Reduction of The Histamine Content and Immunoreactivity of Parvalbumin in by Maillarrd Reaction

View Article Online
Food &
View Journal
Function
Linking the chemistry and physics of food with health and nutrition
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: H. Yang, J. Min, X.
Han, X. Li, J. Hu, H. Liu, M.-J. Cao and G. Liu, Food Funct., 2018, DOI: 10.1039/C8FO01167B.
Volume 7 Number 1 January 2016 Pages 1–612 This is an Accepted Manuscript, which has been through the
Food & Royal Society of Chemistry peer review process and has been
accepted for publication.
Function
Linking the chemistry and physics of food with health and nutrition Accepted Manuscripts are published online shortly after
www.rsc.org/foodfunction
acceptance, before technical editing, formatting and proof reading.

Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the

author guidelines.
Please note that technical editing may introduce minor changes

to the text and/or graphics, which may alter content. The journal’s
ISSN 2042-6496 standard Terms & Conditions and the ethical guidelines, outlined
PAPER
in our author and reviewer resource centre, still apply. In no
T. J. Wooster et al.
Impact of gastric pH proﬁles on the proteolytic digestion of mixed
lg-Xanthan biopolymer gels
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
rsc.li/food-function
Page 1 of 33 Food & Function
View Article Online
DOI: 10.1039/C8FO01167B
1 Reduction of the histamine content and immunoreactivity of parvalbumin in
2 Decapterus maruadsi by a Maillard reaction combined with pressure treatment
3
Published on 27 August 2018. Downloaded by Kaohsiung Medical University on 8/29/2018 8:13:56 AM.
4 Huang Yang#, Juan Min#, Xin-Yu Han#, Xiao-Yan Li, Jia-Wei Hu, Hong Liu, Min-Jie Cao,
Food & Function Accepted Manuscript

5 Guang-Ming Liu*
7 College of Food and Biological Engineering, Xiamen Key Laboratory of Marine Functional Food,
8 Fujian Provincial Engineering Technology Research Center of Marine Functional Food, Fujian
9 Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources,
10 Jimei University, 43 Yindou Road, Xiamen, 361021, Fujian, P.R. China.
11
12 Running title: Reduction of the content of histamine and immunoreactivity of parvalbumin
13
#
14 These authors contributed equally
15 Corresponding author:
16 Guang-Ming Liu
17 College of Food and Biological Engineering, Jimei University
18 Tel: +86-592-6180378
19 Fax: +86-592-6180470
20 Email: gmliu@jmu.edu.cn
21
Food & Function Page 2 of 33
View Article Online
DOI: 10.1039/C8FO01167B
22 Abstract
23 The aim of this study was to develop an effective method for decreasing the content
24 of histamine and the immunoreactivity of parvalbumin in Decapterus maruadsi. As

25 demonstrated by reverse phase high performance liquid chromatography, no effect on

26 histamine content was found when fish were treated by boiling (100 °C),
27 ultrasonication, ultraviolet irradiation, pressure treatment (121 °C, 0.12 MPa).
28 However, the histamine content was reduced by 73.55% when the Maillard reaction
29 was combined with pressure treatment (MPT). Further, the allergenicity of
30 parvalbumin was retained after boiling, ultrasonication and ultraviolet irradiation, but
31 was effectively decreased when fish were treated by MPT. Animal experimental
32 results showed lower levels of IgE, IgG1 and IgG2a and contents of serum histamine
33 when measured in a group of MPT sensitized mice. These results showed that the
34 MPT is an effective method for simultaneously reducing the histamine content and
35 the immunoreactivity of parvalbumin from Decapterus maruadsi.
36
37 Keywords: Decapterus maruadsi; Histamine; Maillard reaction combined with
38 pressure treatment; Parvalbumin; Reduction of allergenicity

View Article Online
DOI: 10.1039/C8FO01167B
39 1. Introduction
40 Blue scad (Decapterus maruadsi) is a highly histamine-rich marine fish belonging to
41 the mackerel family. As an important economic seafood in China, its production

42 reached 600,913 tons in 2016.1 The nutritional value of blue scad is high, and the

43 contents of protein and fat are 18.50% and 3.40%, respectively, and include 18 kinds
44 of amino acid.2, 3
However, safety incidents caused by eating fish are common.
45 Histamine fish poisoning, also known as ‘scombroid poisoning’, occurs commonly
46 worldwide. Histamine is a typical biogenic amine, and it is mainly formed by
47 decarboxylation of histidine or by amination and transamination of aldehydes and
48 ketones, as result of bacterial action during storage.4 A level of 50 mg/kg of histamine
49 has been suggested by the Food and Drug Administration (FDA) as a histamine
50 poisoning limiting value in fish.5 Histamine is widely found in fish and processed
51 aquatic products, and it is one of the most powerful biologically active compounds as
52 it exerts many responses within the body, including anaphylaxis and
53 immunoreactions.6, 7 Histamine poisoning can lead to allergy-like food poisoning,
54 with symptoms such as difficulty in breathing, hives, rash, vomiting, fever, facial
55 swelling and hypertension.8, 9
56 It is equally important to note that aquatic products may not only contain
57 histamine, which causes poisoning, but also parvalbumin (PV), which causes allergic
58 reactions in humans. Elsayed and colleagues first identified PV (called Gad c l) from
59 cod (Gadus callarias) as a fish allergen in 1971.10 It was reported that 92.5%11 of
60 individuals who are allergic to fish have serum-specific IgE against PV. PV
View Article Online
DOI: 10.1039/C8FO01167B
61 represents the major allergen of fish, and it is a Ca2+-binding protein with a molecular
62 mass of about 12.5 kDa and an acidic isoelectric point.12 In general, PV can be
63 divided into α-PV and β-PV, and it is β-PV that mostly causes the allergic reaction in
64 fish allergy sufferers.13 It is notable that the symptoms associated with fish histamine

