Yufeng Lu, Huafeng Cheng, Shaotong Jiang, Lin Lin,*, Jianfeng Lu,*

a Engineering Research Center of Bio-process, MOE, School of Food and Biological Engineering, Hefei University of Technology, Hefei 230000, China

b Key Laboratory for Agricultural Products Processing of Anhui Province, School of Food and Biological Engineering, Hefei University of Technology, Hefei 230000, China

Keywords:Chinese mitten crab Tropomyosin Processing methods Immunoreactivity

A B S T R A C T The tropomyosin (TM) fractions of crab proteins may cause allergic reactions in individuals susceptible to allergies; however, eff icient and safe methods by which to reduce such allergenicity are not currently available. Therefore, in this study, the effects of three different processing methods, i.e., microwave,ultrasound, and high temperature-pressure (HTP) treatments, on the digestion stability of TM from Chinese mitten crab muscle and the allergenicity of TM digestion products were explored. sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that microwaving had little effect on the digestion stability of TM. In contrast, ultrasound and HTP treatments facilitated the degradation of TM.Similarly, Western blotting and inhibition ELISA indicated that the IgE-binding activity of TM was signif icantly reduced after treatment with ultrasound or HTP. Among the three different processing methods,HTP was the most effective method for improving digestibility of TM and reducing immunoreactivity. This f inding provides new insights into treatments for crab allergies.

1. Introduction

Food allergies involve an abnormal response to certain food components after they enter the body [1]. Food allergic reactions have become a common public health concern worldwide as immune responses caused by the ingestion of food containing allergens have increased [2]. According to an epidemiological survey, nearly 5%of adults and 8% of children in Western countries have been affected by adverse immune responses to food [3]. Reports suggested that the percentage of children aged ＜ 18 years in the USA affected by food allergies increased from 3% in 1997 to 6% in 2016 [4], additionally,allergies in the 7-18 age group were mainly derived from crustaceans [5].Unlike other food allergies, symptoms caused by seafood allergies can last for life in up to 90% of patients [1,6].

Eriocheir sinensis, commonly known as the Chinese mitten crab, lives in freshwater but migrates offshore to breed [7]. Chinese mitten crab is considered a traditional “aquatic treasure” in China.This crab is favored by consumers because of its unique flavor and nutritiousness [8]. However, many clinical studies have found that crabs can trigger a series of allergic reactions that cause skin symptoms (urticaria or angioedema) or respiratory symptoms (rhinitis or wheezing) in mild cases but anaphylactic shock or even death in severe cases [9]. Therefore, crustaceans such as shrimps and crabs are listed as one of the eight main sources of food allergens by the Food and Agriculture Organization of the United Nations and World Health Organization [10]. Studies have shown that the major allergen in crabs is tropomyosin (TM), a myofibrillar protein composed of two identical subunits with molecular masses of 35-38 kDa [11,12].TM is an atypical coiled-coil and heat-stable protein formed by two parallelα-helices [13]. Many IgE-binding epitopes have been verif ied in TM [14]. A previous study showed that crab-allergic patients had an immune response to TM , which was combined with crab-specific IgE from these patients [15]. Thus, reducing the antigenicity of TM would be beneficial to crab-allergic individuals [11].

