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Low-pH induced structural changes, allergenicity and in vitro digestibility of lectin from black turtle bean (Phaseolus vulgaris L.)
Food Chemistry 283 (2019) 183–190
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Low-pH induced structural changes, allergenicity and in vitro digestibility of lectin from black turtle bean (Phaseolus vulgaris L.)
T
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Jinlong Zhaoa, Shudong Hea,b, , Mingming Tanga, Xianbao Suna, Zuoyong Zhanga, ⁎ Yongkang Yea,b, Xiaodong Caoa,b, Hanju Suna,b, a
Engineering Research Center of Bio-process of Ministry of Education, School of Food and Biological Engineering, Hefei University of Technology, Hefei 230009, Anhui, PR China b Anhui Province Key Laboratory of Functional Compound Seasoning, Anhui Qiangwang Seasoning Food Co., Ltd., Jieshou 236500, Anhui, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Lectin Phaseolus vulgaris L. bean Low pH treatment Structural changes Allergenicity In vitro digestibility
Lectin was incubated in corresponding acidic buffers (pH 1.0–3.5) for a certain period (0.5, 1, 2, 4, 8, 12 and 24 h) at 25 °C. Low-pH induced changes in structure, allergenicity and in vitro digestibility of lectin from black turtle bean (Phaseolus vulgaris L.) were investigated in the present study. Results indicated that the alteration in structure was a progressive unfolding process mainly depending on pH environment, and the treated lectin attained a stable state at 8 h. Electrophoretic, dynamic light scattering (DLS) and size exclusion chromatography (SEC) analyses suggested that lectin monomers appeared in the solutions of pH < 2.0. Differential scanning calorimetry (DSC) confirmed that thermal stability of lectin weakened in low pH environments. Furthermore, ELISA and in vitro digestion assay showed allergenicity and digestibility significantly decreased with the structural alterations. These results showed low-pH treatments have great potential to reduce the damage of legumes protein consumption.
1. Introduction Lectins are carbohydrate-binding proteins or glycoprotein of nonimmune origin presenting in most legume plants and a wide range of vegetables (He et al., 2018; Kumar et al., 2013; Lagarda-Diaz, GuzmanPartida, & Vazquez-Moreno, 2017). They have shown numerous potential beneficial health effects on human, such as anti-cancer, antiHIV, anti-microbial infection, preventing mucosal atrophy, reducing type 2 diabetes and obesity and promoting gut health (Buul & Brouns, 2014; He et al., 2018). However, nausea, diarrhea, vomiting and abdominal swelling could be caused in both children and adults due to the lectin protein, which has been described as an allergen in food, and the incidence of lectin based food poisoning is increasing in recent years (Buul & Brouns, 2014; He et al., 2018; Lagarda-Diaz et al., 2017; Nasi, Picariello, & Ferranti, 2009). More than 1,000 people had acute gastrointestinal symptoms and 100 people were hospitalized due to consumption of insufficiently boiled white kidney beans (P. vulgaris L.) in Japan in 2006 (Ogawa & Date, 2014). Lectin-intake poisoning accounted for 53.3% of legumes (P. vulgaris L.) food poisoning events in China from the years of 2004 to 2013. As a common legume, black turtle bean (P. vulgaris L.) was consumed worldwide and applied in many foods due to its rich nutrient ⁎
content, but inadvertent exposure of the untreated bean with activated lectin would cause life-threatening anaphylaxis (He et al., 2018; Kumar et al., 2013). Therefore, there is an urgent issue to produce black turtle bean-based foods that are safe for allergic consumers. Previously, the lectin from black turtle bean (P. vulgaris L.) with high haemagglutinating activity was isolated in our laboratory (He, Shi, Walid, Ma, & Xue, 2013; He et al., 2015). Its sequence (NCBI accession no. AHB17899.1), three-dimensional (3-D) structure and epitopes were first identified in our earlier researches (He, Shi, Li, Ma, & Xue, 2015; He et al., 2018). The lectin belongs to the phytohaemagglutinins (PHA) family with highly conserved domain of lectin_legume_LecRK_Arcelin_ConA region, and it is a homodimeric protein where each subunit has a molecular weight of 31 kDa and consists of 275 amino acid residues (Fig. 1) (He et al., 2015; He et al., 2015a, b). Each lectin subunit comprises five indole groups of Trp79, Trp154, Trp176, Trp225 and Trp249, and six indole groups of Tyr72, Tyr133, Tyr135, Tyr150, Tyr190 and Tyr203 (Fig. 1a). It has been suggested four B-cell epitopes renamed B1 (55–66: NVNDNGEPTLSS), B2 (116–126: VGSEPKDKGG), B3 (131–141: NNYKYDSNAHT) and B4 (149–160: LYNVHWDPKPRH) were critical for lectin sensitization (Fig. 1b and c) (He, Zhao, & Elfalleh, 2018). Many previous researches have proven that the antigenicity of
Corresponding authors at: School of Food and Biological Engineering, Hefei University of Technology, Hefei 230009, Anhui, PR China. E-mail addresses: [email protected] (S. He), [email protected] (H. Sun).
https://doi.org/10.1016/j.foodchem.2018.12.134 Received 25 October 2018; Received in revised form 29 December 2018; Accepted 29 December 2018 Available online 14 January 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Ribbon structure of the lectin from black turtle bean (Phaseolus vulgaris L.). Tetrameric structure with the distributions of Tyr and Trp (a); and B-cell epitope regions distributions in monomer (b) and tetramer (c); Epitopes and Tyr/Trp regions were highlighted with black.
China). The lectin from the black turtle bean (P. vulgaris L.) with at least 94% purity was obtained according to our previous studies (He et al., 2013; He et al., 2015). The extracted lectins were freeze-dried and stored in a −80 °C refrigerator until use. Porcine pepsin (2754 U/mg) and trypsin (11356 U/mg) were obtained from Sigma Chemical Co., Ltd (St. Louis, MO, USA). Sodium dodecyl sulphate (SDS), and Coomassie Blue R-250, β-mercaptoethanol, and 8-Anilino-1-naphthalenesulfonate (ANS) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). All chemicals used in the present study were of analytical grade.
allergen protein could be influenced by the structural modification (Lin et al., 2015; Marambe et al., 2015; Rahaman, Vasiljevic, & Ramchandran, 2016). As a simple chemical treatment, low acidic pH is wildly believed to modify functional properties of protein by changing its structure (Foh et al., 2012; Liu et al., 2015). Emulsifying properties of soy protein isolates were markedly improved by the pH-shifting from pH 1.5 (Jiang, Chen, & Xiong, 2009), and enhanced gelling ability was obtained in the soy protein isolates after the treatment under acidic (pH 1.5) conditions combined with proper heating (50 or 60 °C) (Liu et al., 2015). Although knowledge on allergenicity of allergen protein induced by low acidic treatment is poorly documented (Lin et al., 2015; Stănciuc et al., 2016), a speculation would be modestly brought up that the allergenicity of legume lectin might be affected during the acidic processing because of the alteration of protein conformations. Therefore, in the present study, effects of low acidic pH (pH 3.5, 3.0, 2.5, 2.0, 1.5 and 1.0) on the structure and allergenicity of lectin from black turtle bean (P. vulgaris L.) were investigated. Structural alterations were explored by intrinsic/extrinsic fluorescence spectra assays, SDS/Native–PAGE, dynamic light scattering (DLS) and size exclusion chromatography (SEC), respectively. Allergenicity was evaluated with direct enzyme-linked immunosorbent assay (ELISA). In addition, hemagglutination activity and in vitro digestibility of lectin from black turtle bean (P. vulgaris L.) also were assessed, respectively. Our findings would give an insight into relationship between lectin conformational alterations and its allergenicity, which have tremendous application potential in the developments of safe pharmaceuticals and low-allergen legume based foods.
2.2. Low-pH induced process Lectin sample was dissolved into specific pH buffer (pH 3.5, 3.0, 2.5, 2.0, 1.5 and 1.0, respectively) to a centration of 2.0 mg/mL, and was incubated for 0.5, 1, 2, 4, 8, 12 and 24 h at room temperature (25 ± 2 °C) before all measurements, respectively. The lectin sample prepared using phosphate buffer (Na2HPO4-NaH2PO4, 10 mM, pH 7.2) was set as the native control. The buffers used for various treatments were 10 mM of KCl-HCl (pH 1.0–1.5), Gly-HCl (pH 2.0–3.0) and Na2HPO4-citric acid (pH 3.5). All the buffers were filtered through 0.22 μm membrane.
2.3. Intrinsic fluorescence measurements Intrinsic fluorescence measurements were performed on a RF20AXS spectrofluorometer (Horiba Ltd., Kyoto, Japan). Each treated sample was diluted with corresponding buffer to a concentration of 0.2 mg/mL. The changes in tertiary structure were monitored by intrinsic fluorescence emission between 300 and 400 nm at a 280 nm excitation, and the excitation and emission slit width were set at 5 nm, all spectra were corrected by subtraction of corresponding buffer.