65 poisoning are similar to those of an allergic reaction. This is in fact the link between
66 histamine poisoning and food allergy because the ingestion of exogenous histamine
67 in spoiled fish and the degranulated histamine produced during an allergic reaction
68 share the same pathogenetic mechanism.14 Such histamine-induced reactions are
69 often misdiagnosed as IgE-mediated fish allergy. So it is necessary to prevent both
70 scombroid fish poisoning and food allergy due to fishes at the same time.
71 However, biogenic amines have been reported to be heat stable compounds.15
72 Most research has therefore focused on the prevention of histamine formation in
73 stored fish, by means such as temperature control, modified atmosphere packaging,
74 irradiation, addition of preservatives and so on.16 Pressure treatment (PT) does have
75 an effect on the structure and antigenicity of food allergens,17 high pressure (more
76 than 100 MPa) is high in cost and energy consumption in industrial application,
77 however the condition of canning is a very mature production technology that to
78 increase the pressure and temperature (121 °C, 0.12 MPa). Histamine is difficult to
79 eliminate by pressure once it has formed. In another way, The Maillard reaction (MR)
80 seems to be a more effective way of removing histamine. The glucose/histamine
81 Maillard reaction has been proposed for histamine control,18 and histamine can be
82 almost eliminated under appropriate conditions. Moreover, the IgE binding properties
View Article Online
DOI: 10.1039/C8FO01167B
83 of recombinant silver carp PV were partly weakened after the glucose/histamine
84 Maillard reaction because of its effect on PV’s structure and epitopes.19 However,
85 there have been no reports of an effective way to control the histamine content and
86 eliminate allergenicity during food processing and storage in industrial production.

87 And since previous research was based on different reaction conditions, combining
88 the Maillard reaction with pressure treatment was worth considering.
89 The following study compared the differences in reducing the content of
90 histamine and the immunoreactivity of parvalbumin upon boiling treatment (BT),
91 ultrasonication (US), ultraviolet irradiation (UV), PT and MR. In addition, a cell
92 model was constructed to verify the effects of different food processing methods on
93 the rat mast cell line RBL-2H3. Serum and animal experiments were designed to
94 ensure the effects of the Maillard reaction combined with pressure treatment (MPT)
95 could reduce the histamine content and the allergenicity of PV in blue scad at the
96 same time. The method MPT reduced the risk of scombroid fish poisoning and food
97 allergy, and also provided a technical theoretical foundation for the development of
98 low allergy foods.
99
100 2. Materials and Methods
101 2.1 Materials.
102 Frozen blue scad (Decapterus maruadsi) was purchased from the market of Jimei,
103 Xiamen. Fish without heads, tails and viscera were cut into uniform blocks, and the
104 fish were mashed as a control sample reserve. Each sample contains equal amount of
View Article Online
DOI: 10.1039/C8FO01167B
105 histamine or PV and those samples were stored at -30 °C until further use. Standard
106 histamine and dansyl chloride (Dns-Cl) were purchased from Sigma-Aldrich (St.
107 Louis, MO, USA). Fish allergic serum and normal human negative serum were
108 provided by the local Ethics Committee of the First Affiliated Hospital of Xiamen

109 University (XFH, Xiamen, Fujian, China). This study was approved by the XFH
110 Institutional Review Board (XFH2017-EAN014). The patients had a histories of fish
111 anaphylaxis with clear fish-exposure-related symptoms. Their specific IgE antibodies
112 to fish was quantified in vitro with ImmunoCAP (Phadia AB, Uppsala, Sweden) in
113 Table 1. All sera were stored at -30 °C until analysis. Goat anti-rabbit IgG antibody
114 and goat anti-human IgE antibody were provided by Kirkegaard and Perry
115 Laboratories (Gaithersburg, MD, USA). Alum adjuvant was provided by Pierce
116 Biotechnology Inc (Rockford, IL, USA). Unless otherwise indicated, all other
117 chemicals and solvents used were of analytical and chromatographic grade,
118 respectively.
119 2.2 Different processing methods.
120 Various methods were used to evaluate their effects on reducing the histamine and PV
121 contents of blue scad.
122 Boiling treatment: Samples of the blue scad that had been mashed were boiled
123 at 100 °C for 180 min.
124 Ultrasonic treatment: Samples were placed in the ultrasonicator (200 W) for 5
125 h.
126 Ultraviolet treatment: Samples were placed under an ultraviolet lamp for 5 h,
View Article Online
DOI: 10.1039/C8FO01167B
127 with wavelength and power settings of 302 nm and 15 W, respectively.
128 Pressure treatment: Samples were placed in the autoclave sterilizer (121 °C,
129 0.12 MPa) for 20 min as the condition of canning.