Most seafood must be processed before consumption, and food processing (i.e., thermal or non-thermal treatment) can potentially affect allergen structures, e.g., by causing the aggregation or exposure of previously hidden antigenic sites, with possible effects on increasing or decreasing allergenicity [12,16]. In order to improve the organoleptic properties, preservation, and safety of food, thermal treatment is the most commonly applied for industrial production [11],including microwave, boiling, pressure cooking, and high temperature pressure (HTP). To date, various thermal treatments have been shown to effectively reduce the content of specific allergens in certain foods [16].Cabanillas et al. [17] reported that pressure treatment at 256 kPa and 138 °C for 15 or 30 min caused significant fragmentation of walnut protein, which was followed by the reduction of IgEbinding and IgE-crosslinking capacities. In recent years, non-thermal treatment, including enzymatic hydrolysis, ultrasound, and gammairradiation, has also been used in an attempt to inactivate TM [18].Enzymatic hydrolysis may have adverse effects on food quality.One study showed that shrimp allergens were resistant to enzymatic hydrolysis [19]. In addition, although gamma-irradiation has been proved to effectively reduce the sensitization of allergens, it is still controversial whether irradiation will be harmful to humans [18]. It is therefore urgent to explore the most effective methods to reduce the allergenicity of allergens. As a non-thermal processing method,ultrasound is safe and poison-free and maintains the flavor, texture,and nutrition of food products at low or moderate temperatures [20].According to the research of Li et al. [21], the inhibition rate of shrimp treated with ultrasound (30 kHz, 800 W, 50 °C) for 1.5 h was 20% as compared with that of untreated samples (i.e., raw shrimp extracts), which enormously reduced the allergenicity of shrimp.However, it is still unclear whether ultrasound or thermal treatment can affect the digestibility and allergenicity of Chinese mitten crab.The aim of this study is to select appropriate processing methods to control crab allergens by producing a change in allergenicity,which will help to promote the development of hypoallergenic crab products and meet the normal dietary needs of allergic patients.Meanwhile, understanding the relationship between processing methods and allergenicity can help to improve digestion and alleviate immunoreactivity that will help to guarantee consumer safety.

The proteolytic degradation and absorption of food allergens in the gastrointestinal tract determine their allergenicities [6]. Hence,in the present study, the effects of microwave, ultrasonic, and HTP treatments on the digestive stability and immunoreactivity of TM extracted from crabs were investigated. To analyze the mechanism underlying processing method-related changes in the allergenicity of muscle samples,in vitrosimulated gastrointestinal digestion was adopted. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and inhibition ELISA were used to analyze the digestibility and IgE-binding reactivity of TM from digested crude extract of processed crabs.

2. Materials and methods

2.1 Crab and chemicals

Chinese mitten crab (Eriocheir sinensis) was purchased alive from a local fishery market in Wuwei, China. After the crabs were washed,crab muscle was handpicked and stored at -24 °C until experiments were conducted.

Protein standards for SDS-PAGE and immunoblotting,horseradish peroxidase (HRP)-labeled goat anti-human IgE, pepsin(250 U/mg proteins), trypsin (40 U/mg proteins), Coomassie Brilliant Blue R-250, and ECL luminous liquid were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China).N,N,N’,N’-Tetramethylethylenediamine (TEMED) and polyvinylidene fluoride (PVDF) membranes were purchased from Sigma (St. Louis,MO, USA). Rabbit anti-Chinese mitten crab TM polyclonal antibodies were purchased from Abcam Trading Co., Ltd. (Shanghai, China). All other reagents were of analytical grade.

2.2 Patients’ sera

The sera of patients (32748-ZR and 22515-JH) with confirmed specific IgE antibody to crab TM in this study were obtained from PlasmaLab (Everett, MA., USA). The specificity of IgE levels to crab was assessed using the ImmunoCAP system, and individual sera with positive value ≥ 0.35 kU/L were stored at -80 °C until used. Patient was confirmed as being allergic to crustaceans based on his clinical history of immediate hypersensitivity reactions after ingestion of crustacean products. A pool of sera from selected patients was used for IgE immunodetection. Sera samples from nonallergic individuals were employed as negative controls. Demographic details of patients were given in Table 1.

2.3 Crab processing methods

Crab muscle was treated with three different processing methods as follows: (a) microwave heating was done in a microwave oven(NJL07-3, Jiequan Microwave Development Co., Ltd. Nanjing,China) with the power level of 50 W/g for 40 s; (b) ultrasound treatment was carried out using an ultrasound processor (KQ-300VDE,Ultrasonic Instruments Co., Ltd., Kunshan, China) with the power level of 24 W/g at 80 kHz, 30 °C for 60 min; or (c) HTP treatment was done in an autoclave (LDZX-30KBS, Shanghai Shen’an Medical Appliance Factory, China) at 120 °C for 20 min. The power intensity of microwave and ultrasound was determined by pre-test according to Cheng [22] (the data is not shown here). Untreated samples were used as controls. For each processing method, samples were treated in triplicate.

Table 1 Demographic details of two crab-allergic patients.