2. Materials and methods 2.1. Materials Black turtle beans (P. vulgaris L.) cultivated in Heilongjiang province of China were obtained from a local market (Harbin, Heilongjiang, 184
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2.4. Extrinsic fluorescence measurements
expressed as the units of activity per mg of protein (Eq. (1)), and the hemagglutination activity retention (%) could be presented as the ratio of treated lectin to native control.
In the extrinsic fluorescence experiments, four milliliter aliquots of treated lectin samples (0.2 mg/mL) were prepared using the corresponding buffers, and mixed with 50 μL of 8.0 mM of ANS solution for a 5 min reaction. Then, the mixed solutions were performed on a RF20AXS spectrofluorometer (Horiba Ltd., Kyoto, Japan). The excitation was set at 380 nm and the emission spectra were taken in the range of 400–600 nm.
HA(HU/mg) =
2n × 103 C(pr) × V
(1)
where n is the highest well number showing hemagglutination in the Ushaped microtiter plates, and n = 0 means no visible agglutination. C(pr) (mg/mL) is the protein concentration of the lectin solution, while V (μL) is the volume of lectin solution added to each well.
2.5. Gel electrophoresis 2.10. Allergenicity determination The lectin treated with low pH environment was analyzed with gel electrophoresis according to the method of He et al. (2013). The concentration gel and the resolving gel contained 4% and 12% acrylamide, respectively. Lectin samples (2.0 mg/mL) were boiled with loading buffer containing SDS and β-mercaptoethanol at 1:1 (v/v) ratio for 5 min. Then, 15 μL of each sample was loaded in each well of the SDS–PAGE gel. Native gel electrophoresis without SDS, β-mercaptoethanol and boiling for lectin proteins was performed similarly according to the method of SDS-PAGE.
The antigenicity of the lectin sample was determined by ELISA according to our previous method (He et al., 2018). Carbonate bicarbonate buffer (pH 9.6) was added in each well with 1 μg/100 μL of lectin sample and incubated overnight at 4 °C. After washing with PBS (pH 7.2, 10 mM) twice, the plate was blocked with 5% (w/v) defatted milk (200 μL/well) for 1 h at 25 °C, and then was washed five times using PBS (pH 7.2, 10 mM). The recombinant polyclonal anti-lectin antibodies obtained from Nanjing SenBeiJia Biological Technology Co., Ltd. (Nanjing, Jiangsu, China) were subsequently diluted 1:100,000 v/v with PBS (pH 7.2, 10 mM) and added into each well, then the plate was incubated overnight at 4 °C. The plate was following incubated with 1:1000 v/v diluted anti-human-IgE peroxidase antibody (Sigma-Aldrich Chemicals Co., St. Louis, MO, USA) at 37 °C for 2 h and washed five times with PBS. Prior to the detecting, the chromogenic reaction was developed using 3,3′, 5,5′-tetramethylbenzidine (TMB) for 10 min in the dark and was terminated with 0.2 M sulphuric acid. The optical density (OD value) was measured at 450 nm within 15 min by a microplate reader (Tecan Infinite 200Pro, Tecan Group Ltd., Mannedorf, Switzerland). Since the OD value is proportional to the IgE binding capacity of lectin (Lin et al., 2015), the relative IgE binding capacity (%) was used to express the IgE binding capacity of treated lectin with respect to that of native control (100%).
2.6. Dynamic light scattering (DLS) measurements Each treated sample was diluted with corresponding buffers to a concentration of 0.2 mg/mL, and the samples were centrifuged at 12,000 rpm for 10 min and were filtered serially through 0.22 μm Whatman syringe filters. DLS measurements were performed on a Zetasizer µV dynamic light scattering equipment (Malvern Instruments Ltd., Malvern, UK) equipped with a temperature-controlled micro sampler at a scattering angle of 90° at 25 °C. 2.7. Size exclusion chromatography (SEC) SEC experiments of treated lectins were performed on a preparative high performance liquid chromatography (HP PLUS 100D, Lisure Science Co., Ltd., Suzhou, China) using Chromdex 200 prepacked column (16 × 600 mm, Bogelong Bio-Technology Co., Ltd., Shanghai, China). After the prepacked column was equilibrated with desired pH buffers, an aliquot of 5 mL of treated lectin samples (2.0 mg/mL) was injected and monitored at 280 nm, and the elution was carried out at a flow rate of 1.0 mL/min with corresponding pH buffer.
2.11. In vitro digestion assay In vitro simulated gastrointestinal digestion of treated lectin was carried out as previously described (Zhang et al., 2017). For the simulated gastric digestion process, porcine pepsin from porcine gastric mucosa was employed in simulated gastric solution (35 mM NaCl, pH 1.2) at an enzyme: lectin ratio of 1:20 (w/w), and was incubated in a water bath at 37 °C for 10 min prior to the addition of lectin. Aliquots (100 μL) were taken at 0, 10, 20, 30 and 60 min, respectively, for the further determination, and the reaction solution in each aliquots was immediately terminated by adding 30 μL of 0.2 M Na2CO3 solution. The digested protein solution after the 60 min pepsin digestion was adjusted to pH 7.2 with 1.0 M NaOH solution, then was added into the simulated intestinal fluid digestion containing 1.0 M CaCl2, 0.25 M BisTris (pH 7.2) and 0.125 M bile salts for further simulated intestinal digestion. Trypsin was added to the mixture at an enzyme: substrate ratio of 1:100 (w/w) after preheating for 10 min in water bath at 37 °C. Subsequently, aliquots (100 μL) were taken at 5, 10, 30 and 60 min, respectively, and then were stopped the digested reactions by heating in a boiling water bath for 10 min. All protein samples were collected and stored at −20 °C until were analyzed by SDS-PAGE.
2.8. Differential scanning calorimetry (DSC) measurements DSC experiments were carried out on a TA Q100-DSC thermal analyzer (TA Instruments, New Castle, DE, USA). Indium standards were used for DSC calibration prior to all the sample measurements. The treated lectin samples (15 μL, 2.0 mg/mL) were accurately pipetted into aluminum liquid pans, and the calorimetric traces were recorded from 25 °C to 120 °C at a rate of 10 °C/min. A sealed empty pan containing an equal mass of the corresponding pH buffer solution was employed as a reference. All the experiments were performed at least 3 times. The denaturation temperature (Td) and the enthalpy of denaturation (△H) were computed by the TA universal analysis software (Version 4.1D, TA Instrument-Waters LLC, New Castle, DE, USA). 2.9. Hemagglutination activity assay
2.12. Statistical analysis Hemagglutination activity was determined based the method of He et al. (2018). Serial twofold dilutions of 50 μL lectin solutions (2.0 mg/ mL) were added into 96-well U-shaped microtiter plates, followed by addition of 50 μL of rabbit erythrocyte suspension (2%, v/v) to each well. The mixtures were incubated at 4 °C for 1.5 h, and the extent of hemagglutination was determined visually with PBS (pH 7.2, 10 mM) as a negative control. The hemagglutination activity (HA, HU/mg) was
All experiments were repeated in a minimum of triplicates and the values were reported as mean ± standard deviation. Analyses of variance (ANOVA) and Duncan’s means comparison test were applied using SPSS version 11.5 (SPSS Inc. Chicago, IL, USA) with a significance level of 0.05. Ribbon structure of the lectin from black turtle bean (P. vulgaris L.) was generated by using UCSF Chimera (version 1.13, 185
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Fig. 2. The plots of intrinsic fluorescence intensity at the λmax (328 nm) (a) and extrinsic fluorescence intensity at the λmax (480 nm) (b) as a function of incubation time at different pH (pH 3.5, 3.0, 2.5, 2.0, 1.5 and 1.0). The lectin protein concentration was 0.2 mg/mL.
deeper understanding of the pH induced structural changes (Figs. S2 and 2b). Maximum emission wavelength with minimum ANS fluorescence intensity was found in the native control. Low-pH induced lectin showed significant increase in the ANS fluorescence intensity with obvious blue shift (λmax from 518 nm to 480 nm) compared to the native. Similar blue shifts in λmax with increases in ANS fluorescence intensity were early found in the investigations of Arisaema helliborifolium lectin and lima bean trypsin inhibitor at low pH environments for 12 h incubation at room temperature (Khan et al., 2017; Thakur et al., 2015). In addition, there was a significant enhancement in fluorescence intensity of low-pH treated lectin with increasing time to 8 h, indicated that intact structure of lectin became looser due to the unfolding, thus more Trp/Tyr residues were exposed via moving toward to a more nopolar environment, which would lead to the lectin structural changes (Chen et al., 2016). Effects of pH and incubation time on the ANS fluorescence intensity of lectin at 480 nm (λmax) were also further plotted (Fig. 2b), and it found that the increase in ANS fluorescence intensity was accompanied with the reduction of pH values. The maximum ANS fluorescence intensity was obtained in the pH 2.0 treatment for 8 h, indicated that most Trp/Tyr residues have moved toward to more hydrophobic areas (Khan, Qadeer, Ahmad, Ashraf, Bhushan, Chaturvedi, et al., 2013; Yadav et al., 2016). In addition, with the extension of time, the fluorescence intensity and λmax of all treated lectin samples showed little difference compared to the lectin treated for 8 h, suggesting a gradual unfolding of lectin occurred at low pH environments, and a steady state would be obtained at 8 h. Thus, in order to make the following experiments more precise, incubation time 8 h for each lectin sample in the in corresponding low acidic buffer was employed.