130 Maillard reaction treatment: The reaction conditions of Maillard reaction

131 referred to the method of Zhao et al19，and it has been perfected. Samples and 4%
132 glucose solution (v/v, 1:1) were mixed, and then placed in a water bath at 100 °C for
133 30 min (pH 7.0).
134 Maillard reaction combined with pressure treatment: Samples and 4%
135 glucose solution (v/v, 1:1) were mixed. Then the samples were placed in the
136 autoclave sterilizer (121 °C, 0.12 MPa) for 20 min.
137 2.3 Detection of the histamine content.
138 Histamine was quantified using reverse phase high performance liquid
139 chromatography (RP-HPLC) after pre-column derivatization with Dns-Cl.20 A 5-g
140 portion of each fish sample (BT, US, UV, PT, MR, MPT) after homogenization was
141 mixed with 15 mL of trichloroacetic acid (5%, w/v, TCA) in a 50-mL centrifuge tube.
142 Then the homogenate was centrifuged at 10,000 g for 15 min at 4 °C. The
143 supernatant was decanted into a 25-mL volumetric flask. Next, 10 mL TCA (5%) was
144 added to the sediment and the mixture was centrifuged as before. The supernatants
145 were combined in a 25-mL volumetric flask and diluted to the mark with 5% TCA.
146 An aliquot of 300 µL of standard histamine solution (and each extract) was
147 filtered through a water filter membrane. Forty microliter of sodium hydroxide (2 M)
148 and 600 µL of Dns-Cl solution (10 mg/mL in acetone) were gently added to the 2-mL
View Article Online
DOI: 10.1039/C8FO01167B
149 centrifuge tube. The mixture was incubated in the dark at 40 °C for 20 min. After the
150 reaction, the sample was mixed with 100 µL ammonia at room temperature for 30
151 min. Finally, the volume of the reaction mixture was adjusted to 1.5 mL with
152 acetonitrile. The solution was filtered through a 0.22-µm organic filter membrane

153 prior to RT-HPLC analysis.
154 Samples were injected into an RP-HPLC to analyze the histamine
155 concentration.21 The RP-HPLC system consisted of an Agilent 1260 HPLC unit, a
156 pump, a standard automatic sampler and a variable-wavelength detector from Agilent
157 (Palo Alto, CA, USA).The Zorbax Extend-C18 column (5 µm, 4.6 mm I.D. × 150
158 mm; Agilent) was used as the analytical chromatography column. An aliquot of 20
159 µL was injected into the solvent delivery system. The mobile phase of the isocratic
160 elution system was a mixture of water (solvent A) and acetonitrile (solvent B). The
161 flow rate was 1.0 mL/min and the temperature was set at 35 °C. The derivatized
162 histamine was detected at 254 nm. The solvent A/solvent B ratio was held constant at
163 30:70 (v/v) for 7 min. All experiments were performed in duplicate three times at
164 least.
165 2.4 Detection of the immunoreactivity of parvalbumin.
166 The crude sample for the detection of fish PV was prepared using a previously
167 published method.22 A 5-g portion of each fish sample (untreated, BT, US, UV, PT,
168 MR, MPT) was mixed with 15 mL ultrapure water. After homogenization, each
169 homogenate was centrifuged at 12,000 g at 4 °C for 20 min. The supernatants
170 contained the crude protein that was filtered through four layers of silk cloth and
View Article Online
DOI: 10.1039/C8FO01167B
171 stored at 4 °C. The PV in blue scad was purified by heat treatment and
172 DEAE-Sepharose chromatography by the method of Liu et al.23
173 SDS-PAGE and Western blot were conducted according to a previous method
174 with minor modifications.24 All experiments were duplicated, and the variation

175 among three assays was always less than 5%. Analysis of the IgG-/IgE-binding
176 activity by Western blotting/dot blotting was carried out as previously described,19
177 with minor modifications. Briefly, proteins in the polyacrylamide gels were
178 electrophoretically transferred onto a nitrocellulose (NC) membrane. Then, the NC
179 membranes were blocked with 5% skim milk and incubated with rabbit-PV IgG
180 antibodies or human serum (1:4 dilution). The immunoassay was carried out using
181 ECL after incubation with HRP-anti-mouse IgG antibody (1:20,000 dilution) or
182 HRP-anti-human IgE antibody (1:2,000 dilution).
183 2.5 Rat basophilic leukemia cell assay.
184 The rat basophilic leukemia mast cell line (RBL-2H3) was obtained from the
185 American Type Culture Collection (Bethesda, MD, USA) and grown in essential
186 medium with 10% fetal bovine serum and 1% MEM/EBSS at 37 °C. The final cell
187 count was 1 × 106 cell/ mL.
188 The release rate of β-hexosaminidase from RBL-2H3 cells was measured in a
189 model of an IgE-mediated mast cell allergic reaction.25 Sensitized mouse sera (mouse
190 blood from a group sensitized to PV collected on day 15) were stimulated by PV or
191 PV treated by different processing methods (500 ng/mL) for 6 h or 15 min at 37 °C,
View Article Online
DOI: 10.1039/C8FO01167B
192 respectively. The cell supernatant was used to measure the release levels of
193 β-hexosaminidase and histamine using an ELISA kit from IBL (Hamburg, Germany).
194 2.6 Detection of the histamine content and the immunoreactivity of PV in vivo.
195 Female Balb/c mice were purchased from Slaccas Lab Animal Ltd. (Shanghai, China)