2.4 Preparation of crab crude extract

Crude extract from Chinese mitten crab was prepared using the protocol of Zhang et al. [15] but with a minor modification. Raw crab muscles were minced and homogenized with 4-fold (m/V)20 mmol/L PBS buffer (pH 7.5, containing 3% NaCl,m/V) using a homogenizer (PD500; Prima Technology Group, Shanghai, China).The homogenate was boiled for 15 min and immediately cooled in ice water, after which it was centrifuged at 8 200 ×gfor 20 min. The supernatant was regarded as crab muscle crude extract.

2.5 Purification of TM

TM was purified according to the method of Liang et al. [7]with some modifications. Briefly, minced raw samples were homogenized with 10-fold (m/V) 20 mmol/L PBS buffer (pH 7.5). The homogenate was centrifuged at 8 000 ×gfor 15 min, and the supernatant containing soluble sarcoplasmic proteins was discarded. The pellet was resuspended in the aforementioned buffer and then homogenized and centrifuged four more times. The final resulting precipitate was treated with absolute acetone three times and dried overnight [18].The dried powder was subsequently extracted with 10-fold (m/V)20 mmol/L Tris-HCl (1 mol/L KCl and 10 mmol/L mercaptoethanol at pH 7.5) overnight. After centrifugation at 20 000 ×gfor 20 min, the resulting supernatant was subjected to isoelectric precipitation at pH 4.5 with HCl, and the precipitate was dissolved in 20 mmol/L Tris-HCl (pH 7.5). The dissolved precipitate was successively salted out with 41%-60% saturation of (NH4)2SO4and centrifuged at 12 000 ×gfor 10 min. The precipitate was then collected and redissolved in 20 mmol/L Tris-HCl (pH 7.5),after which the residue was filtered out. Finally, small amounts of impurities were removed by heating in a boiling water bath for 15 min, immediately cooling in ice water, and centrifuging at 20 000 ×gfor 20 min. The supernatant containing TM with high purity was used for experiments or stored at -24 °C until use. All purification procedures were conducted at 0 - 4 °C unless otherwise stated.

2.6 Simulated gastric fluid (SGF) digestion stability assay

The digestibility of TM in SGF was estimated according to the methods of Liu et al. [23] and Huang et al. [14] with slight modifications. SGF was prepared following the United States Pharmacopoeia Standard [24]. The total volume of the reaction solution was 1 mL. A ratio of 3.75-U pepsin activity/mg protein was selected for all tests. The pH of gastric digestion was 1.2. Digestion was performed at 37 °C with continuous rocking. SGF was incubated in a water bath at 37 °C for 10 min before added to the test protein. At different time intervals (0, 1, 2, 5, 10, 15, 30 and 60 min), 100 µL of the reaction solution was removed to a separate sampling tube, and the reaction was immediately terminated by adding 30 µL of 200 mmol/L Na2CO3. Samples were then heated at 95 °C for 10 min and analyzed by SDS-PAGE or western blotting. For the 0 min sample preparation,protein samples were mixed with SGF that had been inactivated by neutralization with Na2CO3. In the control experiments, each protein sample was dissolved in the reaction buffer without pepsin and then treated as described above. All experiments were repeated three or more times.

2.7 Simulated intestinal fluid (SIF) digestion stability assay

SIF was prepared following the United States Pharmacopoeia Standard [24]. The total volume of the reaction solution was 1 mL.A ratio of 0.6-U trypsin activity/mg protein was selected for all tests. The pH of intestinal digestion was 7.5. As with SGF, digestion occurred at 37 °C with continuous rocking, and SIF was incubated for 10 min in a 37 °C water bath before added to the test protein.At different time intervals (0, 1, 15, 30, 60, 120, 180 and 240 min),100 µL of the reaction solution was removed to a separate sampling tube, and the reaction was immediately terminated by adding the loading buffer and heating at 95 °C for 10 min. The samples were analyzed by SDS-PAGE or Western blotting. For the 0 min sample preparation, protein samples were added to SIF that was inactivated by heating for 10 min. In the control experiments, each protein sample was dissolved in the reaction buffer without trypsin and then treated as described above. Three or more replicates were used for experiments; differences between replicates were not detected.

2.8 SDS-PAGE and Western blotting analysis

SDS-PAGE was performed according to the method of Huang et al. [14]. Samples were separated in 12% polyacrylamide gels with 5% stacking gel and by employing a vertical electrophoresis system(Bio-Rad, Hercules, CA, USA) according to the manufacturer’s recommendations, the gels were stained for protein with Coomassie Brilliant Blue R-250.

