Investigation on the Anaphylaxis and Anti-Digestive Stable Peptides Identification of Ultrasound-Treated α-Lactalbumin during In-Vitro Gastroduodenal Digestion

Our previous studies indicated that ultrasound treatment can increase the anaphylaxis of protein. However, investigation on the anaphylaxis changes of ultrasound-treated α-lactalbumin (ALA) during digestion is lacking. The anaphylaxis of ultrasound-treated ALA and its digesta was investigated. The anti-digestive stable peptides were identified by high-resolution mass spectrometry. Ultrasound induced the tertiary structure of ALA to unfold and increased its anaphylaxis. During digestion, the anaphylaxis of both gastric and gastroduodenal digesta was further increased. There are two reasons for this phenomenon. On the one hand, linear epitopes played an important role in affecting anaphylaxis compared with the conformational epitope, and some linear epitopes were still retained on the anti-digestive stable peptides produced after gastroduodenal digestion, resulting in increased anaphylaxis after digestion. On the other hand, the presence of intact ALA molecules after digestion still remained strong anaphylaxis. Compared with the digesta of untreated ALA, the digesta of ultrasound-treated ALA possessed higher anaphylaxis. The results indicated that ultrasound increased the anaphylaxis of ALA during digestion.


Introduction
Novel non-thermal food processing technologies are emerging and prospectively maintaining the sensory properties of foodstuff that traditional processing methods such as heating and boiling cannot match [1]. The high-intensity ultrasound technique is specified as a sound wave frequency of 20-100 kHz which is widely applied to microorganism inactivation, components extraction, and cell walls crushing due to its nutrient retention and appreciable acceptability [2]. Recently, ultrasound has also attracted desirable attention for its ability to improve the functional properties (emulsification, foaming properties, and oxidation resistance) of proteins [3,4]. Dong et al. [5]. indicated that the antioxidant activity of ultrasound-processed tropomyosin increases with the prolongation of ultrasound action time. Ultrasound has also drawn the close attention of researchers for its application in protein anaphylactogens [6].

Preparing of Rabbit Anti-ALA IgG
Male Japanese rabbits (three months, permission number was SCXK (Gan) 2014-0005) were purchased from Longping (Nanchang, Jiangxi), which were used to prepare polyclonal rabbit anti-ALA IgG. All procedures were authorized by the ethics committee of Nanchang University (approval number 0064257) and were carried out according to Chinese guidelines for animal welfare (GB/T35892-2018). After acclimatizing in an environmentally controlled breeding room for 7 days, the rabbits were intravenously injected with 0.8 mg/mL of ALA (1 mL) emulsified with the same volume of Freund's complete adjuvant for the first time. After that, ALA was emulsified by Freund's incomplete adjuvant at 1:1 (v/v), and the subsequent booster immunization was carried out at a 6-day interval with a dose of 0.5 mg per rabbit. After the rabbits were anesthetized, the plasma was collected and the rabbit's anti-ALA IgG serum was separated by centrifuging below 4 • C and stored at −80 • C.

Ultrasound Treatment
Considering that ALA accounts for 5% of milk proteins, bovine ALA was suspended in phosphate buffer (pH 7.4, 0.05 mol/L) to a concentration of 1 mg/mL, and 20 mL of ALA solution was treated at a gradually increased intensity (50 and 100 W/cm 2 ) in an ultrasound processor (Scientz-IID, Ningbo, China) equipped with a 3 mm microtip probe. The ultrasound treatment was performed for 10 min with the pulsation of 2 s on, and 2 s off. The samples were in ice-bath conditions to ensure the temperature was below 20 • C. Untreated ALA was named ALA-0. Ultrasound-treated ALA at 50 and 100 W/cm 2 were named ALA-50 and ALA-100, respectively. ALA-0, ALA-50, and ALA-100 were collectively referred to as substrate samples. Other milk components (e.g., fats) that could influence the ultrasound treatment of ALA were not investigated in this study.