196 at 6-8 weeks old. Mice were housed in a specific pathogen-free barrier room (room
197 temperature 23 ± 2 °C, relative humidity 50–70%, 12 h/12 h light/dark cycle). Food
198 and water were available ad libitum. Experiments were performed in conformity with
199 the laws and regulations for treatment of live animals of Jimei University, SCXK
200 2012−0005.
201 Fish samples that contained a low content of histamine were designated FLH;
202 fish samples that contained a high content of histamine were designated FHH. Each
203 group of mice received a different sample, including PBS, FLH, FHH, or FLH, FHH
204 treated by MPT, via intragastric gavage (3 mg/mouse, n=6). Blood was obtained after
205 30 min. Histamine levels in the sera were determined using commercially available
206 ELISA kits from IBL following the manufacturer's instructions.
207 Another batch of mice were exposed to 150 µL of purified PV (150 µg/mouse,
208 n=6) with alum adjuvant by intraperitoneal (i.p.) injection on days 0 and 14. Mouse
209 blood was collected on day 15 after immunization. The sera from individual mice
210 were stored at -30 °C until analysis. On days 21 and 28, mice received 2 mg PV or
211 MPT-treated parvalbumin (MPT-PV) via intragastric gavage (3 mg/mouse, n=6). The
212 samples were dialysed against 10 mM PBS (pH 7.0) after the MRT to remove the
213 remaining reducing sugar. Rectal temperatures were measured for 30 min after the
View Article Online
DOI: 10.1039/C8FO01167B
214 challenge on day 28. Blood was obtained on day 29, and the allergen-specific
215 antibody levels were determined. Also, histamine in the serum was determined.
216 B/T cells were isolated from the spleens of mice on day 29 and stained for CD3
217 and CD19. Cells were cultured in the presence of 25 µg/mL anti-CD3-APC and 25

218 µg/mL anti-CD19 for 30 min, and analyzed by flow cytometry. All flow cytometry
219 experiments were performed using the Guava easyCyte 6-2L system and GuavaSoft
220 3.1.1 software.
221 2.7 Statistical analysis.
222 The reported results were obtained using the software SPSS statistics 17.0 from IBM
223 (Armonk, New York, USA). The differences among samples at different
224 measurement points were determined by one-way analysis of variance (ANOVA) for
225 independent samples at a probability level of 0.05 (P < 0.05). The collection of fish
226 samples spanned many seasons with good reproducibility and the experiments were
227 performed in triplicate and the data are expressed as average ± standard deviation.
228
229 3. Results
230 3.1 Effects of different processing methods on the histamine content.
231 The results for chromatograms of histamine showed that the elution time of histamine
232 was 5.5 min, with a standard peak shape. The linear relationship between peak area
233 and histamine content was good when the histamine content was in the range of
234 1-500 µg/mL (Fig. 1A).

View Article Online
DOI: 10.1039/C8FO01167B
235 First, we investigated the histamine content resulting from different processing
236 methods (Fig. 1B). Samples that were treated by BT, US, UV and PT showed an
237 insignificant effect on the level of histamine compared with the untreated sample
238 (P > 0.05). But the histamine content was reduced significantly by the Maillard

239 reaction (P < 0.01).
240 In fact, the histamine content decreased significantly as the Maillard reaction
241 proceeded. When the Maillard reaction was combined with pressure treatment, the
242 amount of histamine was reduced by 73.55%, and its content was 27.45 mg/kg. These
243 result suggested that an appropriate glucose concentration is critical for the activation
244 of the Maillard reaction.
245 3.2 Effects of different processing methods on the IgG-/IgE-binding activities of
246 parvalbumin.
247 The fish proteins extracted from blue scad after the different processes were analyzed
248 by SDS-PAGE. PV appeared as a band corresponding to a molecular mass of 12.50
249 kDa (Fig. 2A), and did not change significantly after US or UV treatment. Fig. 2A
250 shows that the migration of objective protein bands was obvious in the experimental
251 groups treated by BT and MR, and the protein bands became diffuse for the
252 experimental groups of PT and MPT.
253 Whether processing time shifts caused by different processing methods affected
254 the IgG/IgE binding activity of PV was then determined. The IgG-binding activity of
255 PV was evaluated with Western blotting using anti-fish PV rabbit antibody. The
256 IgG-binding capacities were essentially identical to that of the untreated protein for
View Article Online
DOI: 10.1039/C8FO01167B
257 samples treated by BT, US and UV. Meanwhile, the Maillard reaction was effective
258 in reducing the IgG-binding capacity. However, the maximum effect on the
259 IgG-binding capacity of PV was caused by the PT and MPT processes, which both
260 decreased binding. Fig. 2C shows the PV binding capacity of IgE from the pooled