Western blotting was performed according to the method of Yu et al. [25]. Briefly, protein samples were subjected to SDSPAGE and electrophoretic transfer to PVDF membranes. Nonspecific protein sites were blocked with 5% skim milk in Tris-HCl buffered saline (TBS: 20 mmol/L Tris-HCl, pH 8.0, 0.145 mol/L NaCl). To investigate the IgE-binding activity of TM, the membranes were incubated with rabbit anti-crab polyclonal antibodies (1:2 000 dilution) at room temperature for 2 h. The membranes were then washed five times with TBST (TBS with 0.05% Tween-20) and incubated in HRP-labeled goat anti-human IgE (1: 2 000 dilution)at room temperature for 1 h. The membranes were then washed extensively with TBST. Immunodetection was conducted using ECL luminous liquid as a substrate and recorded with a chemiluminescence imager (ProteinSimple, CA, USA).

2.9 Inhibition ELISA

Inhibition ELISA was performed to determine the specificity of IgE-binding of TM (extracts from processed crab) that had been digested. Polystyrene 96-well ELISA plates were coated with TM(200 ng/well) overnight at 4 °C using 0.16% (m/V) Na2CO3and 0.29%(m/V) NaOH (pH 9.6) as the coating buffer. Coated plates were washed three times with TBST and then blocked with 200 µL/well of blocking buffer (0.5% skim milk in TBST) at 37 °C for 2 h. In another plate, 30 µL of human sera (diluted by one-fifth with 1%nonfat milk in TBS) was mixed with equal volumes of digested TM(with pepsin and trypsin) as an inhibitor. After incubation at 37 °C for 1 h, each 30 µL sample of the sera-TM mixture was transferred to an ELISA plate coated with TM and incubated at 37 °C for 1 h.Plates were later washed with TBST and reacted with HRP-labeled goat anti-human IgE antibody (1:2 000 dilution) for 2 h at 37 °C.After washing with TBST, bound peroxidase activity was developed using 100 µL 3,3’,5,5’-tetramethylbenzidine (TMB), and the reaction was terminated by addition of 2 mol/L sulfuric acid. Absorbance was determined at 450 nm with an automated ELISA plate reader (Thermo Fisher Scientific, Shanghai, China). The hypersensitivities of the digested crude extract and its digestive products were represented by calculating the inhibition rate using the following formula [14,25]:

whereXis the absorbance of patients’ sera without inhibitors, andYandZare the absorbance of patients’ and control sera reacted with various inhibitors, respectively. All determinations of inhibition ratio were duplicated, and mean values were used in analyses.

2.10 Statistical analysis

Experimental data were analyzed by one-way ANOVA using SPSS software (SPSS Inc., Ver. 23, Chicago, IL, USA). Mean differences(P＜ 0.05) were established by Duncan’s multiple range tests.

3. Results and discussions

3.1 Identification and analysis of purified TM

Purified TM was obtained by isoelectric precipitation and ammonium sulfate fractionation. The purified protein was analyzed by SDS-PAGE; the results are shown in Fig. 1A. The molecular mass of the purified TM was approximately 38 kDa (Fig. 1A). The identity of the purified TM was confirmed by immunoblotting with rabbit anti-Chinese mitten crab TM polyclonal antibody (Fig. 1B). A clear 38 kDa protein band was identified as the major allergen of Chinese mitten crab.

Fig. 2 SDS-PAGE results of crude extracts from processed crab by SGF digestion. The crabs were (A) untreated, (B) treated with microwave, (C) treated with ultrasound, or (D) treated with HTP. Enzyme digestion was performed as described in ‘Materials and methods’ followed by SDS-PAGE. After electrophoresis,proteins were visualized by staining with Coomassie Brilliant Blue R-250. In the control experiments, proteinase was replaced with 20 mmol/L PBS (pH 7.5).Molecular masses of the protein markers (M) were shown on the left edges. The black arrow represents the target protein (TM).

Fig. 1 SDS-PAGE and immunoblotting of purified TM from Chinese mitten crab muscle. (A) SDS-PAGE. (B) Immunoblotting using rabbit anti-crab TM polyclonal antibody. The black arrow represents the target protein (TM).