Gastroduodenal Digestion In-Vitro
To better simulate the physiological parameters of the infant gastroduodenal tract, the digestive behavior of substrate samples was performed by a previous method, with some adaptations [24]. In the process of simulated gastric juice (SGJ) digestion, substrate samples were mixed with 10 mL of SGJ (Ph = 3.0) embodied with pepsin (22.75 U/mg sample) and NaCl (0.15 mol/L). After performing for 2 h at 37 • C, the gastric digestive behavior was suspended by monitoring the pH to 7.0. Afterward, 10 mL of simulated duodenal juice (SDJ) was added to digest for 2 h, and the components of SDJ were as follows: 2 mmol/L of bile salts (sodium glycodeoxycholate and sodium taurocholate), 3.45 U trypsin/mg sample, and 0.04 U chymotrypsin/mg sample. It was stopped by adding 4 mmol/L of protease inhibitor 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride. Samples were placed below −20 • C until used. ALA-0, ALA-50, and ALA-100 digested by SGJ were named ALA-0G, ALA-50G, and ALA-100G, respectively, and ALA-0G, ALA-50G, and ALA-100G were called gastric digesta; ALA-0, ALA-50, and ALA-100 digested by SGJ followed SDJ was denoted as ALA-0D, ALA-50D, and ALA-100D, respectively, and ALA-0D, ALA-50D, and ALA-100D were called gastroduodenal digesta. The intrinsic fluorescence was analyzed according to a previous report with some modifications [25]. 0.5 mg/mL of substrate samples, gastric digesta, and gastroduodenal digesta were prepared for intrinsic fluorescence spectra detection by a fluorescence spectrophotometer (Hitachi F-7000, Tokyo, Japan). The excitation wavelength was 280 nm, and the emission spectrum was recorded from 300 nm to 600 nm, with 2 nm bandpass filters in both excitation and emission.

Analysis of Tricine SDS PAGE
Tricine SDS PAGE analysis was carried out according to a previous study with some modifications [26]. To detect the pattern of ALA-0, ALA-50, ALA-100, ALA-0G, ALA-50G,  ALA-100G, ALA-0D, ALA-50D, and ALA-100D, separating gel (16.5%), spacer gel (10%), and stacking gel (4%) were prepared for electrophoresis (15 µL of samples were applied) with an initial voltage of 30 V (1-2 h) until the indicator entered separating gel, then 100 V was employed. Following electrophoresis, gels were fixed with fixation fluid (0.5% glutaraldehyde in 30% alcohol) for 30 min and dyed with coomassie brilliant blue R-250 for 20 min, and finally destained until the background was transparent. The samples' distribution in tricine SDS PAGE gel was transferred to a nitrocellulose membrane for western blotting. The analysis of western blotting was carried out according to a method previously described with some modifications [27].

Indirect Competitive ELISA
The IgG/IgE-binding abilities were analyzed by icELISA with rabbit anti-ALA IgG and CMA patients' sera. Briefly, the incubation circumstance was at 37 • C for 1 h and the washing solution was PBST (containing 0.05% Tween-20). The incubation sequence was as follows: each well of the microplate was coated with 2 µg/mL of native ALA (100 µL/well); 1% (w/v) of fish gelatin blocking solution (250 µL) was blocked; 50 µL of rabbit anti-ALA IgG (1:10,000) or CMA patients' sera (1:20) and 50 µL of samples were blended in the wells; HRP-labeled goat anti-rabbit IgG (1:5000) or HRP-labeled goat anti-human IgE (1:800) was followed; TMB two-component substrate solution was used for color reaction (15 min); sulfuric acid (2 mol/L) was added to stop the reaction. The binding rate was calculated following the equation previously described [28]. The inhibition rate was calculated as: where B and B 0 are the OD values with and without samples, respectively. B c was the OD values without rabbit anti-ALA IgG or CMA patients' sera. IC50 is the concentration of samples that causes a 50% inhibition of IgG recognition (µg/mL). Each sample was performed in triplicate.

Degranulation Assay in KU812 Cells
The KU812 cells were maintained in the medium RPMI-1640, which contained 10% of fetal bovine sera and 105 U/L of penicillin/streptomycin. The culture condition was 37 • C with CO 2 (5%). The sensitizing condition was as follows: KU812 cells were activated by 10 µL of patients' sera (24 h); then 100 µL/well of samples (0.5 mg/mL) were added for activation (3 h). The degranulation assay (the release of β-Hex, IL-6, and histamine) was analyzed by ELISA assays, following the instruction of commercial kits.

