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INTRODUCTION
Buckwheat has traditionally been used as a food source in Asian countries, including Korea, Japan, and China. Although buckwheat has been used more frequently in Asian countries than in Western countries, the use of buckwheat has increased globally because of its health functional effects. The known beneficial effects include abundant flavonoids, phytosterols, dietary fiber, vitamins, minerals, and antioxidants. It also showed cholesterol-lowering effects [1]. In addition, due to its gluten-free characteristics, buckwheat could even be offered to patients with celiac disease as an alternative to wheat [2].
However, buckwheat can cause an immediate and IgE-mediated type food allergy, and sometimes only a tiny amount of buckwheat can induce a very severe reaction, such as anaphylaxis [3]. One study demonstrated that scrubbing a small quantity of buckwheat on lips or licking it by tongue (< 1 g) could induce allergic symptoms [4]. Approximately 0.22% of Japanese school children (194/92,680) had buckwheat allergy [5], and approximately 10% of patients with buckwheat allergy experienced anaphylaxis after ingestion of buckwheat [6]. In Korea, 0.1% (44/42,202) of school age children have buckwheat allergy [7]. Recently, buckwheat allergy has often been reported even in western countries, including the UK [8], Germany [9], and the United States [2]. Presence of buckwheat as a hidden allergen in most common Italian foods, such as pizza and pasta, has also been reported [10].
Development of hypoallergenic food may be helpful in improvement of the quality of life for patients and reduce the occurrence of dangerous allergic reactions. A number of studies have been conducted in an effort to reduce allergenicity of buckwheat. Use of Maillard-type glycosylation [11], buckwheat seed fraction [12], and salting method [13,14] resulted in partial reduction of the allergenicity. In addition, treatment with heat, acid, alkali, and reduced carboxymethylation had positive effects on reduction of allergenicity [15]. Handoyo et al. [16] reported that allergenic 22 kDa buckwheat protein was completely degraded after fermentation with Rhizopus oligosporus, although it was limited to only part of allergens. According to Satoh et al. [17], buckwheat has sequential epitopes, which are resistant to heat and acid. Enzymatic hydrolysis is associated with a greater chance of breaking sequential epitopes [18], and Terracciano et al. [19] demonstrated that the total and allergenic proteins were significantly degraded after treatment with enzymatic hydrolysis. Hydrolyzed cow's milk has already been used as a hypoallergenic food [20]. Previous studies found that buckwheat allergens cannot be completely hydrolyzed by pepsin or chymotrypsin through immunoassay with sera of buckwheat-sensitive patients [21]. Hence, in the current study, after enzymatic hydrolysis using seven commercial proteases, analysis of the hydrolysates was performed using SDS-PAGE, competitive inhibition ELISA, immunoblotting with rabbit antiserum developed against buckwheat protein, and direct ELISA with pooled serum of buckwheat-sensitive patients.
MATERIALS AND METHODS
Patients' sera
Twenty one sera samples were obtained from buckwheat-sensitive patients (five females and 16 males ranging in age from one to 19 years old) with atopic dermatitis who were treated at Samsung Medical Center (Korea). Specific IgE levels measured using the Phadia UniCAP-system (Sweden) ranged from 1.11 to 58.2 kUA/L (Table 1). Sera from healthy and non-atopic individuals who had never shown allergic symptoms were used as negative control. The serum samples were stored at -80 ℃ until use. This study was approved by the Samsung Medical Center Institutional Review Board (SMC 2012-06-047).
Extraction of buckwheat protein
Buckwheat protein was extracted from buckwheat powder purchased from the National Agricultural Cooperative Federation (Fagopyrum esculentum, Korea) with minor modifications of the method of Tanaka et al [22]. Buckwheat powder (5 g) was mixed in 50 ml of 0.086 M NaCl (Showa, Japan) solution containing 0.033M NaHCO3(Duksan, Korea) and placed overnight at 4℃ with stirring. After centrifugation at 12,000 × g for 30 min, the supernatant was lyophilized at -80℃, and stored at -70℃ until use. Protein concentration was determined using the BCA protein assay kit (Pierce, U.S.A.) with bovine serum albumin as the standard.