3.2 Digestibility of processed crab crude extract by SGF

The digestibility of crude extract was detected by SDS-PAGE.A band with a molecular mass of about 38 kDa was observed(Fig. 2). In the SGF digestion assay system, the original TM band was gradually digested over time, and this was accompanied by the generation of other protein bands. A new degradation band (34 kDa)appeared at 1 min, and a fragment with a size of approximately 34 kDa progressively intensified and remained after 60 min (Fig. 2A-C).As SGF progressed, the original TM band produced another weak fragment of 32 kDa after microwave and ultrasound treatments (Fig. 2B and C).Compared with other processing methods, the original TM band and degradation fragments treated with HTP became so unclear that the variation tendency could not be confirmed (Fig. 2D); this may indicate that HTP causes aggregation of myofibrillar proteins [6,12]. However,we noted that the band of TM after treatment with HTP was degraded rapidly and all the bands disappeared completely after incubation for 15 min.

Since the crude extract contained nonallergic proteins besides the allergic protein (TM), it was difficult to ascertain by SDSPAGE whether the degraded fragments with low molecular weights originated from TM or nonallergic proteins. To clarify the identities of the degraded protein fragments and fully understand the digestion of TM, western blotting was conducted with polyclonal antibodies.

Western blotting figures corresponding to Fig. 2 showed that, under digestion with pepsin, a main degraded fragment with a molecular mass of 34 kDa remained immunoreactive to anti-crab-specific rabbit antibodies from 0 to 60 min (Fig. 3A-C); this suggests that the 34 kDa fragment was resistant to digestion. After treatment with HTP, the concentration of the original band and degraded bands decreased significantly (Fig. 3D). As shown in Fig. 3, the original band of TM disappeared entirely at 60 (A), 60 (B), 60 (C), and 15 min (D). Considering these results, HTP seems to be more effective as a method for improving the digestibility of the crab allergen TM in SGF.

Compared with untreated samples, TM treated with microwave and ultrasound produced some new degradation bands; however,these treatments did not seem to affect digestibility. In a previous study, 7 stable heat/digested IgE-binding linear epitopes of TM were identified inScylla paramamosainTM, and it was confirmed that linear epitopes were related to the immunoreactivity of allergens [26].This might be because linear epitopes of TM treated with microwave and ultrasound had not been destroyed by gastric digestion and therefore still maintained a high IgE-binding capacity. Similarly,in a previous study, no significant difference in the digestion stability of allergens from wheat gliadin was observed following microwave treatment (70, 200, and 500 W for 5 min) [27]. Venkatachalam et al. [28]also found that the content of three allergens (Ana o 1, Ana o 2, and Ana o 3) in cashew nuts remained stable after microwave heating at 500-1 000 W for 1-2 min. The unchanged digestive stability might be because protein denaturation and polymerization caused by long-time microwave heating did not affect the related epitopes in allergenic proteins. However, in another study, more fragments and strips of shrimp were generated after microwave treatments(75, 100, and 125 °C for 5, 10, and 15 min), leading to the effective degradation of proteins, especially in treatments at 125 °C [29].The results of this latter study differ from our results. Thus, the effect of microwave processing on the digestive stability of allergenic proteins from different sources is different, and further studies are still needed. After HTP treatment, crab muscle was loosened in terms of tissue structure; the restriction enzyme cleavage sites were exposed, so it was decomposed into more peptides and amino acids by digestive enzymes, which was more conducive to digestion. This might explain why the digestibility of TM from crab crude extract increased significantly. Therefore, the capacity of any processing method to alter the immunoreactivity of the allergen depends on the properties of the allergen and the types and conditions of processing.Notably, IgE reactivity was not observed against the lower fragment (32 kDa) (Fig. 3B and C). This might be attributable to IgE-binding epitopes of the degraded fragment having been destroyed by pepsin digestion.

Fig. 3 Western blotting using polyclonal antibodies to detect the degradation of the TM of crude extracts in SGF. The crabs were (A) untreated, (B) treated with microwave, (C) treated with ultrasound, or (D) treated with HTP. Samples were separated by SDS-PAGE followed by immunological detection. Positions of molecular mass standards (M) were labeled on the left. The black arrow represents the target protein (TM).