Gel Filtration Chromatography Isolation
Gastroduodenal digesta were isolated according to the previous study with some modifications [29]. Freeze-dried ALA-0D and ALA-100D were dissolved in ultrapure water (2 mL). After being filtered through nylon filter membrane (0.45 mm), ALA-0D and ALA-100D were loaded onto Sephadex G-25 column (Φ 16 mm × 50 cm). An HD-21-88 ultraviolet detector (QiTe, Shanghai, China) coupled with an automatic fraction collector (QiTe, Shanghai, China) was used. The isolated condition was as follows: the eluent was ultrapure water; the flow rate was 0.4 mL/min; the elution solution was collected every 8 min; the detected wavelength was 220 nm; the detection sensitivity was 0.5 A.

Results and Discussion
3.1. Structural Characterization 3.1.1. Structure of Ultrasound-Treated ALA Aromatic amino acids in ALA, especially tryptophan, are the basis of intrinsic fluorescence. The intrinsic fluorescence spectra evolution during ultrasound treatment of ALA was analyzed, due to the appropriability of intrinsic fluorescence spectra for investigating the tertiary structure changes in proteins. The fluorescence emission intensity was detected at a wavelength range of 200-600 nm. When excited at 280 nm, ALA-0 presented a fluorescence emission maximum (λmax) at 346 nm ( Figure 1A). ALA-50 and ALA-100 displayed an increase in fluorescence intensity at the same λmax with an increase in ultrasound intensity. The results indicated that ultrasound could induce the tertiary structure of ALA unfolding and expose aromatic amino acids, thus enhancing the fluorescence intensity. A similar phenomenon was observed by Shao et al. [30].

Structure of Ultrasound-Treated ALA
Aromatic amino acids in ALA, especially tryptophan, are the basis of intrinsic fluorescence. The intrinsic fluorescence spectra evolution during ultrasound treatment of ALA was analyzed, due to the appropriability of intrinsic fluorescence spectra for investigating the tertiary structure changes in proteins. The fluorescence emission intensity was detected at a wavelength range of 200-600 nm. When excited at 280 nm, ALA-0 presented a fluorescence emission maximum (λmax) at 346 nm ( Figure 1A). ALA-50 and ALA-100 displayed an increase in fluorescence intensity at the same λmax with an increase in ultrasound intensity. The results indicated that ultrasound could induce the tertiary structure of ALA unfolding and expose aromatic amino acids, thus enhancing the fluorescence intensity. A similar phenomenon was observed by Shao et al. [30].
The molecular weight distribution of samples obtained from the tricine SDS PAGE pattern is outlined in Figure 1B. Obvious bands of ALA-0, ALA-50, and ALA-100 approaching 14.4 kDa were visible (Lanes 1-3 in Figure 1B), and the three bands (Lanes 1-3) had a similar migration distance. No major changes of lanes 1-3 were discovered in the tricine SDS PAGE pattern, revealing ultrasound treatment could not cause ALA aggregate to polymers or degrade to low-molecular-weight products [31]. Therefore, combined with the analysis of intrinsic fluorescence spectra, after ultrasound treatment, the tertiary structure of ALA was unfolded but the primary structure was not destroyed. The molecular weight distribution of samples obtained from the tricine SDS PAGE pattern is outlined in Figure 1B. Obvious bands of ALA-0, ALA-50, and ALA-100 approaching 14.4 kDa were visible (Lanes 1-3 in Figure 1B), and the three bands (Lanes 1-3) had a similar migration distance. No major changes of lanes 1-3 were discovered in the tricine SDS PAGE pattern, revealing ultrasound treatment could not cause ALA aggregate to polymers or degrade to low-molecular-weight products [31]. Therefore, combined with the analysis of intrinsic fluorescence spectra, after ultrasound treatment, the tertiary structure of ALA was unfolded but the primary structure was not destroyed.