Hydrolysis of buckwheat protein
Alkaline protease (Genencor, Finland), GC106 (Genencor, Japan), papain (Enzyme Solutions, Australia), bromelain (Great Food, Thailand), collupulin (Gist-brocades, France), Flavourzyme, and Protamex (Novo, Denmark) were used as proteolytic enzymes. The characteristics and test conditions of each enzyme are described in Table 2. The extracted buckwheat protein was hydrolyzed according to the method of Clemente et al. [23] with minor modification of digestion time using seven enzymes (an enzyme-to-buckwheat protein ratio of 10%) with 10 mM PBS (phosphate buffered saline) at individual optimum pH and temperature for four hours.
SDS-PAGE and Immunoblotting analysis
SDS-PAGE was performed according to the method of Laemmli [24]. The protein was denatured with the sample buffer containing reducing agent for 5 min at 95℃, followed by analysis on 4-12% Bis-Tris gradient gels (NuPAGE, Invitrogen, U.S.A.) for 35 min at 200 V, and then visualized by staining with Coomassie Brilliant Blue (Bio-Rad, UK). Precision plus protein standards (product No. 161-0374, Bio-Rad, UK) were used as molecular weight markers. Buckwheat protein was transferred from the SDS-PAGE gel to a polyvinylidenedifluoride (PVDF) membrane using the iBlot Dry Blotting System (Invitrogen, Israel). After blocking with 2% nonfat dried milk for one hour at room temperature, the membrane was incubated with rabbit antiserum diluted 1:1,000 in 2% nonfat dried milk overnight at room temperature. The membrane was then washed three times with 0.03% PBST for 10 minutes each and incubated with goat anti-rabbit IgG-HRP (Thermo, U.S.A.) diluted 1:10,000 in 2% nonfat dried milk for one hour at room temperature. After washing three times again, the membrane was reacted with Amersham ECL reagent (GE Healthcare, Sweden) for two minutes. The blotted membrane was then exposed to high-performance chemiluminescence film (GE Healthcare, UK), after which the film was developed.
Enzyme-linked immunosorbent assay (ELISA)
In order to evaluate the reduction of buckwheat allergenicity, a direct ELISA was performed with pooled serum of 21 buckwheat-sensitive patients. The plate was coated with 100 µl of each enzymatic hydrolyzed buckwheat protein sample containing 50 µg/ml protein diluted in 50 mM carbonate-bicarbonate buffer (pH 9.6) (Sigma, U.S.A.), and incubated at 4℃ overnight. After washing the plate three times with PBST (Tween20 (Sigma, U.S.A.) diluted 0.05% in PBS (Solon, U.S.A.)), the plate was blocked by addition of 200 µl/well of 2% BSA (Roth Chemical, Germany) in 0.05% PBST and incubated for one hour at room temperature for prevention of unspecific linking. Pooled serum was diluted 1:40 in 2% BSA-PBST. The plate was washed three times and incubated with 100 µl of diluted pooled serum for two hours at room temperature. After washing three times, adsorbed IgE was detected with 100 µl of goat anti-human IgE-HRP (1 µg/ml) (KPL, U.S.A.) diluted 1:2,500 in 2 % BSA-PBST. The plate was further washed and developed with 100 µl of TMB peroxidase (3,3'5,5'-tetramethylbenzidine) (KPL, U.S.A.). Absorbance was measured using a microplate reader (Bio-Rad, Japan) at 595 nm in duplicate. The allergenicity of hydrolysate was compared with that of buckwheat protein.