261 sera (Table 1) of fish allergic patients. When PV was processed by PT or MPT, the
262 inhibitory effect on IgE binding was very obvious, whereas the other experimental
263 groups still displayed substantial immunoreactivity.
264 3.3 Effects of processing on the degranulation of RBL-2H3 cells.
265 The effects of PV allergenicity after the different processing methods on the
266 degranulation of RBL-2H3 cells are shown in Fig. 2D. The β-hexosaminidase release
267 rate of the negative control (PBS) was 13.24%, and the β-hexosaminidase release rate
268 of the positive control (raw fish) was 45.46%. The β-hexosaminidase release rates
269 were 42.50%, 45.39% and 44.26% (P > 0.05) when the fish were processed by BT,
270 US and UV methods, respectively. In contrast, the β-hexosaminidase release rates
271 were 17.10%, 28.33% and 15.67% (P < 0.01) when the fish were processed by PT,
272 MR and MPT, respectively.
273 3.4 The toxicity of Maillard-reacted histamine in vivo.
274 The toxicity potential of Maillard-reacted histamine was tested in a histamine
275 experimental protocol (Fig. 3A). The histamine contents in the fish extracts of FLH,
276 FHH and their Maillard-reacted products were 14.01, 150.51, 6.08 and 11.99 mg/kg,
277 respectively (determined by RP-HPLC). These results suggested that the level of
278 histamine (Fig. 3B) was significantly higher in the group of mice fed with spoiled
View Article Online
DOI: 10.1039/C8FO01167B
279 fish extracts, and the histamine came back to a normal level after the Maillard
280 reaction. It was also noted that mice had diarrhea only after feeding with spoiled fish
281 extracts, but there were no significant differences in body temperature or body
282 weight.

283 3.5 Allergic responses after intragastric exposure to Maillard-reacted
284 parvalbumin.
285 The sensitizing and allergenic potential of food allergens is commonly tested in
286 different animal models of food allergy.26-28 Therefore, a challenge animal model of
287 fish allergy was used to test the allergenicity of MR-PV in vivo (Fig. 4A). First, the
288 rectal temperature is commonly used to assess anaphylaxis in mice. Significant
289 differences in the above index were identified among these mouse groups (Fig. 4B).
290 Second, exposure of the mice to PV led to a similar increase in the levels of
291 PV-specific IgE in their serum. The levels of IgE, IgG1 and IgG2a (Fig. 4C-E) after
292 re-stimulation with PV were higher than those of PBS-exposed mice. The levels of
293 IgE, IgG1 and IgG2a in MR-PV-exposed mice were reduced. What is more, the level
294 of histamine was significantly lower in MR-PV-exposed mice compared to
295 PV-exposed mice (Fig. 4F).
296 Among the splenocytes (Fig. 5), the percentage of CD19+ B cells increased
297 from 45.06% (PBS group) to 78.22% (PV group). However, the amount of B cells in
298 the MPT-PV group was significantly decreased by 28.13% compared with the PV
299 group.
300
View Article Online
DOI: 10.1039/C8FO01167B
301 4. Discussion
302 Histamine poisoning is the most common cause of scombroid fish poisoning
303 worldwide and results from the ingestion of histamine-contaminated fish. At the same
304 time, fish is a highly allergenic food responsible for inducing allergic reactions in

305 both children and adults. However, in spite of the high prevalence of histamine fish
306 poisoning and fish allergy, there is a lack of in-depth knowledge of ways of reducing
307 and controling the associated risks in the food industry.
308 It is important to note that, once histamine is formed, it is highly resistant to
309 modification, so cooking, smoking and freezing cannot prevent histamine fish
310 poisoning reactions. In this study, we found that the histamine content was stable
311 when it was extracted from fish treated by HT, PT, UV and US, and the level of
312 histamine was not decreased by simple physical processing methods (Fig. 1B).
313 However, the Maillard reaction could reduce histamine levels effectively. When
314 glucose was added to the fish, a Maillard reaction that was affected by the
315 temperature, concentration of glucose and reaction time could reduce the amount of
316 histamine.29 Although the Maillard reaction of Free Amino acids in food processing is
317 the manifestation of nutrient los，fortunately, fish are rich in proteins that can provide
318 essential amino acids for the body after digestion in the gastrointestinal tract.
319 On the other hand, PV is the main allergen in fish.30 Firstly, we explorated the
320 effects of the processing methods which shifted the IgG/IgE-binding activity of PV.
321 The IgG binding activity of PV was not reduced after boiling (100 °C), due to the
322 presence of Ca2+ in the PV structure which can stabilize the tertiary structure of PV.
View Article Online
DOI: 10.1039/C8FO01167B
323 In our study, MPT was the most effective processing method. On the one hand,
324 intensively dry heated has been reported to affect the structures of allergenic
325 proteins.31 On the other hand, according to research,32, 33 the Maillard reaction can
326 reduce the allergenicity of PV effectively. After a Maillard reaction with glucose, PV

327 position was rising and dispersion, along with the degree of the Maillard reaction.
328 During the Maillard reaction the antigenic epitopes of PV may have been inhibited or
329 masked according to Almond.34 The Maillard reaction has many benefits in fish
330 processing，such as improving sensory flavor and color of food. Furmore, the
331 glycated fish peptide is good for the intestinal flora.35
332 To verify the influence of the MPT treatment on PV immunoreactivity in vivo, a
333 widely used mast mice model was then constructed. Type I hypersensitivity is
334 mediated by IgE binding with FcεRI, which is located on the surface of mast cells.
335 Activated mast cells will release various mediators, including histamine,
336 β-hexosaminidase and other proinflammatory cytokines.12 The serum IgE antibodies
337 of PV were obtained by injecting PV intraperitoneally into mice.36 The effect of PV
338 on the degranulation of RBL-2H3 cells was studied by the cell-allergic model.37 We
339 found that less β-hexosaminidase was released in the group treated with MPT. These
340 results suggested that PV binds to glucose through the Maillard reaction at canned
341 processing conditions, which influences the structures of histamine and PV. It was
342 noted that MPT was the best method for reducing β-hexosaminidase release, so MPT
343 was considered in subsequent animal experiments. The regulation of IgE production
344 by B cells is very poorly understood. In this study, we found that the total percentage
View Article Online
DOI: 10.1039/C8FO01167B
345 of B cells was still higher in MPT-PV sensitized mice, which explains the higher
346 levels of IgE than in the group administered PBS. More clinical data may be needed
347 to verify the safety of low-sensitivity products so far, we hope to see such products
348 on the market at an early date for the benefit of allergic consumers. There is a wide