Structure Characteristic of Ultrasound-Treated ALA during Gastroduodenal Digestion In-Vitro
To investigate the variation in the structure of ALA after gastric/gastroduodenal digestion, ALA-0G, ALA-50G, ALA-100G, ALA-0D, ALA-50D, and ALA-100D was analyzed by tricine SDS PAGE (Lanes 4-9 in Figure 1B). In comparison to substrate samples, pepsin hydrolysis caused the tricine SDS-PAGE pattern of gastric digesta (Lanes 4-6 in Figure 1B) to form three hydrolysis fragments (distributed in 3.3-14.4 kDa), implying that the tertiary and primary structure of ALA was destroyed under simulated gastric digestion. The band that appeared near 14.4 kDa was completely undigested ALA. This observation confirmed that ALA was not completely digested because of the simulated immature gastric environment of infants [32]. Three bands were also discovered in ALA-0D, ALA-50D, and ALA-100D (Lanes 7-9 in Figure 1B), and the one near 3.3 kDa was smeared. This might account for further hydrolysis induced by trypsin and α-chymotrypsin accelerating the digestion of ALA-0D, ALA-50D, and ALA-100D, causing them to become loose and form small peptide fragments. Therefore, we suggested that gastric digestion destroyed the tertiary and primary structure of ALA-0, ALA-50, and ALA-100, and gastroduodenal digestion further damaged their tertiary and primary structure.

IgG/IgE-Binding Abilities and Western Blotting
ALA is a major whey protein anaphylactogen that can bind with anaphylactic ALAspecific IgG/IgE, leading to various food anaphylactic symptoms. The IgG/IgE-binding abilities of substrate samples, gastric and gastroduodenal digesta were evaluated by icELISA and shown in Figure 2. Figure 2A shows the IgG IC 50 value of ALA-50 and ALA-100 shifting to 50.07 and 44.63 µg/mL, respectively. However, the IgG IC 50 value of ALA-0 was 52.99 µg/mL. Comparatively, 1.19 folds of the IgG IC 50 value were observed when ALA was treated with the ultrasound intensity of 100 W/cm 2 . These results indicated that ultrasound unfolded the tertiary structure of ALA, and exposed the IgG-binding epitopes, resulting in increased anaphylaxis [23]. Furthermore, with the increase of ultrasound intensity, the tertiary structure of ALA gradually unfolded and exposed more IgG-binding epitopes, leading to higher anaphylaxis.
The IgG IC 50 value of gastric digesta tended toward reduced values (increased anaphylaxis), compared with the substrate samples. The IgG IC 50 value of ALA-0G was 38.27 µg/mL, and that of ALA-50G and ALA-100G successively declined when the ultrasound intensities were increased to 50 (33.15 µg/mL, 1.15 folds), and 100 W/cm 2 (32.00 µg/mL, 1.20 folds), indicating an increase of IgG-binding ability in ALA-50G and ALA-100G. The tertiary structure of gastric digesta was destroyed after digestion ( Figure 1B), so the conformational epitopes were destroyed. However, compared with the substrate samples, the anaphylaxis was enhanced in the gastric digesta, which probably explained that linear epitopes played a more important role in affecting the anaphylaxis of ALA, compared with the conformational epitopes. Furthermore, the presence of intact ALA molecules after digestion still remained strong anaphylaxis. Gazme et al. [33] showed that the in-vitro digestion caused the egg white proteins to remain residual immune-reactivity due to the presence of intact proteins such as lysozyme. The IgG IC 50 value of ALA-0D (24.39 µg/mL), ALA-50D (24.65 µg/mL), and ALA-100D (21.13 µg/mL) was reduced again, and ALA-100D was reduced dramatically, with the highest anaphylaxis. made rabbit anti-ALA IgG (1:1000 dilution). HRP-labeled goat anti-rabbit IgG was diluted to 1:1000. Given the fact that the anaphylaxis of protein anaphylactogens increased after ultrasound treatment, only gastric and gastroduodenal digesta were embodied for western blotting analysis [30]. As shown in Figure 2C, in comparison with line 1 (ALA-0), lines 2-7 (ALA-0G, ALA-50G, ALA-100G, ALA-0D, ALA-50D, and ALA-100D) also bound with rabbit anti-ALA IgG, showing both digesta had strong anaphylaxis.