To evaluate the reduction of buckwheat antigenicity, a competitive indirect ELISA (ciELISA) was performed with rabbit antiserum to buckwheat protein. A microplate was coated with 100 µl of extracted buckwheat protein (2 µg/ml) and incubated at 4℃ overnight. After washing the plate three times with 0.05% PBST, rabbit antiserum to buckwheat protein (diluted 1:500 in PBST) and buckwheat protein were mixed at a 1:1 ratio. The mixture (100 µl) was added to each well and the plate was incubated for one hour for induction of competitive binding. After washing, 100 µl of goat anti-rabbit IgG-HRP (Sigma, U.S.A.) diluted 1:10,000 in PBS as secondary antibody was added, followed by incubation for one hour. The plate was further washed and developed with 100 µl of TMB substrate solution for 30 min, and the reaction was stopped by addition of 50 µl of 2 M H2SO4 (Sigma, U.S.A.). Absorbance was measured at 450 nm in duplicate. The half maximal inhibitory concentration (IC50) was used as a measure of the effectiveness of hydrolysis in inhibition of antigen-antibody binding reaction.
RESULTS
SDS-PAGE and Immunoblotting analysis
Analysis of the proteolytic effect of seven enzymes on buckwheat allergens was performed using SDS-PAGE (Fig. 1 A) and immunoblotting with rabbit antiserum to buckwheat protein (Fig. 1 B). The intact buckwheat protein (BW) showed several bands at 30-50 kDa, a distinct band at 20-25 kDa, and a smeared band below 15 kDa on SDS-PAGE. All buckwheat protein bands were recognized as antigens by rabbit antiserum. After hydrolysis with alkaline protease (AK), all buckwheat protein bands of the intact buckwheat sample were no longer observed on SDS-PAGE, and they were no longer recognized as antigens by the antiserum. Bromelain (BRO), collupulin (COL), papain (PAP), and Protamex (PRO) partially hydrolyzed buckwheat proteins, and proteins less than 25 kDa remained. Flavourzyme (FLA) and GC106 (GC) barely digested buckwheat protein, and they were fully recognized as antigen by the antiserum.
Change of antigenicity and allergenicity after hydrolysis
Competitive Inhibition ELISA (ciELISA) was performed for evaluation of the change of antigenicity after hydrolysis with seven commercial proteolytic enzymes (Table 3). The antigenicity showed a significant decrease, to 1/92.6, 1/92.6, and 1/82.5 when measured as IC50 after hydrolysis with alkaline protease, bromelain, and papain, respectively. Hydrolysis of buckwheat protein with Flavourzyme and GC106 barely changed its antigenicity. Hydrolysis with Protamex and collupulin resulted in a moderate change in antigenicity.
Direct ELISA was performed with pooled serum of buckwheat-sensitive patients for evaluation of its allergenicity (Fig. 2). Hydrolysis of buckwheat protein with alkaline protease resulted in decreased IgE reactivity to 41% of unhydrolyzed buckwheat protein. The allergenicity of hydrolyzed buckwheat protein with Protamex also fell to 51.6% of the unhydrolyzed protein, and approximately 40% reduction of allergenicity was observed when buckwheat protein was hydrolyzed with collupulin or papain. After hydrolysis with bromelain, the allergenicity of hydrolysates showed a slight decrease. The allergenicity even showed a slight increase after hydrolysis of buckwheat protein with Flavourzyme and GC106.
DISCUSSION
Seven commercial proteases were examined in order to determine the effect of hydrolysis of buckwheat protein on changes of antigenicity and allergenicity of buckwheat allergens. Enzymes examined could be grouped based on its characteristics as serine-type peptidases, cysteine-type peptidases, aminopeptidase, and aspartic-type peptidase.
Enzymatic hydrolysis has generally been known to reduce the antigenicity of proteins. Traditional fermented soybean products have shown less antigenicity than raw soybean, and hydrolyzed infant formulas have already been used for children with allergy to cow milk protein [19,25]. Both alkaline protease and Protamex, classified as serine peptidases (EC 3.4.21.62), particularly subtilisin-like type, appeared to be relatively effective in reduction of buckwheat allergenicity. Food allergens are water-soluble glycoproteins, 10-70 kDa in size, and, in general, it should be more than 10 kDa for binding of IgE [18,26]. Alkaline protease degraded all of the buckwheat proteins to low molecular proteins below 10 kDa, and showed the most effective result in reducing IgE-binding. Subtilisin-like type serine proteases such as alkaline protease and Protamex are broadly specific for aromatic or hydrophobic residues, and have generally been used in traditional fermented soybean products [27]. One study reported that proteases from Bacillus subtilis, such as Protease N and Proleather, destroyed most soybean proteins, and reduced the allergenicity [28]. Major soybean allergens were selectively hydrolyzed with Proleather, and the hydrolysate showed reduced allergenicity without a decline in gel forming ability [29]. Soybean allergens were significantly reduced and no longer recognized by soybean-sensitive patients after fermentation with protease from B. subtilis [30]. Frias et al. [31] also reported a decrease in immunoreactivity of soybean samples evaluated with human plasma of more than 80% after fermentation with B. subtilis.