349 market for low sensitivity products，to date, fish with low sensitivity products are
350 relatively rare.
351 In addition, different animal models were constructed to analyze the difference
352 between histamine poisoning (scombroid food poisoning) and fish allergy. Fish with
353 higher histamine content may cause a more severe allergic reaction, so this part of the
354 experiment was already being planned. Histamine fish poisoning is usually mild, of
355 short duration and self-limiting.38 Given that symptoms result from excess amounts
356 of histamine, the physical manifestations of histamine fish poisoning are similar to
357 those of an allergic reaction. Ideally, fish could be processed by the Maillard reaction
358 so that the bacteria cannot grow and histidine decarboxylase is not activated. It is
359 necessary to analyze these two homologous risks，which can help consumers identify
360 the root causes of adverse food reactions.
361
362 5. Conclusions
363 In this study, on the basis of the method of the Maillard reaction combined with
364 pressure treatment, a canned processing method for Decapterus maruadsi was
365 developed and a Chinese patent (No. CN104855489A) was applied for. In general, a
366 method was found to control the histamine content of and reduce the PV
View Article Online
DOI: 10.1039/C8FO01167B
367 hypersensitivity caused by fish, and animal experiments showed concurrent
368 reductions in the intake of extraneous histamine and degranulation release in vivo.
369 This method provides a new basis for producing low allergy fish products in the food
370 industry.

371
372 Conflicts of interest
373 There are no conflicts to declare.
374
375 Acknowledgements
376 This work was supported by grants from the National Natural Scientific
377 Foundation of China (U1405214), the Marine Scientific Research Special Foundation
378 for Public Sector Program (201505026-03).
379
380 Abbreviations:
381 BT, boiling treatment;
382 FLH, the fish contained low content of histamine;
383 FHH, the fish contained high content of histamine;
384 PT, pressure treatment ;
385 MR, Maillard reaction;
386 MPT: Maillard reaction combined with pressure treatment ;
387 PV, parvalbumin;
388 US, ultrasonic;

Page 19 of 33
389
UV, ultraviolet;;
Food & Function
DOI: 10.1039/C8FO01167B
View Article Online

View Article Online
DOI: 10.1039/C8FO01167B
390 References
391 1 Anonymous China fisheries yearbook. China Agriculture Press, 2017, 41.
392 2 C. Zhong, Q. F. Cai, G. M. Liu, L. C. Sun, K. Hara, W. J. Su and M. J. Cao, Purification and
393 characterisation of cathepsin L from the skeletal muscle of blue scad (Decapterus maruadsi)

394 and comparison of its role with myofibril-bound serine proteinase in the degradation of
395 myofibrillar proteins. Food Chem. 2012, 133, 1560–1568.
396 3 A. Artharn, S. Benjakul, T. Prodpran and M. Tanaka, Properties of a protein-based film from
397 round scad (Decapterus maruadsi) as affected by muscle types and washing. Food Chem.
398 2007, 103, 867-874.
399 4 S. Qiu, Y. Wan, Y. Cheng, Y. Liu, M. P. Yadav and L. J. Yin, Reduction of biogenic amines in
400 sufu by ethanol addition during ripening stage. Food Chem. 2017, 239, 1244-1252.
401 5 Decomposition and histamine in raw, frozen tuna and mahi-mahi, canned tuna and related
402 species. Compliance Policy Guide. 7108.240. FDA. 1996.
403 6 L. O. Mahony, M. Akdis and C. A. Akdis, Regulation of the immune response and
404 inflammation by histamine and histamine receptors. J. Allergy Clin. Immunol. 2011, 128,
405 1153-1164.
406 7 C. A. Akdis, K. Blaser, Histamine in the immune regulation of allergic inflammation. J.
407 Allergy Clin. Immunol. 2003, 112, 15-22.
408 8 C. Feng, S. Teuber and M. E. Gershwin, Histamine (Scombroid) Fish Poisoning: a
409 Comprehensive Review. Clin. Rev. Allergy Immunol. 2016, 50, 64-69.
410 9 A. Naila, S. Flint, G. Fletcher, P. Bremer and G. Meerdink, Control of Biogenic Amines in
411 Food-Existing and Emerging Approaches. J. Food Sci. 2010, 75, 139-150.
View Article Online
DOI: 10.1039/C8FO01167B
412 10 Y. Ma, U. Griesmeier, M. Susani, C. Radauer, P. Briza, A. Erler, M. Bublin, S. Alessandri, M.
413 Himly, S. Vczquez-Cortcbs, I. S. Rincon-de-Arellano, E. Vassilopoulou and P.
414 Saxoni-Papageorgiou, Comparison of natural and recombinant forms of the major fish
415 allergen parvalbumin from cod and carp. Mol. Nutr. Food Res. 2008, 52, 196-207.