University of California San Francisco, San Francisco, CA, USA). 3. Results and discussion 3.1. Intrinsic fluorescence The structural changes of the low-pH treated lectin for different time were shown in Figs. S1 and 2a using the intrinsic fluorescence emission. The excitation fluorescence spectra of proteins are resulting from the functions of Trp, Tyr, and Phe residues, and the fluorescence emission spectrum at 280 nm is mainly due to the existing of Trp and Tyr residues (He et al., 2015a, b). The maximum fluorescence emission of the native lectin (pH 7.2) was found at 328 nm, which was similar with the report of He et al. (2014), and suggested that Trp and Tyr residues might be mainly located in the hydrophobic regions of the protein molecule. The maximum fluorescence intensity (157 a.u) was also found in native lectin spectrogram (Figs. S1 and 2a). In our previous study (He et al., 2018), Trp249 and Tyr72, 190 as well as 203 were found to be buried in the hydrophobic regions of protein molecule which might contribute to the high fluorescence intensity. As presented in Fig. 2a, there was a evident blue shift in the maximum emission with a considerable decrease in fluorescence intensity of treated lectin in the range of 0–8 h, indicated that the protein surface amino acids, such as Trp79, 154, 176 and 225, and Tyr133, 135 and 150, might gradually move to the non-polar environments, and the decrease in the fluorescence intensity could be attribute to the deprotonation of the neighboring basic amino acids (He et al., 2015a, b). These observations indicated that the tertiary structure of low-pH treated lectin was disrupted, and the degree of these alterations mainly depended on the functions of pH environments. Earlier studies also demonstrated that the amino acid groups on the proteins surface would be protonated when the environmental pH was below the isoelectric point, thus the native lectin structure might be disrupted due to the electric charge effects (Khan, Ahmad, & Khan, 2007; Lin et al., 2015). Similar results were also obtained in the investigation for lima bean trypsin inhibitor incubated at acidic buffers for 12 h (Khan et al., 2017). The change in intrinsic fluorescence intensity at the λmax (328 nm) was plotted as a function of time at different pH (Fig. 2a). Significantly decreased fluorescence intensity could be clearly observed in the range of 0–8 h, while no significant difference was found in fluorescence intensity of low-pH treated lectin with the further increase in incubation time, indicated that a steady state of the tertiary structure of lectin was formed up to 8 h with low-pH treatment.
3.3. Gel electrophoretic characterization, DLS and SEC analysis Structural changes of low-pH treated lectin from black turtle bean (P. vulgaris L.) were further investigated by gel electrophoretic, DLS and SEC analyses (Fig. 3). SDS-PAGE patterns showed a single band corresponding to the molecular mass of 31 kDa (Fig. 3a), and no significance was observed over pH range of 3.5–2.0, indicated the stability of primary structure. However, the original band decreased mildly in intensity with the formation of minor low molecular weight fragments over low pH range of 1.5–1.0, which might be due to acidic hydrolysis of lectin protein. As shown in Fig. 3b, the lectin without boiling and βmercaptoethanol treatments showed a single band on the top of resolving gel (Lane 1–5), which was similar to native lectin band, while a single band on the middle of resolving gel could be observed in Lanes 6 and 7, respectively, indicated that lectin homotetrameric might dissociate to the monomers (31 kDa) in lower acidic (pH 1.5–1.0) environments.
3.2. Extrinsic fluorescence Extrinsic fluorescence analysis was performed to obtain a much 186
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Fig. 3. SDS–PAGE (a) and native-PAGE (b) profiles of native and low-pH treated lectin from black turtle bean (Phaseolus vulgaris L.) for 8 h. Lane M, protein marker; Lanes 1–7, different low pH treatment for pH 7.2 (native), 3.5, 3.0, 2.5, 2.0, 1.5 and 1.0, respectively. Size exclusion chromatography (SEC) profiles (c) of native and low pH (pH 3.5, 3.0, 2.5, 2.0, 1.5 and 1.0) treated lectin from black turtle bean (Phaseolus vulgaris L.) for 8 h.
228.4 min was obtained from the treated lectin at pH 1.0, indicated the full monomerization of the protein (Fig. 3c). Taken together the above results, the lectin protein retained its tetramer states until pH 2.0, and further decrease in pH would make protein dissociate to monomer states because of the intramolecular electrostatic repulsions (Fitch, Whitten, Hilser, & Garcia, 2010; Khan et al., 2013).
The influence of low pH incubation on the hydrodynamic radius (Rh) of lectin was investigated using DLS (Table S1). Compared with the native control, a significant increase in Rh was found with the pH decreasing from 3.5 to 2.0, which could be attributed to lectin structure unfolding. The intact structure gradually became looser due to the intramolecular charge-charge repulsion in low pH environments, which could result in the unfolding of structure (Khan, Qadeer, & Ahmad, 2013; Thakur et al., 2015). These results also are good agreement with the intrinsic and extrinsic fluorescence analysis, indicating some subtle changes in the lectin structure. With the further decrease of pH, the Rh of lectin exhibited a prominent reduction of 57.8% and 69.6% in pH 1.5 and 1.0 treatments compared to that of the initial Rh, respectively, which further confirmed the dissociation of lectin tetramers. Fig. 3c showed the SEC profiles of treated lectins under different pH buffers. Native lectin was eluted as a single peak around 123.1 min, and the elution times of lectin treated with pH 3.5–1.0 buffers presented a progressive decreased trend, which could be attributed to the increase of molecule size (Khan et al., 2013). While two peaks center around 123.1 and 228.4 min were observed in the elution profile of lectin after pH 1.5 treatment, and the former peak (123.1 min) was gentle, which might attribute to the gradual monomerization of lectin at pH 1.5. Some others multimeric lectin proteins also present a similar pH-dependent dissociation behavior in acidic solutions, such as soybean agglutinin (Molla et al., 2009), banana lectin (Khan et al., 2013), turmeric root lectin (Biswas & Chattopadhyaya, 2014). A single peak around
3.4. Thermal stability DSC curves of lectin from the black turtle bean (P. vulgaris L.) after low-pH treatments were shown in Fig. 4. All treated lectin samples showed a prominent endothermic peak. Widest and deepest endothermic peak with the highest peak temperature (Td) was obtained in the native control, and the peak width and depth decreased with the decreasing of pH value. According to the endotherm area analysis, both Td and denaturation enthalpy (△H) values after low-pH treatments were decreased (Fig. 4a), which could be attributed to possible structural alterations of the protein, implying the loss of thermal stability (He et al., 2015a, b). 3.5. Hemagglutination activity Special carbohydrates on the red blood cell surface could be recognized by the active lectin, and cross-linked network in suspension could be formed based on the function, which was called 187
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high surface accessibility and flexibility (Fig. 1b and c). Based on the above studies, lectin protein underwent gradual unfolding and dissociation in low acidic conditions which could terminate the surface exposure of epitopes, and cause the loss in surface accessibility and flexibility for IgE recognition (Marambe et al., 2015; Rahaman et al., 2016; Stănciuc et al., 2016). In other words, exposed surface epitopes were buried, destroyed or degradation owing to these structural alterations, which were no longer recognized by IgE antibodies or able to stimulate an immune response, ultimately reducing the allergenicity (Shen et al., 2015; Stănciuc et al., 2016).
3.7. In vitro digestion stability Fig. 4. DSC curves of native and low pH (pH 3.5, 3.0, 2.5, 2.0, 1.5 and 1.0) treated lectin from black turtle bean (Phaseolus vulgaris L.) for 8 h. The upside down arrow indicated the exothermic direction.