With SGF digestion for 60 min (Fig. 2A-C), TM remained in the SGF digestion products, indicating that it was strongly resistant to gastric digestion. Previously, other crustacean allergens, such as the TM allergens from shrimp (Exopalaemon modestus) [15] and crab(S. serrata) [14], exposed to SGF digestion for 60 min were shown to be resistant to pepsin. Resistance of proteins to gastric digestion is regarded as an indicator of potential allergenicity because many food allergens are commonly resistant to pepsin [30,31]. Thus, the allergenicity of TM in this study was evaluated by measuring the resistance to pepsin digestion.

3.3 Digestibility of processed crab crude extract by SIF

The effects of microwave, ultrasonic and HTP treatments on the digestion stability of crab crude extract are shown in Fig. 4. Under digestion of trypsin, the original TM band with a molecular weight of 38 kDa as well as other protein bands were shallower and weaker on the electropherogram as digestion time increased; this suggested that the sample was gradually digested (Fig. 4). In the digestion pattern of SIF, TM was susceptible to trypsin digestion, and a fragment of 34 kDa was detected after 1 min, whereas the 34 kDa fragment of TM disappeared within a short time (Fig. 4A-C). Interestingly, the original TM treated by ultrasound produced a faint fragment at about 23 kDa, but this fragment quickly disappeared after 30 min of trypsin digestion (Fig. 4C). In comparison with the untreated crab, microwave heating had no effect on the digestion stability of TM, whereas the original TM and its proteolytic fragments were all degraded rapidly after treatment with ultrasound (Fig. 4A-C). However, the original TM band after HTP treatment was faint, so it was difficult to recognize the degradation fragments (Fig. 4D).

Fig. 4 SDS-PAGE results of crude extracts from processed crab by SIF digestion. The crabs were (A) untreated, (B) treated with microwave, (C) treated with ultrasound, or (D) treated with HTP. Enzyme digestion was performed as described in ‘Materials and methods’ followed by SDS-PAGE as in Fig. 2. The black arrow represents the target protein (TM).

To identify the IgE-binding proteins in crab muscle crude extract,western blotting using specific rabbit polyclonal antibodies against TM was applied. Fig. 5 showed the Western blotting figures that correspond to Fig. 4. The digestion conditions were found to be similar to those obtained with SDS-PAGE. In the digestion pattern of SIF, the original TM band was rapidly degraded to the main fragment with a size of approximately 34 kDa (Fig. 5A-C). Western blotting results demonstrated that IgE antibodies had a positive reaction with the original TM band (38 kDa) and the proteolytic fragment with a molecular mass of 34 kDa (Fig. 5A-C). As shown in Fig. 5, the original TM and degraded fragments were undetectable when digested for 60 (A), 60 (B), 30 (C), and 15 min (D). In the presence of trypsin,all of the digestive stable proteins treated with HTP were completely degraded in the shortest time (Fig. 5D). The high pressure may have loosened the structure of the protein and caused the destruction of IgE-binding epitopes in degraded fragments, resulting in the decreased immunoreactivity of TM.

Fig. 5 Western blotting using polyclonal antibodies to detect the degradation of the TM of crude extracts in SIF. The crabs were (A) untreated, (B) treated with microwave, (C) treated with ultrasound, or (D) treated with HTP. Samples were separated by SDS-PAGE followed by immunological detection. Positions of molecular mass standards (M) were labeled on the left. The black arrow represents the target protein (TM).

As shown in Fig. 5, compared with that in untreated and microwave-treated samples, the digestion stability of TM after samples was ultrasound- and HTP-treated was significantly reduced,which suggested that TM was not resistant to trypsin digestion.Similarly, high-intensity ultrasound (800 W for 15 min) has previously been shown to degrade TM and generate more protein fragments, thereby improving the digestibility of TM from shrimp muscle [15]. This result might be due to ultrasound forming cavitation bubbles followed by the cyclic generation and collapse of cavities resulting in chemical and physical changes [20], which in turn modifies the conformation of proteins, loosens the protein structure,and alters the susceptibility of protein to trypsin through the exposure of cleavage sites. In addition, the proteolytic fragments of TM were degraded fastest when treated with HTP in comparison to the other methods. This result could have arisen because HTP processing contributed to the destruction of protein structure, thereby loosening the crab muscle protein, resulting in more regular and slender fiber bundles, and ultimately facilitating digestion [12]. It was found that thermal processing (including boiling, steaming, and high pressure)made muscle proteins ofS. paramamosainform a more compact and orderly structure, which meant it was digested more easily [11].According to this research, high pressure treatment among the three different processing methods could change the secondary and tertiary structures of proteins by affecting noncovalent action (hydrogen bonds and water dispersing bonds) and adjusting digestibility and immunoreactivity [18]. This can explain the fact of that TM treated by HTP treatment was digested more easily than TM in the raw samples.