416 11 M. F. Sharp, J. N. Stephen, L. Kraft, T. Weiss, S. D. Kamath and A. L. Lopata,
417 Immunological cross-reactivity between four distant parvalbuminsimpact on allergen
418 detection and diagnostics. Mol. Immunol. 2015, 63, 437-448
419 12 A. Kuehn, I. Swoboda, K. Arumugam, C. Hilger and F. Hentges, Fish allergens at a glance:
420 Variable allergenicity of parvalbumins, the major fish allergens. Front. Immunol. 2014, 5,
421 1-8.
422 13 Y. Hamada, T. A. Sato, S. I. Shizaki, Y. Nagashima and K. Shiomi, Expression and evaluation
423 of IgE-binding capacity of recombinant pacific mackerel parvalbumin. Allergology Int. 2004,
424 53, 271-278.
425 14 E. Ridolo, I. Martignago, G. Senna and G. Ricci, Scombroid syndrome: it seems to be fish
426 allergy but. it isn't. Curr. Opin. Allergy Clin. Immunol. 2016, 16, 516-521.
427 15 W. Tapingkae, S. Tanasupawat, K. L. Parkin, S. Benjaku and W. Visessanguan, Degradation
428 of histamine by extremely halophilic archaea isolated from high salt-fermented fishery
429 products. Enzyme Microb. Tech. 2010, 46, 92-99.
430 16 Y. Hu, Z. Huang, J. Li and H. Yang, Concentrations of biogenic amines in fish, squid and
431 octopus and their changes during storage. Food Chem. 2012, 135, 2604-2611.
432 17 J. Somkuti, and L. Smeller, High pressure effects on allergen food proteins. Biophys Chem.
433 2013, 183, 19-29.

View Article Online
DOI: 10.1039/C8FO01167B
434 18 W. Jiang, X. X. He, H. C. Yang, X. W. Xiang, S. W. Hu, S. J. Li and Y. Liu, Histamine
435 reduction by Maillard reaction with glucose. Food Control, 2017, 136-144.
436 19 Y. J. Zhao, Q. F. Cai, T. C. Jin, L. J. Zhang, L. J. Zhang, D. X. Fei, G. M. Liu and M. J. Cao,
437 Effect of Maillard reaction on the structural and immunological properties of recombinant

438 silver carp parvalbumin. LWT−Food Sci. Technol, 2017, 75, 25-33.
439 20 M. Saaid, B. Saad and N. H. Hashim, Abdussalamsalhin, M. A., Muhammadidiris, S.
440 Determination of biogenic amines in selected malaysian food. Food Chem. 2009, 113,
441 1356-1362.
442 21 J. W. Hu, M .J. Cao, S. C. Guo, L. J. Zhang, W. J. Su and G. M. Liu, Identification and
443 inhibition of histamine forming bacteria in blue scad (Decapterus maruadsi) and chub
444 mackerel (Scomber japonicus). J. Food Protect. 2015, 78, 383–389.
445 22 G. M. Liu, N. Wang, Q. F. Cai, T. Li, L. C. Sun, W. J. Su and M. J. Cao, Purification and
446 characterization of parvalbumins from silver carp (Hypophthalmichthy molitris). J. Sci. Food
447 Agr, 2010, 90, 1034-1040.
448 23 M. J. Cao, L. L. Guo, L. J. Zhang, W. J. Su, Y. L. Liang and G. M. Liu, Purification and
449 Allergenicity Identification of Carp Parvalbumin. Food Sci, 2009, 30, 187-190.
450 24 Y. Shen, M. J. Cao, Q. F Cai, W. J. Su, H. L. Yu, W. W. Ruan and G. M. Liu, Purification,
451 cloning, expression and immunological analysis of Scylla serrata arginine kinase, the crab
452 allergen. J. Sci. Food Agr. 2011, 91, 1326-1335.
453 25 N. J. Plundrich, R. R. Bansode, E. A. Foegeding, L. L. Williams and M. A. Lila,
454 Protein-bound Vaccinium fruit polyphenols decrease IgE binding to peanut allergens and
455 RBL-2H3 mast cell degranulation in vitro. Food Funct. 2017, 8, 1611-1621.
View Article Online
DOI: 10.1039/C8FO01167B
456 26 M. Bol-Schoenmakers, R. Bleumink, M. Marcondes-Rezende, E. Mouser, I. Hassing, I.
457 Ludwig, J. J. Smit and R. H. H. Pieters, Diclofenac enhances allergic responses in a mouse
458 peanut allergy model. Clin. Exp. Allergy. 2011, 41, 424–433.
459 27 F. Van-Wijk, S. Nierkens, W. de-Jong, E. J. Wehrens, L. Boon, P. van-Kooten, L. M. Knippels