SDS-PAGE was used to evaluate the digestibility, and the results were shown in Fig. 6 with a single band around 31 kDa (Lane 1) observed in native lectin. In simulated gastric digestion process, there were no significance in band intensity for 60 min simulated gastric digestion, and no digested fragments were observed (Fig. 6a), indicated that lectin allergens presented a consistent resistance to proteolysis by pepsin. An earlier study also suggested that red kidney bean (P. vulgaris L.) lectin (PHA-L) showed high resistance (60 min) to proteolytic enzyme, and undigested lectin might cause nausea, diarrhea and vomiting by provoking the immune system after reaching the intestinal mucosa (He et al., 2018; Kumar, Sharma, Das, Jain, & Dwivedi, 2014). Althgough all the bands from low-pH induced lectin could be clearly visible with the increase of digestion time under simulated gastric digestion process (Lane 1–5), the band intensity gradually decreased. The decrease degree of band seemed to be depended on various pH environments, and a new digested fragment was generated around 18 kDa (Fig. 6b-d), suggesting that the treated lectin was slowly digested under simulated gastric digestion process, which could be attributed to the lectin protein structural alteration (He et al., 2015a, b; Zhang et al., 2017). In continuous simulated intestinal digestion process, significantly decrease was observed in the intensity of native lectin band, and it would be almost invisible after 60 min digestive process (Fig. 6a), which demonstrated that the lectin allergen mainly was digested in small intestine. The result was good agreement with our previous investigations of the in vitro digestibility of black turtle bean lectin (P. vulgaris L.) and PEGylated black turtle bean (P. vulgaris L.) protein isolate (He et al., 2018; He et al., 2015a, b). Fig. 6b showed that lectin with pH 3.0 treatment was digested after a 30 min incubation, while intact lectins with pH 2.0 and 1.0 were not detectable after only 10 and 5 min digestion process, respectively (Fig. 6c-d). Lectin protein structure might be looser when lectin was exposed in low acidic solution, and complete monomerization would be obtained in pH 1.0
hemagglutination activity of lectin (Buul & Brouns, 2014). Effect of low-pH treatment on the hemagglutination activity of lectin was shown in Fig. 5a. Full hemagglutination activity of lectin from the black turtle bean (P. vulgaris L.) was retained down to pH 2.0, indicated that the structural perturbation was far away the regions of sugar binding sites. While the hemagglutination activity was dropped abruptly (P < 0.05) with 50% loss over pH range of 1.5–1.0, suggesting that some sugarbinding sites might be disrupted because of the protein dissociation. Similar pH stabilities were also observed in white kidney bean lectin (P. vulgaris), french bean (P. vulgaris) lectin and Anasazi bean (P. vulgaris) lectin for 30 min or 60 min incubation in low acidic conditions (Chan, Wong, & Ng, 2011; Chan, Xia, & Ng, 2016; Sharma et al., 2009).
3.6. Allergenicity The IgE binding capacity of lectin from the black turtle bean (P. vulgaris L.) with low-pH treatment was evaluated by ELISA assays (Fig. 5b). Generally, the IgE binding capacity decreased significantly with the reduction of pH from 7.2 to 3.0 (P ≤ 0.05), indicating that low-pH treatment would be beneficial to the antigenicity decrease of lectin. The IgE binding capacity still decreased significantly with further decreasing of pH, and the relative IgE binding capacity at pH 1.0 dropped to 40.93% of native control, suggested lectin monomeric state might have lower sensitization. Similarly, carrot major allergen Dau c 1 (Daucus carota) monomers were also proven to have lower allergenic potency (Reese et al., 2007). According to our previous study (He et al., 2018), four B-cell epitopes in native lectin were situated at the protein surface and shared
Fig. 5. The hemagglutination activity retention (%) (a) and relative IgE binding capacity (%) (b) of native and low pH (pH 3.5, 3.0, 2.5, 2.0, 1.5 and 1.0) treated lectin from black turtle bean (Phaseolus vulgaris L.) for 8 h. Different letters (a–f) on the columns indicated significant difference between each other at P < 0.05 level. 188
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Fig. 6. The digestion stabilities of native (a) and low pH (b, pH 3.0; c, pH 2.0; d, pH 1.0) treated lectin from black turtle bean (Phaseolus vulgaris L.) for 8 h in simulated gastric and intestinal digestion, respectively, evaluated by SDS–PAGE. Lane M, protein marker; Lane P, pepsin; Lane T, trypsin; Lanes 1–5, Simulated gastric digestion for 0, 10, 20, 30 and 60 min, respectively; Lanes 6–9, Simulated intestinal digestion for 5, 10, 30 and 60 min, respectively.
Appendix A. Supplementary data
environment. Thus, more cleavage sites for trypsin might be exposed due to the structural alterations, making the protein more susceptible to proteolytic attack (He et al., 2015a, b; Marambe et al., 2015). Our results demonstrated that in vitro digestibility was correlated well with potential allergenicity.
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2018.12.134. References
4. Conclusion Biswas, H., & Chattopadhyaya, R. (2014). Thermal, chemical and pH induced unfolding of turmeric root lectin: Modes of denaturation. PloS One, 9(8), e103579. Buul, V. J. V., & Brouns, F. J. P. H. (2014). Health effects of wheat lectins: A review. Journal of Cereal Science, 59(2), 112–117. Chan, Y. S., Wong, J. H., & Ng, T. B. (2011). A glucuronic acid binding leguminous lectin with mitogenic activity toward mouse splenocytes. Protein & Peptide Letters, 18(2), 194–202. Chan, Y. S., Xia, L., & Ng, T. B. (2016). White kidney bean lectin exerts anti-proliferative and apoptotic effects on cancer cells. International Journal of Biological Macromolecules, 85, 335–345. Chen, Y., Tu, Z., Wang, H., et al. (2016). Glycation of β-lactoglobulin under dynamic high pressure microfluidization treatment: Effects on IgE-binding capacity and conformation. Food Research International, 89(Pt 1), 882–888. Fitch, C. A., Whitten, S. T., Hilser, V. J., & Garcia, M. E. B. (2010). Molecular mechanisms of pH-driven conformational transitions of proteins: Insights from continuum electrostatics calculations of acid unfolding. Proteins Structure Function & Bioinformatics, 63(1), 113–126. Foh, M. B. K., Xia, W., Amadou, I., et al. (2012). Influence of pH shift on functional properties of protein isolated of tilapia (Oreochromis niloticus) muscles and of soy protein isolate. Food & Bioprocess Technology, 5(6), 2192–2200. He, Q., Sun, X., He, S., et al. (2018). PEGylation of black kidney bean (Phaseolus vulgaris L.) protein isolate with potential functironal properties. Colloids & Surfaces B Biointerfaces, 164, 89–97. He, S., Shi, J., Li, X., et al. (2015). Identification of a lectin protein from black turtle bean (Phaseolus vulgaris) using LC-MS/MS and PCR method. LWT – Food Science and Technology, 60(2), 1074–1079. He, S., Shi, J., Ma, Y., et al. (2014). Kinetics for the thermal stability of lectin from black turtle bean. Journal of Food Engineering, 142(142), 132–137. He, S., Shi, J., Walid, E., et al. (2013). Extraction and purification of a lectin from small black kidney bean (Phaseolus vulgaris) using a reversed micellar system. Process Biochemistry, 48(4), 746–752. He, S., Shi, J., Walid, E., et al. (2015). Reverse micellar extraction of lectin from black turtle bean (Phaseolus vulgaris): Optimisation of extraction conditions by response
In conclusion, our present investigation indicated black turtle bean (P. vulgaris L.) lectin at low pH environments presented a progressive unfolding process and attained its stable state at 8 h. Structural alterations and monomerization could be caused by the low-pH induced process. The structural alterations might bury, destroy or degrade surface epitopes, and expose cleavage sites to protease, which would lead to a noticeable reduction in potential allergenicity and in vitro digestibility of lectin allergens after low-pH induced treatments. Low-pH induced process may be an efficient method to reduce the risks of legumes protein consumption, and future studies should focus on in vivo experiments to explore the allergenicity and digestibility changes. Acknowledgements The authors are grateful for the financial support from the National Natural Science Foundation of China (No. 31701524), the Anhui Provincial Natural Science Foundation (No. 1708085MC70), the Fundamental Research Funds for the Central Universities (No. JZ2018HGTB0245), and the Financial Grant from China Postdoctoral Science Foundation (No. 2017M611208, No. 2018T110211). Conflict of interest statement The authors declare no competing financial interest. 189
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Marambe, H. K., Mcintosh, T. C., Cheng, B., et al. (2015). Structural stability and Sin a 1 anti-epitope antibody binding ability of yellow mustard (Sinapis alba L.) napin during industrial-scale myrosinase inactivation process. Food & Function, 6(7), 2384–2395. Molla, A. R., Maity, S. S., Ghosh, S., et al. (2009). Organization and dynamics of tryptophan residues in tetrameric and monomeric soybean agglutinin: Studies by steadystate and time-resolved fluorescence, phosphorescence and chemical modification. Biochimie, 91(7), 857–867. Nasi, A., Picariello, G., & Ferranti, P. (2009). Proteomic approaches to study structure, functions and toxicity of legume seeds lectins. Perspectives for the assessment of food quality and safety. Journal of Proteomics, 72(3), 527–538. Ogawa, H., & Date, K. (2014). The “White Kidney Bean Incident” in Japan. Methods in molecular biology, 1200, 39–45. Rahaman, T., Vasiljevic, T., & Ramchandran, L. (2016). Effect of processing on conformational changes of food proteins related to allergenicity. Trends in Food Science & Technology, 49, 24–34. Reese, G., Ballmer-Weber, B. K., Wangorsch, A., et al. (2007). Allergenicity and antigenicity of wild-type and mutant, monomeric, and dimeric carrot major allergen Dau c 1: Destruction of conformation, not oligomerization, is the roadmap to save allergen vaccines. Journal of Allergy and Clinical Immunology, 119(4), 944–951. Sharma, A., Tzibun, N., Wong, H., et al. (2009). Purification and characterization of a lectin from Phaseolus vulgaris cv. (Anasazi beans). Journal of Biomedicine & Biotechnology, 2009(1), 1–9. Shen, L. L., Zhu, Q. Q., Huang, F. W., et al. (2015). Effect of heat treatment on structure and immunogenicity of recombinant peanut protein Ara h 2.01. LWT – Food Science and Technology, 60(2), 964–969. Stănciuc, N., Banu, I., Turturică, M., et al. (2016). pH and heat induced structural changes of chicken ovalbumin in relation with antigenic properties. International Journal of Biological Macromolecules, 93(Pt A), 572–581. Thakur, K., Kaur, M., Rabbani, G., et al. (2015). Structural variations and molten globule state in Arisaema helliborifolium lectin under various treatments as monitored by spectroscopy. Protein & Peptide Letters, 23(2), 107–109. Yadav, P., Shahane, G., Ramasamy, S., et al. (2016). Structural-functional insights and studies on saccharide binding of Sophora japonica seed lectin. International Journal of Biological Macromolecules, 91, 75–84. Zhang, Z., Zhang, X., Chen, W., et al. (2017). Conformation stability, in vitro digestibility and allergenicity of tropomyosin from shrimp (Exopalaemon modestus) as affected by high intensity ultrasound. Food Chemistry, 245, 997–1009.