Bioactive Mediators Release Activity
ALA would combine with fragment crystallizable (Fc) of the specific IgE that binds to the high-affinity IgE receptor FcεRI of mast cells or basophils during the effector phase, arousing cell degranulation and bioactive mediators, such as histamine, IL-6, and β-Hex generating, resulting in diarrhea, gastroduodenal bleeding, and other anaphylactic symptoms [35]. To investigate the influence of ultrasound on the release of bioactive mediators  Figure 2B showed the IgE-binding rates of the nine samples mentioned above, sharing similar trends such as the IgG-binding abilities. In general, the anaphylaxis of the substrate samples increased with the increase of ultrasound intensity, and the anaphylaxis of the gastric and gastroduodenal digesta was further increased, especially for gastroduodenal digesta. ALA-100, ALA-100G, and ALA-100D showed the highest IgE binding rates in substrate samples, gastric digesta, and gastroduodenal digesta (p < 0.05), respectively. Guimaraes et al. [34] found that Cry1Ab protoxin still conserved its immunoreactivity during digestion.

Bioactive Mediators Release Activity
ALA would combine with fragment crystallizable (Fc) of the specific IgE that binds to the high-affinity IgE receptor FcεRI of mast cells or basophils during the effector phase, arousing cell degranulation and bioactive mediators, such as histamine, IL-6, and β-Hex generating, resulting in diarrhea, gastroduodenal bleeding, and other anaphylactic symptoms [35]. To investigate the influence of ultrasound on the release of bioactive mediators of ALA-0, ALA-50, ALA-100, and their digesta, KU812 cells were applied in the experiment. After the experimental KU812 cells were sensitized with CMA patients' sera, FcεRI, a high-affinity IgE receptor that was believed to be closely related to cell degranulation, was expressed on the KU812 cells surface. The cells were then in contact with ALA-0, ALA-50, ALA-100, and their digesta, releasing bioactive mediators.
Panels A, B, and C of Figure 3 show, respectively, the release of β-Hex, IL-6, and histamine of substrate samples, as well as those of their gastric and gastroduodenal digesta. Stimulating with ALA-0 caused KU812 cell degranulation, resulting in the release of β-Hex (3.33 pg/mL). Dramatically, ultrasound-treated ALA (ALA-50, ALA-100) showed increased degranulation ability and led to the increased release of β-Hex, demonstrating that the anaphylaxis was boosted via ultrasound treatment ( Figure 3A). The results were consistent with the IgG/IgE-binding abilities analysis, which was due to the unfolded tertiary structure of ALA treated by ultrasound, leading to epitopes exposure. Besides, when simulated gastric digestion was carried out, the β-Hex release of gastric digesta were in the rank of ALA-0G (3.11 pg/mL) < ALA-50G (3.48 pg/mL) < ALA-100G (4.19 pg/mL), of which could also be authenticated in gastroduodenal digesta, increased release of β-Hex was discovered in the digesta of ultrasound-treated ALA. A similar phenomenon could be found in the release of IL-6 and histamine ( Figure 3B,C). In general, the anaphylaxis of ultrasoundtreated ALA was strengthened. We hypothesized the reason was that ultrasound unfolded the tertiary structure of ALA and exposed increased antibody-binding epitopes, thus promoting cell degranulation. The anaphylaxis of both the gastric/gastroduodenal digesta of ultrasound-treated ALA was still higher, compared with the gastric/gastroduodenal digesta of untreated ALA, further confirming that ultrasound increased the anaphylaxis of ALA during digestion.
Foods 2021, 10, x FOR PEER REVIEW 8 of of ALA-0, ALA-50, ALA-100, and their digesta, KU812 cells were applied in the expe ment. After the experimental KU812 cells were sensitized with CMA patients' sera, FcεR a high-affinity IgE receptor that was believed to be closely related to cell degranulatio was expressed on the KU812 cells surface. The cells were then in contact with ALA ALA-50, ALA-100, and their digesta, releasing bioactive mediators. Panels A, B, and C of Figure 3 show, respectively, the release of β-Hex, IL-6, an histamine of substrate samples, as well as those of their gastric and gastroduodenal d gesta. Stimulating with ALA-0 caused KU812 cell degranulation, resulting in the relea of β-Hex (3.33 pg/mL). Dramatically, ultrasound-treated ALA (ALA-50, ALA-10 showed increased degranulation ability and led to the increased release of β-Hex, demo strating that the anaphylaxis was boosted via ultrasound treatment ( Figure 3A). The r sults were consistent with the IgG/IgE-binding abilities analysis, which was due to t unfolded tertiary structure of ALA treated by ultrasound, leading to epitopes exposu Besides, when simulated gastric digestion was carried out, the β-Hex release of gast digesta were in the rank of ALA-0G (3.11 pg/mL) < ALA-50G (3.48 pg/mL) < ALA-100 (4.19 pg/mL), of which could also be authenticated in gastroduodenal digesta, increas release of β-Hex was discovered in the digesta of ultrasound-treated ALA. A similar ph nomenon could be found in the release of IL-6 and histamine ( Figure 3B,C). In gener the anaphylaxis of ultrasound-treated ALA was strengthened. We hypothesized the re son was that ultrasound unfolded the tertiary structure of ALA and exposed increas antibody-binding epitopes, thus promoting cell degranulation. The anaphylaxis of bo the gastric/gastroduodenal digesta of ultrasound-treated ALA was still higher, compar with the gastric/gastroduodenal digesta of untreated ALA, further confirming that ultr sound increased the anaphylaxis of ALA during digestion.