The TM band was relatively stable in SGF, and its degraded fragment (34 kDa) could still be detected after 60 min (Fig. 2).However, in the presence of SIF, both TM and its proteolytic fragments were completely digested within a short period (Fig. 4).Because of their different cleavage specificities for peptide bonds, TM digestion patternsin vitrocaused by pepsin and trypsin are robustly different [14]. Pepsin tends to cleave peptide bonds next to Phe or Tyr residues [32]. Trypsin has an explicit specificity; it preferably cleaves next to the hydrophilic amino acid residues Lys and Arg [33].Zhang et al. [15] measured the amino acid content of TM; their results suggested that the low Phe and Try content (1.82% and 2.01%, respectively) and high Lys and Arg content (10.97% and 10.03%, respectively) might be one of the reasons why TM was resistant to pepsin digestion but relatively susceptible to trypsin. Notably, the results of TM analysis from crab crude extract by SDS-PAGE were consistent with the results obtained by western blotting (Fig. 2vs. Fig. 3;Fig. 4vs. Fig. 5), suggesting that the digestion pattern of TM by SGF and SIF was not affected by the presence of other myofibrillar proteins. Liu et al. [23] provided a similar conclusion in their research.

3.4 Inhibition ELISA

To analyze the IgE-binding abilities of TM digested and its degraded fragments, inhibition ELISA was used to detect the sensitivity of proteolytic fragments with low IgE reactivity. According to the digestion pattern of SDS-PAGE, the capacity for IgE-binding was indirectly measured by ascertaining the extent of inhibition of IgE reactivity: where inhibition was increased, this showed stronger IgEbinding abilities and digested proteins that were more allergenic [34].As shown in Fig. 6, after using different processing methods to treat crab muscle, TM from crab muscle was digested with pepsin (60 min)and trypsin (240 min); the allergenicity of TM and its degraded fragments was reduced but still existed. TM digested by pepsin for 1 h or trypsin for 4 h displayed less inhibition than did undigested TM. Compared with untreated samples, microwave-treated samples were not significantly affected in terms of the allergenicity of TM;however, the TM-specific inhibition rate was reduced greatly after ultrasound and HTP processing. In addition, the digested samples treated with HTP displayed the lowest IgE-binding activities,suggesting that HTP technology greatly affected the allergenicity of TM from crab muscle.

TM and its degraded fragments retained IgE-binding activity,which indicated that inhibition ELISA was more sensitive than SDSPAGE or Western blotting for detecting proteolytic fragments with immunoreactivity. In addition, IgE-binding measured by inhibition in the present study might not completely equate to allergenicity. Upon binding with allergenic proteins, IgE antibodies present on the surface of mast cells or basophil membranes are crosslinked, which is a requirement for allergenicity determination [23]. Therefore, to reveal changes in TM allergenicity after proteinase digestion, it is necessary that experiments analyzing the release of histamine from basophils and involving the skin prick test are conducted.

Fig. 6 Inhibition ELISA analysis of digestive samples from raw and processed crabs. Different superscript letters mean significant differences between values with different processing methods (P ＜ 0.05) (1, untreated crab; 2, crab treated with microwave; 3, crab treated with ultrasound, 4,crab treated with HTP). Samples digested by pepsin or trypsin for 1 or 4 h,respectively. Pooled patients’ serum (1:5 dilution) and HRP-labeled goat anti-human IgE antibody (1:2 000 dilution) were used. Data are presented as mean ± SD deviations (n = 6).