surface methodology. Food Chemistry, 166, 93–100. He, S., Simpson, B. K., Ngadi, M. O., et al. (2015a). In vitro studies of the digestibility of lectin from black turtle bean (Phaseolus vulgaris). Food Chemistry, 173(173), 397–404. He, S., Simpson, B. K., Ngadi, M. O., et al. (2015b). pH stability study of lectin from black turtle bean (Phaseolus vulgaris) as influenced by guanidinium-HCl and thermal treatment. Protein & Peptide Letters, 22(1), 45–51. He, S., Simpson, B. K., Sun, H., et al. (2018). Phaseolus vulgaris lectins: A systematic review of characteristics and health implications. Critical Reviews in Food Science and Nutrition, 58(1), 70–83. He, S., Zhao, J., Elfalleh, W., et al. (2018). In silico identifcation and in vitro analysis of B and T-cell epitopes of the black turtle bean (Phaseolus vulgaris L.) lectin. Cellular Physiology and Biochemistry, 49(4), 1600–1614. Jiang, J., Chen, J., & Xiong, Y. L. (2009). Structural and emulsifying properties of soy protein isolate subjected to acid and alkaline pH-shifting processes. Journal of Agricultural and Food Chemistry, 57(16), 7576–7583. Khan, F., Ahmad, A., & Khan, M. I. (2007). Chemical, thermal and pH-induced equilibrium unfolding studies of Fusarium solani lectin. Iubmb Life, 59(1), 34–43. Khan, J. M., Alsenaidy, M. A., Khan, M. S., et al. (2017). pH induced single step shift of hydrophobic patches followed by formation of an MG state and an amyloidogenic intermediate in Lima Bean Trypsin Inhibitor (LBTI). International Journal of Biological Macromolecules, 103, 111–119. Khan, J. M., Qadeer, A., Ahmad, E., et al. (2013). Monomeric banana lectin at acidic pH overrules conformational stability of its native dimeric form. PloS One, 8(4), e62428. Kumar, S., Sharma, A., Das, M., et al. (2014). Leucoagglutinating phytohemagglutinin: Purification, characterization, proteolytic digestion and assessment for allergenicity potential in BALB/c mice. Immunopharmacology and Immunotoxicology, 36(2), 138–144. Kumar, S., Verma, A. K., Das, M., et al. (2013). Clinical complications of kidney bean (Phaseolus vulgaris L.) consumption. Nutrition, 29(6), 821–827. Lagarda-Diaz, I., Guzman-Partida, A. M., & Vazquez-Moreno, L. (2017). Legume lectins: Proteins with diverse applications. International Journal of Molecular Sciences, 18(6), 1242–1259. Lin, H., Li, Z., Lin, H., et al. (2015). Effect of pH shifts on IgE-binding capacity and conformational structure of tropomyosin from short-neck clam (Ruditapes philippinarum). Food Chemistry, 188, 248–255. Liu, Q., Geng, R., Zhao, J., et al. (2015). Structural and gel textural properties of soy protein isolate when subjected to extreme acid pH-shifting and mild heating processes. Journal of Agricultural & Food Chemistry, 63(19), 4853–4861.
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Low-pH induced structural changes, allergenicity and in vitro digestibility of lectin from black turtle bean (Phaseolus vulgaris L.)
T
⁎
Jinlong Zhaoa, Shudong Hea,b, , Mingming Tanga, Xianbao Suna, Zuoyong Zhanga, ⁎ Yongkang Yea,b, Xiaodong Caoa,b, Hanju Suna,b, a
Engineering Research Center of Bio-process of Ministry of Education, School of Food and Biological Engineering, Hefei University of Technology, Hefei 230009, Anhui, PR China b Anhui Province Key Laboratory of Functional Compound Seasoning, Anhui Qiangwang Seasoning Food Co., Ltd., Jieshou 236500, Anhui, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Lectin Phaseolus vulgaris L. bean Low pH treatment Structural changes Allergenicity In vitro digestibility
Lectin was incubated in corresponding acidic buffers (pH 1.0–3.5) for a certain period (0.5, 1, 2, 4, 8, 12 and 24 h) at 25 °C. Low-pH induced changes in structure, allergenicity and in vitro digestibility of lectin from black turtle bean (Phaseolus vulgaris L.) were investigated in the present study. Results indicated that the alteration in structure was a progressive unfolding process mainly depending on pH environment, and the treated lectin attained a stable state at 8 h. Electrophoretic, dynamic light scattering (DLS) and size exclusion chromatography (SEC) analyses suggested that lectin monomers appeared in the solutions of pH < 2.0. Differential scanning calorimetry (DSC) confirmed that thermal stability of lectin weakened in low pH environments. Furthermore, ELISA and in vitro digestion assay showed allergenicity and digestibility significantly decreased with the structural alterations. These results showed low-pH treatments have great potential to reduce the damage of legumes protein consumption.
1. Introduction Lectins are carbohydrate-binding proteins or glycoprotein of nonimmune origin presenting in most legume plants and a wide range of vegetables (He et al., 2018; Kumar et al., 2013; Lagarda-Diaz, GuzmanPartida, & Vazquez-Moreno, 2017). They have shown numerous potential beneficial health effects on human, such as anti-cancer, antiHIV, anti-microbial infection, preventing mucosal atrophy, reducing type 2 diabetes and obesity and promoting gut health (Buul & Brouns, 2014; He et al., 2018). However, nausea, diarrhea, vomiting and abdominal swelling could be caused in both children and adults due to the lectin protein, which has been described as an allergen in food, and the incidence of lectin based food poisoning is increasing in recent years (Buul & Brouns, 2014; He et al., 2018; Lagarda-Diaz et al., 2017; Nasi, Picariello, & Ferranti, 2009). More than 1,000 people had acute gastrointestinal symptoms and 100 people were hospitalized due to consumption of insufficiently boiled white kidney beans (P. vulgaris L.) in Japan in 2006 (Ogawa & Date, 2014). Lectin-intake poisoning accounted for 53.3% of legumes (P. vulgaris L.) food poisoning events in China from the years of 2004 to 2013. As a common legume, black turtle bean (P. vulgaris L.) was consumed worldwide and applied in many foods due to its rich nutrient ⁎
content, but inadvertent exposure of the untreated bean with activated lectin would cause life-threatening anaphylaxis (He et al., 2018; Kumar et al., 2013). Therefore, there is an urgent issue to produce black turtle bean-based foods that are safe for allergic consumers. Previously, the lectin from black turtle bean (P. vulgaris L.) with high haemagglutinating activity was isolated in our laboratory (He, Shi, Walid, Ma, & Xue, 2013; He et al., 2015). Its sequence (NCBI accession no. AHB17899.1), three-dimensional (3-D) structure and epitopes were first identified in our earlier researches (He, Shi, Li, Ma, & Xue, 2015; He et al., 2018). The lectin belongs to the phytohaemagglutinins (PHA) family with highly conserved domain of lectin_legume_LecRK_Arcelin_ConA region, and it is a homodimeric protein where each subunit has a molecular weight of 31 kDa and consists of 275 amino acid residues (Fig. 1) (He et al., 2015; He et al., 2015a, b). Each lectin subunit comprises five indole groups of Trp79, Trp154, Trp176, Trp225 and Trp249, and six indole groups of Tyr72, Tyr133, Tyr135, Tyr150, Tyr190 and Tyr203 (Fig. 1a). It has been suggested four B-cell epitopes renamed B1 (55–66: NVNDNGEPTLSS), B2 (116–126: VGSEPKDKGG), B3 (131–141: NNYKYDSNAHT) and B4 (149–160: LYNVHWDPKPRH) were critical for lectin sensitization (Fig. 1b and c) (He, Zhao, & Elfalleh, 2018). Many previous researches have proven that the antigenicity of
Corresponding authors at: School of Food and Biological Engineering, Hefei University of Technology, Hefei 230009, Anhui, PR China. E-mail addresses: [email protected] (S. He), [email protected] (H. Sun).
https://doi.org/10.1016/j.foodchem.2018.12.134 Received 25 October 2018; Received in revised form 29 December 2018; Accepted 29 December 2018 Available online 14 January 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Ribbon structure of the lectin from black turtle bean (Phaseolus vulgaris L.). Tetrameric structure with the distributions of Tyr and Trp (a); and B-cell epitope regions distributions in monomer (b) and tetramer (c); Epitopes and Tyr/Trp regions were highlighted with black.