Isolation and Anaphylaxis Analysis of Gastroduodenal Digesta Groups
We found that gastric/gastroduodenal digesta still had strong anaphylaxis, especia for ALA-100D. We therefore took ALA-100D as an example to isolate by gel filtrati chromatography. ALA-0D and ALA-100D were isolated by the Sephadex G-25 colum

Isolation and Anaphylaxis Analysis of Gastroduodenal Digesta Groups
We found that gastric/gastroduodenal digesta still had strong anaphylaxis, especially for ALA-100D. We therefore took ALA-100D as an example to isolate by gel filtration chromatography. ALA-0D and ALA-100D were isolated by the Sephadex G-25 column  Figure 4). Based on A 220 , the chromatogram was divided into three groups (I, II, and III), and the molecular weight of the three groups followed the order: group I > group II > group III.
Foods 2021, 10, x FOR PEER REVIEW 9 of 16 ( Figure 4). Based on A220, the chromatogram was divided into three groups (I, II, and III), and the molecular weight of the three groups followed the order: group I > group II > group III. The results in Figure 3 showed that the anaphylaxis increased after gastroduodenal digestion, and ultrasound treatment raised the anaphylaxis ability of ALA after gastroduodenal digestion. Therefore, the KU812 degranulation assay for the three groups (I, II, and III) in ALA-0D and ALA-100D was performed. The release of histamine and IL-6 of each group is shown in Figure 5. The histamine release of group I in ALA-0D and ALA-100D was 8.37 and 8.21 μg/L, and there was no significant difference between them. Compared with ALA-0D, the histamine release of group II in ALA-100D was 7.23 μg/L and higher ( Figure 5A). A similar phenomenon was observed in group III in ALA-0D and ALA-100D. That was to say, the cell degranulation of group III in ALA-100D was higher than that in ALA-0D (p < 0.05). When the KU812 cells were exposed to group I in ALA-D, the β-Hex release was 404.15 pg/mL and was lower than that of ALA-100D (p < 0.05). Furthermore, when it came to group II and III, the β-Hex release content of ALA-100D was higher than ALA-0D (p < 0.05) ( Figure 5B). In general, gastroduodenal digesta groups still retained anaphylaxis, and groups II and III had a low release activity of bioactive mediators. However, they still retained anaphylaxis. The results in Figure 3 showed that the anaphylaxis increased after gastroduodenal digestion, and ultrasound treatment raised the anaphylaxis ability of ALA after gastroduodenal digestion. Therefore, the KU812 degranulation assay for the three groups (I, II, and III) in ALA-0D and ALA-100D was performed. The release of histamine and IL-6 of each group is shown in Figure 5. The histamine release of group I in ALA-0D and ALA-100D was 8.37 and 8.21 µg/L, and there was no significant difference between them. Compared with ALA-0D, the histamine release of group II in ALA-100D was 7.23 µg/L and higher ( Figure 5A). A similar phenomenon was observed in group III in ALA-0D and ALA-100D. That was to say, the cell degranulation of group III in ALA-100D was higher than that in ALA-0D (p < 0.05). When the KU812 cells were exposed to group I in ALA-D, the β-Hex release was 404.15 pg/mL and was lower than that of ALA-100D (p < 0.05). Furthermore, when it came to group II and III, the β-Hex release content of ALA-100D was higher than ALA-0D (p < 0.05) ( Figure 5B). In general, gastroduodenal digesta groups still retained anaphylaxis, and groups II and III had a low release activity of bioactive mediators. However, they still retained anaphylaxis. Letters (a-f) demonstrate significant differences (p < 0.05). ALA-0D and ALA-100D: untreated ALA and ultrasound-treated ALA at 100 W/cm 2 digested by simulated gastric juice followed simulated duodenal juice.