In comparison with the SGF digestion assay, the inhibition rate of the digestion products in the SIF rapidly dropped to a lower level (Fig. 6),indicating that the substantial linear epitopes and conformational epitopes of TM were hydrolyzed by trypsin. For ultrasound and especially the HTP treatment, the inhibition rate of the digestion products was much lower than that observed with untreated crabs.Studies have shown that higher temperature might permit masking of some IgE-binding epitopes through protein-chemical crosslinking,which altered the recognition of crab allergens by IgE and thereby potentially reduced lgE-binding [35]. Similar results were reported in a previous study in which TM in a crude extract obtained from the mud crab (S. paramamosain), which was heated at 115 °C and 0.14 MPa for 15 min in an autoclave, showed a reduced inhibition rate compared with that observed in raw crabs [12]. In a similar study, consistent with our findings, Yu et al. [25] reported that high pressure steaming(121 °C and 0.14 MPa for 20 min in an autoclave) could significantly reduce the allergenicity of TM in comparison to boiling or ultrasound and boiling combined. In recent years, a number of studies have shown that numerous chemical interactions may be induced in the food matrix during processing, thereby affecting the allergic potential of proteins in the whole food body [11]. High temperature caused the Maillard reaction between proteins and reducing sugars in the complex matrix, which changed the morphology in the matrix, caused the conformational changes of allergen, and the exposure of peptide bonds, resulting in the decrease of IgE-binding activities [26]. Further,according to the research of Liu et al. [11], high pressure unfolded theα-helices of TM and the heat stable epitope peptides were found inα-helix of the allergen. It could be inferred that high pressure broke epitope peptides which would be available for reaction with antibodies [12], thereby alleviating immunoreactivity. In addition, it has shown that high pressure treatment at high temperature was able to decrease IgE-binding of walnut proteins more efficiently than high pressure treatment at low temperature in another study [17]. Thus,HTP treatment as a processing method could potentially be applied to reduce the allergenicity of crab. Interestingly, the allergenicity of TM following treatment with HTP still existed after pepsin digestion or trypsin digestion; therefore, HTP did not completely remove the immunoreactivity of certain allergens, although the extent of antigen-antibody binding required to cause the allergic reaction was significantly reduced [36]. According to some reports, even boiling or baking at 180 °C did not remove the allergenicity of apple peel,nor was allergenicity destroyed following severe thermal processing methods, such as sterilization (121 °C for 30 min), applied to peach juice [37]. These findings reflect the fact that, even under harsh processing conditions and despite IgE-binding ability being greatly reduced, a proportion of the protein molecules likely remain in the native state, or some hidden epitopes are still able to bind IgE.

IgE response to the digested samples treated with ultrasound was significantly higher than those of untreated. One of the reasons might be that during sonication, high shear energy waves and turbulence in the cavitation zone induced the changes of TM spatial structure, leading to exposure of pepsin and trypsin cleavage sites, therefore reducing the sensitization by changing the IgE reaction conformation or linear epitope [15]. For the SGF and SIF digestion, microwave treatment had no significant difference(P＜ 0.05) compared with the untreated crab. The reason for the unchanged allergenicity may be that the related epitopes in crab allergenic proteins were not susceptible to microwave radiation.It indicated that microwave as thermal processing did not show a potential application in reducing the allergenicity of crab. Whereas, a significant decrease in the antigenicity of TM when fish frame protein was radiated by microwave [38]. Previous studies have shown that microwave treatment could cause the destructions or conformational changes in the linear epitopes of TM, resulting in the reduction of allergenicity [29]. But this conclusion did not reflect in our research,the explanation for this phenomenon needs to be further studied. Due to the diversity and complexity of the thermal process, it is necessary to explore the structural changes of allergens extracted from heattreated crab muscle.

4. Conclusions

We assessed the effects of different processing methods(microwave, ultrasound, and HTP treatments) on the digestive stability and allergenicity of TM from the Chinese mitten crab. We found that microwaving had no obvious impact on the digestion of TM. However, ultrasound treatment promoted the degradation of TM in SIF. In addition, HTP treatment reduced allergenicity markedly.Indeed, HTP was the most effective method by which to accelerate the digestion of TM via digestive enzymes and reduce the capacity for IgE-binding. These results therefore provide a theoretical basis on which to develop hypoallergenic crab products in the future.

Declaration of competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the earmarked fund for the Anhui Provincial Modern Agri-industry Technology Research System(AHCYJSTX-08) and the China Agriculture Research System of MOF and MARA (CARS-48).