China). The lectin from the black turtle bean (P. vulgaris L.) with at least 94% purity was obtained according to our previous studies (He et al., 2013; He et al., 2015). The extracted lectins were freeze-dried and stored in a −80 °C refrigerator until use. Porcine pepsin (2754 U/mg) and trypsin (11356 U/mg) were obtained from Sigma Chemical Co., Ltd (St. Louis, MO, USA). Sodium dodecyl sulphate (SDS), and Coomassie Blue R-250, β-mercaptoethanol, and 8-Anilino-1-naphthalenesulfonate (ANS) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). All chemicals used in the present study were of analytical grade.
allergen protein could be influenced by the structural modification (Lin et al., 2015; Marambe et al., 2015; Rahaman, Vasiljevic, & Ramchandran, 2016). As a simple chemical treatment, low acidic pH is wildly believed to modify functional properties of protein by changing its structure (Foh et al., 2012; Liu et al., 2015). Emulsifying properties of soy protein isolates were markedly improved by the pH-shifting from pH 1.5 (Jiang, Chen, & Xiong, 2009), and enhanced gelling ability was obtained in the soy protein isolates after the treatment under acidic (pH 1.5) conditions combined with proper heating (50 or 60 °C) (Liu et al., 2015). Although knowledge on allergenicity of allergen protein induced by low acidic treatment is poorly documented (Lin et al., 2015; Stănciuc et al., 2016), a speculation would be modestly brought up that the allergenicity of legume lectin might be affected during the acidic processing because of the alteration of protein conformations. Therefore, in the present study, effects of low acidic pH (pH 3.5, 3.0, 2.5, 2.0, 1.5 and 1.0) on the structure and allergenicity of lectin from black turtle bean (P. vulgaris L.) were investigated. Structural alterations were explored by intrinsic/extrinsic fluorescence spectra assays, SDS/Native–PAGE, dynamic light scattering (DLS) and size exclusion chromatography (SEC), respectively. Allergenicity was evaluated with direct enzyme-linked immunosorbent assay (ELISA). In addition, hemagglutination activity and in vitro digestibility of lectin from black turtle bean (P. vulgaris L.) also were assessed, respectively. Our findings would give an insight into relationship between lectin conformational alterations and its allergenicity, which have tremendous application potential in the developments of safe pharmaceuticals and low-allergen legume based foods.
2.2. Low-pH induced process Lectin sample was dissolved into specific pH buffer (pH 3.5, 3.0, 2.5, 2.0, 1.5 and 1.0, respectively) to a centration of 2.0 mg/mL, and was incubated for 0.5, 1, 2, 4, 8, 12 and 24 h at room temperature (25 ± 2 °C) before all measurements, respectively. The lectin sample prepared using phosphate buffer (Na2HPO4-NaH2PO4, 10 mM, pH 7.2) was set as the native control. The buffers used for various treatments were 10 mM of KCl-HCl (pH 1.0–1.5), Gly-HCl (pH 2.0–3.0) and Na2HPO4-citric acid (pH 3.5). All the buffers were filtered through 0.22 μm membrane.
2.3. Intrinsic fluorescence measurements Intrinsic fluorescence measurements were performed on a RF20AXS spectrofluorometer (Horiba Ltd., Kyoto, Japan). Each treated sample was diluted with corresponding buffer to a concentration of 0.2 mg/mL. The changes in tertiary structure were monitored by intrinsic fluorescence emission between 300 and 400 nm at a 280 nm excitation, and the excitation and emission slit width were set at 5 nm, all spectra were corrected by subtraction of corresponding buffer.
2. Materials and methods 2.1. Materials Black turtle beans (P. vulgaris L.) cultivated in Heilongjiang province of China were obtained from a local market (Harbin, Heilongjiang, 184
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2.4. Extrinsic fluorescence measurements
expressed as the units of activity per mg of protein (Eq. (1)), and the hemagglutination activity retention (%) could be presented as the ratio of treated lectin to native control.
In the extrinsic fluorescence experiments, four milliliter aliquots of treated lectin samples (0.2 mg/mL) were prepared using the corresponding buffers, and mixed with 50 μL of 8.0 mM of ANS solution for a 5 min reaction. Then, the mixed solutions were performed on a RF20AXS spectrofluorometer (Horiba Ltd., Kyoto, Japan). The excitation was set at 380 nm and the emission spectra were taken in the range of 400–600 nm.
HA(HU/mg) =
2n × 103 C(pr) × V
(1)
where n is the highest well number showing hemagglutination in the Ushaped microtiter plates, and n = 0 means no visible agglutination. C(pr) (mg/mL) is the protein concentration of the lectin solution, while V (μL) is the volume of lectin solution added to each well.
2.5. Gel electrophoresis 2.10. Allergenicity determination The lectin treated with low pH environment was analyzed with gel electrophoresis according to the method of He et al. (2013). The concentration gel and the resolving gel contained 4% and 12% acrylamide, respectively. Lectin samples (2.0 mg/mL) were boiled with loading buffer containing SDS and β-mercaptoethanol at 1:1 (v/v) ratio for 5 min. Then, 15 μL of each sample was loaded in each well of the SDS–PAGE gel. Native gel electrophoresis without SDS, β-mercaptoethanol and boiling for lectin proteins was performed similarly according to the method of SDS-PAGE.
The antigenicity of the lectin sample was determined by ELISA according to our previous method (He et al., 2018). Carbonate bicarbonate buffer (pH 9.6) was added in each well with 1 μg/100 μL of lectin sample and incubated overnight at 4 °C. After washing with PBS (pH 7.2, 10 mM) twice, the plate was blocked with 5% (w/v) defatted milk (200 μL/well) for 1 h at 25 °C, and then was washed five times using PBS (pH 7.2, 10 mM). The recombinant polyclonal anti-lectin antibodies obtained from Nanjing SenBeiJia Biological Technology Co., Ltd. (Nanjing, Jiangsu, China) were subsequently diluted 1:100,000 v/v with PBS (pH 7.2, 10 mM) and added into each well, then the plate was incubated overnight at 4 °C. The plate was following incubated with 1:1000 v/v diluted anti-human-IgE peroxidase antibody (Sigma-Aldrich Chemicals Co., St. Louis, MO, USA) at 37 °C for 2 h and washed five times with PBS. Prior to the detecting, the chromogenic reaction was developed using 3,3′, 5,5′-tetramethylbenzidine (TMB) for 10 min in the dark and was terminated with 0.2 M sulphuric acid. The optical density (OD value) was measured at 450 nm within 15 min by a microplate reader (Tecan Infinite 200Pro, Tecan Group Ltd., Mannedorf, Switzerland). Since the OD value is proportional to the IgE binding capacity of lectin (Lin et al., 2015), the relative IgE binding capacity (%) was used to express the IgE binding capacity of treated lectin with respect to that of native control (100%).
2.6. Dynamic light scattering (DLS) measurements Each treated sample was diluted with corresponding buffers to a concentration of 0.2 mg/mL, and the samples were centrifuged at 12,000 rpm for 10 min and were filtered serially through 0.22 μm Whatman syringe filters. DLS measurements were performed on a Zetasizer µV dynamic light scattering equipment (Malvern Instruments Ltd., Malvern, UK) equipped with a temperature-controlled micro sampler at a scattering angle of 90° at 25 °C. 2.7. Size exclusion chromatography (SEC) SEC experiments of treated lectins were performed on a preparative high performance liquid chromatography (HP PLUS 100D, Lisure Science Co., Ltd., Suzhou, China) using Chromdex 200 prepacked column (16 × 600 mm, Bogelong Bio-Technology Co., Ltd., Shanghai, China). After the prepacked column was equilibrated with desired pH buffers, an aliquot of 5 mL of treated lectin samples (2.0 mg/mL) was injected and monitored at 280 nm, and the elution was carried out at a flow rate of 1.0 mL/min with corresponding pH buffer.