Anti-Digestive Stable Peptides Identification
The anti-digestive stable peptides of groups II and III in ALA-0D and ALA-10 were analyzed by HPLC-MS/MS. Figure 6A-D shows the MS and MS/MS spectra of pe tides DTQAIVQNNDSTEY (AA 37-50) and KFLDDDLTDDIM (AA 79-90). The m/z AA 37-50 was 798.3477, and the peptide AA 37-50 was identified according to the diff ence between the values of b and y ions. The amino acid sequences of AA 79-90 and oth peptides were identified in the same way. As shown in Table 1, it was found that 22 a 20 anti-digestive stable peptides were identified from group II in ALA-0D and ALA-100 respectively, and identified peptides of group III in ALA-0D and ALA-100D were 16 a 10. However, it is difficult to assess if the differences on the number of identified peptid represent a significant difference on digestibility between ALA-0D and ALA-100D.The anti-digestive stable peptides of group II mainly distributed in 32-43 (32-43, 33- Figure 7 shows the an digestive stable peptides in the 3D structure of ALA. It could be seen that most of the an digestive stable peptides were distributed on the α-helix. (A) Figure 5. The KU812 cells degranulation of groups I, II, and III isolated from ALA-0D and ALA-100D. The release of histamine (A) and β-Hex (B). Letters (a-f) demonstrate significant differences (p < 0.05). ALA-0D and ALA-100D: untreated ALA and ultrasound-treated ALA at 100 W/cm 2 digested by simulated gastric juice followed simulated duodenal juice.

Anti-Digestive Stable Peptides Identification
The anti-digestive stable peptides of groups II and III in ALA-0D and ALA-100D were analyzed by HPLC-MS/MS. Figure 6A-D shows the MS and MS/MS spectra of peptides DTQAIVQNNDSTEY (AA 37-50) and KFLDDDLTDDIM (AA 79-90). The m/z of AA 37-50 was 798.3477, and the peptide AA 37-50 was identified according to the difference between the values of b and y ions. The amino acid sequences of AA 79-90 and other peptides were identified in the same way. As shown in Table 1, it was found that 22 and 20 anti-digestive stable peptides were identified from group II in ALA-0D and ALA-100D, respectively, and identified peptides of group III in ALA-0D and ALA-100D were 16 and 10. However, it is difficult to assess if the differences on the number of identified peptides represent a significant difference on digestibility between ALA-0D and ALA-100D.These anti-digestive stable peptides of group II mainly distributed in 32-43 (32- (108) were only discovered in group III of ALA-100D. This might be ultrasound unfolded the structure of ALA, leading peptide bond 98-99 and 107-108 (108-109) to be hydrolyzed and formed peptide 99-107 and 99-108. Figure 7 shows the anti-digestive stable peptides in the 3D structure of ALA. It could be seen that most of the anti-digestive stable peptides were distributed on the α-helix. Letters (a-f) demonstrate significant differences (p < 0.05). ALA-0D and ALA-100D: untreated ALA and ultrasound-treated ALA at 100 W/cm 2 digested by simulated gastric juice followed simulated duodenal juice.

Conclusions
In this work, it was exhibited that ultrasound unfolded the structure of ALA, thereby increasing the IgG/IgE-binding abilities and also raising the release of the bioactive mediators. The anaphylaxis of untreated and ultrasound-treated ALA further increased after digestion, which might be that linear epitopes play an important role in affecting anaphylaxis compared with the conformational epitopes. Furthermore, some linear epitopes were still retained on the anti-digestive stable peptides produced after gastroduodenal digestion. Among gastric/gastroduodenal digesta, the digesta of ultrasound-treated ALA embodied high anaphylaxis. Therefore, ultrasound could increase the anaphylaxis of ALA during digestion.