to biochemical analysis indicate temperature/time-dependent differences in the type of obtained MRPs as well as in the protein structure influenced by the temperature. Those results were reflected in biological activity of processed Ara h 1. We showed that high temperature itself (60 and 145 °C) did not alter the DH of Ara h 1 significantly (Fig.
4) while glycation influenced the susceptibility of Ara h 1 to pepsin hydrolysis in a condition-dependent way. The addition of glucose to the system led to more pronounced changes in the Ara h 1 structure caused by the modification of amino acids such as phenylalanine, tyrosine and tryptophan [
48‐
50] that are involved in maintaining tertiary structure of Ara h 1 [
8,
48]. The glycation-induced conformational changes of Ara h 1 modified the access of pepsin to its cleavage sites resulting in 41 % decrease of DH in the case of treatment at 60 °C and a reduction of DH to 5 % in the case of glycation at 145 °C. It has been previously shown that advanced stages of MR promote the cross-linking of Ara h 1 which makes the protein progressively more insoluble and unextractable [
10,
11,
15,
48]. Enzymatic hydrolysis of cross-linked Ara h 1 most probably generated so-called ‘limit-peptide fragments’ which prevents further hydrolysis because the peptide around the cross-linking site are inaccessible to the hydrolytic enzymes [
51]. The literature data on the influence of MR on susceptibility of Ara h 1 to enzymatic hydrolysis are not consistent [
10,
11]; however, the studies of Seiquer et al. [
52] provide a new point of view on the effects of consumption of MRP-rich diets on dietary nitrogen utilization. Authors showed a negative effect of diet rich in MR products on protein digestibility and pointed particularly at the long-term effects of dietary MRPs on nitrogen utilization [
52].
Data from in vitro and in vivo studies suggest that most of AGEs escape digestion in the upper gastrointestinal tract and are mainly recovered in the faeces [
53]. Reaching the intestines, glycopeptides may influence the metabolic activity of enterocytes as well as physiological response of intestinal microbiota thus having an impact on the overall health status [
17]. We showed a significant anti-proliferative activity of non-hydrolysed Ara h 1 on Caco-2 cells which increased at higher protein concentrations (data not shown). The anti-proliferative potential of native Ara h 1 was significantly reduced by hydrolysis of the protein. This fact may be due to damage to protein sequences that display an anti-proliferative effect. Moreover, the peptides formed during the hydrolysis are a source of nutrients for Caco-2 cells what may additionally reduce the anti-proliferative effect. The anti-proliferative mode of action of peanut 7S globulin seems to be consistent with data obtained by Jia et al. [
54] who demonstrated a decreased proliferation rate of intestinal epithelial cells isolated from 28-day-old piglets as the result of incubation with soy 7S globulin. Caco-2 proliferation rate expressed by the cell viability (direct cell counting, measurement of total cell protein or DNA) or its enzymatic activity (MTT and WST-1 tests) often forms the basis for an assessment of cytotoxic potential of examined substances [
14,
22,
28,
29,
55]. Gerlier and Thomasset observed that the increase in mitochondrial activity was independent of the increase in DNA synthesis [
56], which proves that the proliferative events can be independent from mitochondrial activity thus complicating comparison of different studies. Some authors imply additional methods, for example cell membrane damage tests: crystal violet assay and lactate dehydrogenase (LDH) leakage [
27] leading to a more accurate assessment of cell physiological condition. However, these classical methods measure both cell viability and membrane permeabilization but do not quantify the proportion of apoptotic target cells compared to normal vital cells and dead cells and are not able to distinguish early apoptotic cells from highly apoptotic and necrotic ones [
57,
58]. More precise and consistent methods for assessment of food compounds cytotoxicity are needed to enable the comparison of results presented by different research groups. Therefore, based on anti-proliferative effects of native Ara h 1, we can speculate that Ara h 1 possess cytotoxic properties against intestinal epithelia cells. Our results indicate that protein-bound MRPs obtained during glycation of Ara h 1 at 60 and 145 °C may attenuate the anti-proliferative effect of native Ara h 1. To clarify this hypothesis, independently of BrdU test, more precise cytometric methods should be applied [
59,
60].