2.11. In vitro digestion assay In vitro simulated gastrointestinal digestion of treated lectin was carried out as previously described (Zhang et al., 2017). For the simulated gastric digestion process, porcine pepsin from porcine gastric mucosa was employed in simulated gastric solution (35 mM NaCl, pH 1.2) at an enzyme: lectin ratio of 1:20 (w/w), and was incubated in a water bath at 37 °C for 10 min prior to the addition of lectin. Aliquots (100 μL) were taken at 0, 10, 20, 30 and 60 min, respectively, for the further determination, and the reaction solution in each aliquots was immediately terminated by adding 30 μL of 0.2 M Na2CO3 solution. The digested protein solution after the 60 min pepsin digestion was adjusted to pH 7.2 with 1.0 M NaOH solution, then was added into the simulated intestinal fluid digestion containing 1.0 M CaCl2, 0.25 M BisTris (pH 7.2) and 0.125 M bile salts for further simulated intestinal digestion. Trypsin was added to the mixture at an enzyme: substrate ratio of 1:100 (w/w) after preheating for 10 min in water bath at 37 °C. Subsequently, aliquots (100 μL) were taken at 5, 10, 30 and 60 min, respectively, and then were stopped the digested reactions by heating in a boiling water bath for 10 min. All protein samples were collected and stored at −20 °C until were analyzed by SDS-PAGE.
2.8. Differential scanning calorimetry (DSC) measurements DSC experiments were carried out on a TA Q100-DSC thermal analyzer (TA Instruments, New Castle, DE, USA). Indium standards were used for DSC calibration prior to all the sample measurements. The treated lectin samples (15 μL, 2.0 mg/mL) were accurately pipetted into aluminum liquid pans, and the calorimetric traces were recorded from 25 °C to 120 °C at a rate of 10 °C/min. A sealed empty pan containing an equal mass of the corresponding pH buffer solution was employed as a reference. All the experiments were performed at least 3 times. The denaturation temperature (Td) and the enthalpy of denaturation (△H) were computed by the TA universal analysis software (Version 4.1D, TA Instrument-Waters LLC, New Castle, DE, USA). 2.9. Hemagglutination activity assay
2.12. Statistical analysis Hemagglutination activity was determined based the method of He et al. (2018). Serial twofold dilutions of 50 μL lectin solutions (2.0 mg/ mL) were added into 96-well U-shaped microtiter plates, followed by addition of 50 μL of rabbit erythrocyte suspension (2%, v/v) to each well. The mixtures were incubated at 4 °C for 1.5 h, and the extent of hemagglutination was determined visually with PBS (pH 7.2, 10 mM) as a negative control. The hemagglutination activity (HA, HU/mg) was
All experiments were repeated in a minimum of triplicates and the values were reported as mean ± standard deviation. Analyses of variance (ANOVA) and Duncan’s means comparison test were applied using SPSS version 11.5 (SPSS Inc. Chicago, IL, USA) with a significance level of 0.05. Ribbon structure of the lectin from black turtle bean (P. vulgaris L.) was generated by using UCSF Chimera (version 1.13, 185
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Fig. 2. The plots of intrinsic fluorescence intensity at the λmax (328 nm) (a) and extrinsic fluorescence intensity at the λmax (480 nm) (b) as a function of incubation time at different pH (pH 3.5, 3.0, 2.5, 2.0, 1.5 and 1.0). The lectin protein concentration was 0.2 mg/mL.
deeper understanding of the pH induced structural changes (Figs. S2 and 2b). Maximum emission wavelength with minimum ANS fluorescence intensity was found in the native control. Low-pH induced lectin showed significant increase in the ANS fluorescence intensity with obvious blue shift (λmax from 518 nm to 480 nm) compared to the native. Similar blue shifts in λmax with increases in ANS fluorescence intensity were early found in the investigations of Arisaema helliborifolium lectin and lima bean trypsin inhibitor at low pH environments for 12 h incubation at room temperature (Khan et al., 2017; Thakur et al., 2015). In addition, there was a significant enhancement in fluorescence intensity of low-pH treated lectin with increasing time to 8 h, indicated that intact structure of lectin became looser due to the unfolding, thus more Trp/Tyr residues were exposed via moving toward to a more nopolar environment, which would lead to the lectin structural changes (Chen et al., 2016). Effects of pH and incubation time on the ANS fluorescence intensity of lectin at 480 nm (λmax) were also further plotted (Fig. 2b), and it found that the increase in ANS fluorescence intensity was accompanied with the reduction of pH values. The maximum ANS fluorescence intensity was obtained in the pH 2.0 treatment for 8 h, indicated that most Trp/Tyr residues have moved toward to more hydrophobic areas (Khan, Qadeer, Ahmad, Ashraf, Bhushan, Chaturvedi, et al., 2013; Yadav et al., 2016). In addition, with the extension of time, the fluorescence intensity and λmax of all treated lectin samples showed little difference compared to the lectin treated for 8 h, suggesting a gradual unfolding of lectin occurred at low pH environments, and a steady state would be obtained at 8 h. Thus, in order to make the following experiments more precise, incubation time 8 h for each lectin sample in the in corresponding low acidic buffer was employed.
University of California San Francisco, San Francisco, CA, USA). 3. Results and discussion 3.1. Intrinsic fluorescence The structural changes of the low-pH treated lectin for different time were shown in Figs. S1 and 2a using the intrinsic fluorescence emission. The excitation fluorescence spectra of proteins are resulting from the functions of Trp, Tyr, and Phe residues, and the fluorescence emission spectrum at 280 nm is mainly due to the existing of Trp and Tyr residues (He et al., 2015a, b). The maximum fluorescence emission of the native lectin (pH 7.2) was found at 328 nm, which was similar with the report of He et al. (2014), and suggested that Trp and Tyr residues might be mainly located in the hydrophobic regions of the protein molecule. The maximum fluorescence intensity (157 a.u) was also found in native lectin spectrogram (Figs. S1 and 2a). In our previous study (He et al., 2018), Trp249 and Tyr72, 190 as well as 203 were found to be buried in the hydrophobic regions of protein molecule which might contribute to the high fluorescence intensity. As presented in Fig. 2a, there was a evident blue shift in the maximum emission with a considerable decrease in fluorescence intensity of treated lectin in the range of 0–8 h, indicated that the protein surface amino acids, such as Trp79, 154, 176 and 225, and Tyr133, 135 and 150, might gradually move to the non-polar environments, and the decrease in the fluorescence intensity could be attribute to the deprotonation of the neighboring basic amino acids (He et al., 2015a, b). These observations indicated that the tertiary structure of low-pH treated lectin was disrupted, and the degree of these alterations mainly depended on the functions of pH environments. Earlier studies also demonstrated that the amino acid groups on the proteins surface would be protonated when the environmental pH was below the isoelectric point, thus the native lectin structure might be disrupted due to the electric charge effects (Khan, Ahmad, & Khan, 2007; Lin et al., 2015). Similar results were also obtained in the investigation for lima bean trypsin inhibitor incubated at acidic buffers for 12 h (Khan et al., 2017). The change in intrinsic fluorescence intensity at the λmax (328 nm) was plotted as a function of time at different pH (Fig. 2a). Significantly decreased fluorescence intensity could be clearly observed in the range of 0–8 h, while no significant difference was found in fluorescence intensity of low-pH treated lectin with the further increase in incubation time, indicated that a steady state of the tertiary structure of lectin was formed up to 8 h with low-pH treatment.
3.3. Gel electrophoretic characterization, DLS and SEC analysis Structural changes of low-pH treated lectin from black turtle bean (P. vulgaris L.) were further investigated by gel electrophoretic, DLS and SEC analyses (Fig. 3). SDS-PAGE patterns showed a single band corresponding to the molecular mass of 31 kDa (Fig. 3a), and no significance was observed over pH range of 3.5–2.0, indicated the stability of primary structure. However, the original band decreased mildly in intensity with the formation of minor low molecular weight fragments over low pH range of 1.5–1.0, which might be due to acidic hydrolysis of lectin protein. As shown in Fig. 3b, the lectin without boiling and βmercaptoethanol treatments showed a single band on the top of resolving gel (Lane 1–5), which was similar to native lectin band, while a single band on the middle of resolving gel could be observed in Lanes 6 and 7, respectively, indicated that lectin homotetrameric might dissociate to the monomers (31 kDa) in lower acidic (pH 1.5–1.0) environments.
3.2. Extrinsic fluorescence Extrinsic fluorescence analysis was performed to obtain a much 186
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Fig. 3. SDS–PAGE (a) and native-PAGE (b) profiles of native and low-pH treated lectin from black turtle bean (Phaseolus vulgaris L.) for 8 h. Lane M, protein marker; Lanes 1–7, different low pH treatment for pH 7.2 (native), 3.5, 3.0, 2.5, 2.0, 1.5 and 1.0, respectively. Size exclusion chromatography (SEC) profiles (c) of native and low pH (pH 3.5, 3.0, 2.5, 2.0, 1.5 and 1.0) treated lectin from black turtle bean (Phaseolus vulgaris L.) for 8 h.
228.4 min was obtained from the treated lectin at pH 1.0, indicated the full monomerization of the protein (Fig. 3c). Taken together the above results, the lectin protein retained its tetramer states until pH 2.0, and further decrease in pH would make protein dissociate to monomer states because of the intramolecular electrostatic repulsions (Fitch, Whitten, Hilser, & Garcia, 2010; Khan et al., 2013).