Papain, collupulin, and bromelain, classified as cysteine-type peptidases (EC 3.4.22.2), also caused a partial decrease in the allergenicity of buckwheat. The band at 20-25 kDa that remained after papain hydrolysis appeared to be Fag e 1 (22-24 kDa) because it was reported to show resistance to papain [32]. Hydrolysis with papain reduced the antigenicity, similar to the level hydrolyzed with alkaline protease, and the allergenicity of the hydrolysate with papain did not fall as much as when hydrolyzed with alkaline protease. The hydrolytic effects of hydrolysis with collupulin on buckwheat allergens were similar to those with papain, although the antigenicity was decreased more effectively with papain. Bromelain showed an effective decrease in antigenicity, however, allergenicity decreased by only 12%. As shown in these results, it appeared that the change of allergenicity evaluated with human sera did not match the change of antigenicity evaluated with rabbit antisera (Table 3 and Fig. 2). In general, antigenicity has been evaluated with rabbit antisera in order to determine the allergenicity of proteins because it has many limitations in evaluation of allergenicity with human sera, such as ethical issues and lack of quantity. However, similar to this result, Mine and Zhang [33] reported that human and rabbit antibodies recognized egg white proteins differently, and strong correlation was observed between antibody recognition and antigen structure when sensitized. It is considered that the differences were caused by the fact that the rabbit antisera was produced by injection of purified and emulsified buckwheat allergens, while patients were sensitized when the antigens were absorbed through the gastrointestinal tract in type of processed foods. It is suggested that the allergenicity of the proteins should be confirmed by measurement of human IgE.
Flavourzyme and GC 106 barely affected the change of buckwheat allergens. Flavourzyme is classified as an aminopeptidase (EC 3.4.11.1), and GC106 is included in aspartic-type peptidase (EC 3.4.23.18). Both enzymes are derived from Aspergillus spp., although they are included in different classifications. A previous study reported that hydrolysis with Flavourzyme somewhat increased the allergenicity. The IgE reactivity of patients with peanut allergy showed an increase within the first 30 min after treatment with Flavourzyme [34], and another study also showed an increment in allergenicity when chickpea was hydrolyzed with Flavourzyme [23]. It could be assumed that enzymatic hydrolysis might be helpful in exposure of the interior hidden epitopes of buckwheat, resulting in slightly increased allergenicity (Fig. 2). Treatments such as heating and proteolysis may result in formation of new epitopes, and may promote the interaction of IgE with the immune system in the intestine and lead to a potential increase in protein allergenicity [35].
None of the enzymes could completely eliminate the allergenicity, although the antigenicity appeared to have decreased. Serine-peptidases such as alkaline protease and Protamex appeared to be relatively effective in hydrolysis of buckwheat allergens, and, in particular, alkaline protease decreased the allergenicity by more than 50%. This reducing effect is similar to that of soybean allergens degraded by fermentation with subtilisin. In this study, the antigenicity measured using rabbit antiserum did not correspond well to the allergenicity measured using human sera when buckwheat protein was hydrolyzed with various proteolytic enzymes. This result indicates that the allergenicity may remain unchanged, even though the antigenicity appeared to have decreased significantly. Measurement of IgE reactivity of human sera may be required for evaluation of the hypoallergenic characteristics of buckwheat allergens. There is a lack of information on buckwheat allergens, and studies for characterization of enzymatic hydrolysis of buckwheat appear to be prerequisite to reduction of buckwheat allergenicity.
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