We demonstrated a stimulation of IL-8 production by Caco-2 cells incubated in the presence of native Ara h 1. These results seem to be consistent with an effect of Ara h 1 on the proliferation of Caco-2 cells. An increase of IL-8 secretion by Caco-2 cells may be a cellular response to compensate for anti-proliferative effects of this protein. Those findings confirm the ability of Ara h 1 to induct a local inflammatory response in gut mucosa what may be related to the high allergenic potential of this molecule. The stimulatory properties of the native form of Ara h 1 were retained after pepsin hydrolysis which suggests that the fragments responsible for this effect were protected from digestion. MRPs formed during glycation of Ara h 1, at all studied time/temperature conditions (37, 60, 145 °C), acted as an inhibitor of IL-8 secretion by Caco-2 cells. Moreover, the results observed for Ara h 1 glycated at 37 and 60 °C demonstrate a consistent effect as no differences between the mode of action of those samples were observed. However, we demonstrated different stage of MR for each tested condition what was also confirmed on SDS-PAGE. Therefore, inhibition of IL-8 secretion observed as the result of incubation of Caco-2 cells with Ara h 1 glycated under all three tested conditions may be caused by different substances from a chemical point of view. However, in the light of obtained results, a more detailed chemical analysis of the products of glycation of Ara h 1 together with the determination of their anti-inflammatory effect is needed. Our results suggest that an inhibitory effect is more prominent before pepsin digestion than after. So most probably, the bigger structures play a crucial role in IL-8 inhibition. However, the glycopeptides obtained after pepsin hydrolysis did not lose their biological properties responsible for IL-8 inhibition. Our findings are consistent with the previous results which showed that low-molecular MR fractions may reduce the inflammation in Caco-2 cells induced with IFN-γ + PMA by the transcriptional down-regulation of genes involved in the NF-κB pathway as well as the translational inhibition of iNOS and IL-8 expression [
21,
22]. Other studies showed that transcription factor NF-κB which plays a critical role in inflammation and cancer development [
61] may be activated via the receptor for advanced glycation end products (RAGE) [
62]. Therefore, the advanced MRPs, formed due to glycation of Ara h 1, may exert their effect on Caco-2 through RAGE which is indeed expressed by Caco-2 cells [
23]. As it was shown for protein-linked CML (
N-carboxymethyl-lysine), MRPs may induce the activation state of p44/42 (ERK1/2) mitogen-activated protein kinases (MAPKs) in Caco-2 cells via interaction with RAGE [
23]. MAPKs were shown to regulate cellular proliferation in Caco-2 cells [
63] making it possible that AGEs obtained during glycation of Ara h 1 stimulated intestinal epithelial cell proliferation via MAPK activation. However, this speculation requires further studies. Some authors showed an anti-proliferative action of MRPs [
22,
50,
64], while Jing and Kitts [
14] showed no significant toxicity of casein glycated at 55 °C during 28 days to Caco-2 cells. These findings suggest that the way of action of MRPs on the proliferation of Caco-2 cells depends on the biochemical structure of a protein and the type of obtained products.
We showed that glycation of Ara h 1 at high temperatures (60 and 145 °C) led to advanced stages of MR which decreases the degree of protein hydrolysis and modulates the interaction of Ara h 1 with Caco-2 cells. However, glycation of Ara h 1 at 37 °C, which was shown to cause least biochemical changes, also modulated the interaction of modified Ara h 1 with gut epithelium by inhibition of IL-8 secretion. It is important to note that conclusions concerning the bioactive properties of MRPs, susceptibility to pepsin hydrolysis and interaction with gut epithelium underestimate the importance of the temperature/time conditions of MR. Our studies showed that the MRPs of Ara h 1 obtained in all three studied time/temperature conditions may influence the “pro-inflammatory network” in the human intestinal mucosa, creating the need of further studies to determine the chemical structure of these products, their anti-inflammatory mechanisms of action as well as interaction with gut microbiota.