The influence of low pH incubation on the hydrodynamic radius (Rh) of lectin was investigated using DLS (Table S1). Compared with the native control, a significant increase in Rh was found with the pH decreasing from 3.5 to 2.0, which could be attributed to lectin structure unfolding. The intact structure gradually became looser due to the intramolecular charge-charge repulsion in low pH environments, which could result in the unfolding of structure (Khan, Qadeer, & Ahmad, 2013; Thakur et al., 2015). These results also are good agreement with the intrinsic and extrinsic fluorescence analysis, indicating some subtle changes in the lectin structure. With the further decrease of pH, the Rh of lectin exhibited a prominent reduction of 57.8% and 69.6% in pH 1.5 and 1.0 treatments compared to that of the initial Rh, respectively, which further confirmed the dissociation of lectin tetramers. Fig. 3c showed the SEC profiles of treated lectins under different pH buffers. Native lectin was eluted as a single peak around 123.1 min, and the elution times of lectin treated with pH 3.5–1.0 buffers presented a progressive decreased trend, which could be attributed to the increase of molecule size (Khan et al., 2013). While two peaks center around 123.1 and 228.4 min were observed in the elution profile of lectin after pH 1.5 treatment, and the former peak (123.1 min) was gentle, which might attribute to the gradual monomerization of lectin at pH 1.5. Some others multimeric lectin proteins also present a similar pH-dependent dissociation behavior in acidic solutions, such as soybean agglutinin (Molla et al., 2009), banana lectin (Khan et al., 2013), turmeric root lectin (Biswas & Chattopadhyaya, 2014). A single peak around
3.4. Thermal stability DSC curves of lectin from the black turtle bean (P. vulgaris L.) after low-pH treatments were shown in Fig. 4. All treated lectin samples showed a prominent endothermic peak. Widest and deepest endothermic peak with the highest peak temperature (Td) was obtained in the native control, and the peak width and depth decreased with the decreasing of pH value. According to the endotherm area analysis, both Td and denaturation enthalpy (△H) values after low-pH treatments were decreased (Fig. 4a), which could be attributed to possible structural alterations of the protein, implying the loss of thermal stability (He et al., 2015a, b). 3.5. Hemagglutination activity Special carbohydrates on the red blood cell surface could be recognized by the active lectin, and cross-linked network in suspension could be formed based on the function, which was called 187
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high surface accessibility and flexibility (Fig. 1b and c). Based on the above studies, lectin protein underwent gradual unfolding and dissociation in low acidic conditions which could terminate the surface exposure of epitopes, and cause the loss in surface accessibility and flexibility for IgE recognition (Marambe et al., 2015; Rahaman et al., 2016; Stănciuc et al., 2016). In other words, exposed surface epitopes were buried, destroyed or degradation owing to these structural alterations, which were no longer recognized by IgE antibodies or able to stimulate an immune response, ultimately reducing the allergenicity (Shen et al., 2015; Stănciuc et al., 2016).
3.7. In vitro digestion stability Fig. 4. DSC curves of native and low pH (pH 3.5, 3.0, 2.5, 2.0, 1.5 and 1.0) treated lectin from black turtle bean (Phaseolus vulgaris L.) for 8 h. The upside down arrow indicated the exothermic direction.
SDS-PAGE was used to evaluate the digestibility, and the results were shown in Fig. 6 with a single band around 31 kDa (Lane 1) observed in native lectin. In simulated gastric digestion process, there were no significance in band intensity for 60 min simulated gastric digestion, and no digested fragments were observed (Fig. 6a), indicated that lectin allergens presented a consistent resistance to proteolysis by pepsin. An earlier study also suggested that red kidney bean (P. vulgaris L.) lectin (PHA-L) showed high resistance (60 min) to proteolytic enzyme, and undigested lectin might cause nausea, diarrhea and vomiting by provoking the immune system after reaching the intestinal mucosa (He et al., 2018; Kumar, Sharma, Das, Jain, & Dwivedi, 2014). Althgough all the bands from low-pH induced lectin could be clearly visible with the increase of digestion time under simulated gastric digestion process (Lane 1–5), the band intensity gradually decreased. The decrease degree of band seemed to be depended on various pH environments, and a new digested fragment was generated around 18 kDa (Fig. 6b-d), suggesting that the treated lectin was slowly digested under simulated gastric digestion process, which could be attributed to the lectin protein structural alteration (He et al., 2015a, b; Zhang et al., 2017). In continuous simulated intestinal digestion process, significantly decrease was observed in the intensity of native lectin band, and it would be almost invisible after 60 min digestive process (Fig. 6a), which demonstrated that the lectin allergen mainly was digested in small intestine. The result was good agreement with our previous investigations of the in vitro digestibility of black turtle bean lectin (P. vulgaris L.) and PEGylated black turtle bean (P. vulgaris L.) protein isolate (He et al., 2018; He et al., 2015a, b). Fig. 6b showed that lectin with pH 3.0 treatment was digested after a 30 min incubation, while intact lectins with pH 2.0 and 1.0 were not detectable after only 10 and 5 min digestion process, respectively (Fig. 6c-d). Lectin protein structure might be looser when lectin was exposed in low acidic solution, and complete monomerization would be obtained in pH 1.0
hemagglutination activity of lectin (Buul & Brouns, 2014). Effect of low-pH treatment on the hemagglutination activity of lectin was shown in Fig. 5a. Full hemagglutination activity of lectin from the black turtle bean (P. vulgaris L.) was retained down to pH 2.0, indicated that the structural perturbation was far away the regions of sugar binding sites. While the hemagglutination activity was dropped abruptly (P < 0.05) with 50% loss over pH range of 1.5–1.0, suggesting that some sugarbinding sites might be disrupted because of the protein dissociation. Similar pH stabilities were also observed in white kidney bean lectin (P. vulgaris), french bean (P. vulgaris) lectin and Anasazi bean (P. vulgaris) lectin for 30 min or 60 min incubation in low acidic conditions (Chan, Wong, & Ng, 2011; Chan, Xia, & Ng, 2016; Sharma et al., 2009).
3.6. Allergenicity The IgE binding capacity of lectin from the black turtle bean (P. vulgaris L.) with low-pH treatment was evaluated by ELISA assays (Fig. 5b). Generally, the IgE binding capacity decreased significantly with the reduction of pH from 7.2 to 3.0 (P ≤ 0.05), indicating that low-pH treatment would be beneficial to the antigenicity decrease of lectin. The IgE binding capacity still decreased significantly with further decreasing of pH, and the relative IgE binding capacity at pH 1.0 dropped to 40.93% of native control, suggested lectin monomeric state might have lower sensitization. Similarly, carrot major allergen Dau c 1 (Daucus carota) monomers were also proven to have lower allergenic potency (Reese et al., 2007). According to our previous study (He et al., 2018), four B-cell epitopes in native lectin were situated at the protein surface and shared
Fig. 5. The hemagglutination activity retention (%) (a) and relative IgE binding capacity (%) (b) of native and low pH (pH 3.5, 3.0, 2.5, 2.0, 1.5 and 1.0) treated lectin from black turtle bean (Phaseolus vulgaris L.) for 8 h. Different letters (a–f) on the columns indicated significant difference between each other at P < 0.05 level. 188
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Fig. 6. The digestion stabilities of native (a) and low pH (b, pH 3.0; c, pH 2.0; d, pH 1.0) treated lectin from black turtle bean (Phaseolus vulgaris L.) for 8 h in simulated gastric and intestinal digestion, respectively, evaluated by SDS–PAGE. Lane M, protein marker; Lane P, pepsin; Lane T, trypsin; Lanes 1–5, Simulated gastric digestion for 0, 10, 20, 30 and 60 min, respectively; Lanes 6–9, Simulated intestinal digestion for 5, 10, 30 and 60 min, respectively.
Appendix A. Supplementary data
environment. Thus, more cleavage sites for trypsin might be exposed due to the structural alterations, making the protein more susceptible to proteolytic attack (He et al., 2015a, b; Marambe et al., 2015). Our results demonstrated that in vitro digestibility was correlated well with potential allergenicity.
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2018.12.134. References
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In conclusion, our present investigation indicated black turtle bean (P. vulgaris L.) lectin at low pH environments presented a progressive unfolding process and attained its stable state at 8 h. Structural alterations and monomerization could be caused by the low-pH induced process. The structural alterations might bury, destroy or degrade surface epitopes, and expose cleavage sites to protease, which would lead to a noticeable reduction in potential allergenicity and in vitro digestibility of lectin allergens after low-pH induced treatments. Low-pH induced process may be an efficient method to reduce the risks of legumes protein consumption, and future studies should focus on in vivo experiments to explore the allergenicity and digestibility changes. Acknowledgements The authors are grateful for the financial support from the National Natural Science Foundation of China (No. 31701524), the Anhui Provincial Natural Science Foundation (No. 1708085MC70), the Fundamental Research Funds for the Central Universities (No. JZ2018HGTB0245), and the Financial Grant from China Postdoctoral Science Foundation (No. 2017M611208, No. 2018T110211). Conflict of interest statement The authors declare no competing financial interest. 189
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