460 and R. Pieters, The CD28/CTLA-4-B7 signaling pathway is involved in both allergic
461 sensitization and tolerance induction to orally administered peanut proteins. J. Immunol.
462 2007, 178, 6894 - 6900.
463 28 V. Ahuja, M. Quatchadze, D. Stelter, A. Albrecht and R. Stahlmann, Evaluation of
464 biotechnology-derived novel proteins for the risk of food-allergic potential: advances in the
465 development of animal models and future challenges. Arch. Toxicol. 2010, 84, 909 - 917.
466 29 Z. Jiang, L. Wang, H. Che and B. Tian, Effect of temperature and pH on
467 angiotensin-I-converting enzyme inhibitory activity and physicochemical properties of
468 bovine casein peptide in aqueous Maillard reaction system. LWT-Food Sci. Technol. 2014,
469 59, 35-42.
470 30 L. Tordesillas, M. C. Berin and H. A. Sampson, Immunology of Food Allergy. Immunity.
471 2017, 47, 32-50.
472 31 F. Liua, M. Teodorowicz, M. A. J. S. van-Boekela, H. J. Wichersbc and K. A. Hettinga, The
473 decrease in the IgG-binding capacity of intensively dry heated whey proteins is associated
474 with intense Maillard reaction, structural changes of the proteins and formation of
475 RAGE-ligands. Food Funct. 2016, 7, 239-249.
476 32 T. Takai, E. Mizuuchi, Y. Kikuchi, T. Nagamune, K. Okumura and H. Ogawa, Glycosylation
477 of Recombinant Proforms of Major House Dust Mite Allergens Der p 1 and Der f 1
View Article Online
DOI: 10.1039/C8FO01167B
478 Decelerates the Speed of Maturation. Int. Arch. Allergy Imm. 2006, 139, 181-187.
479 33 H. J. Jongh, S. L. Taylor and S. J. Koppelman, Controlling the aggregation propensity and
480 thereby digestibility of allergens by maillardation as illustrated for cod fish parvalbumin. J.
481 Biosci. Bioeng. 2011, 111, 204-211.

482 34 R. J. Almond, B. F. Flanagan, A. Antonopoulos, S. M. Haslam, A. Dell, I. Kimber and R. J.
483 Dearman, Differential immunogenicity and allergenicity of native and recombinant human
484 lactoferrins: role of glycosylation. Eur. Immunol. 2013, 43, 170-181.
485 35 W. Jin, K. Han, S. Dong, Y. Yang, Z. Mao, M. Su and M. Zeng, Modifications in gut
486 microbiota and fermentation metabolites in the hindgut of rats after the consumption of
487 galactooligosaccharide glycated with a fish peptide. Food Funct. 2018.
488 36 K. D. Garcia-Orozco, E. Aispuro-Hernandez, G. Yepiz-Plascencia, A. M. Calderondelabarca
489 and R. R. Sotelomundo, Molecular characterization of arginine kinase, an allergen from the
490 shrimp (Litopenaeus vannamei). Int. Arch. Allergy Imm. 2007, 144, 535-542.
491 37 J. A. Warner, C. Kroegel, Pulmonary immune cells in health and disease: mast cells and
492 basophils. Eur. Respir. J. 1994, 7, 1326-1341.
493 38 C. Feng, S. Teuber and M. E. Gershwi, Histamine (Scombroid) Fish Poisoning: a
494 Comprehensive Review. Clin. Rev. Allergy Immunol. 2015, 50, 64-69.
495
View Article Online
DOI: 10.1039/C8FO01167B
496 Table 1. Specific IgE levels in patients in relation to the fish.
497 F: female, M: male, A serum with the specific IgE > 0.35 (kU/L) is defined as positive.
498
499 Figure legends
500 Fig. 1. The effects of different processing methods in reducing histamine by RP-HPLC.

501 A, A linear relationship between peak area and histamine content was demonstrated when the
502 content of histamine was 1-500 µg/mL.
503 B, The histamine concentration in a sample was determined using reverse phase high
504 performance liquid chromatograph (RP-HPLC).
505 CON: negative control; BT: boiling treatment; US: ultrasonic; UV: ultraviolet; PT: pressure
506 treatment; MR: Maillard reaction; MPT: Maillard reaction combined with pressure
507 treatment.
508 *P < 0.05, **P < 0.01. The data represent the mean ± SD of triplicate determinations.
509
510 Fig. 2. The effects of different processing on reducing PV.
511 A-B, SDS-PAGE or Western blot of the effects of different processing methodsl in reducing PV.
512 Using the rabbit anti-PV IgG antibody.
513 C, Dot blot showing the effects of different processing methods in reducing PV. Patient serum
514 pool was used as the antibody.
515 D, The effects of different processing methods on stimulation of rat basophil (RBL-2H3) release
516 by β-hexosaminidase. β-Hexosaminidase release rate was calculated as (A1 - A2)/[(A1 - A2)
517 +(A3 - A4)], in which A is the absorbance of each well, A1 is the absorbance of the
518 supernatant well, A2 is the absorbance of a blank of supernatant, A3 is the absorbance of cell
519 lysate, A4 is the absorbance of a blank of cell lysate.
520 Con: negative control; BT: boiling treatment; US: ultrasonic; UV: ultraviolet; PT: pressure
521 treatment; MR: Maillard reaction; MPT: Maillard reaction combined with pressure
522 treatment.
View Article Online
DOI: 10.1039/C8FO01167B
524
525 Fig. 3. The effect of the Maillard product in the mouse histamine poisoning experiment.
526 A, Histamine poisoning experimental protocol. Six- to eight-week-old female mice were used.
527 Each group of mice received a different sample, including PBS, and the fish contained a low
528 content of histamine (FLH) or a high content of histamine (FHH). FLH or FHH were treated

529 by MPT via intragastric gavage (3 mg/mouse, n=6), and the blood was obtained after 30
530 min.
531 B, The levels of histamine in the sera were measured.
533
534 Fig.. 4. Sensitizing potential in the mouse sensitization experiment.
535 A, Sensitization protocol. Mice were exposed to 150 µL of purified PV (150 µg/mouse, n=6) with
536 alum adjuvant by intraperitoneal (i.p.) injection on days 0 and 14. Mice received 2 mg PV or
537 the PV after the Maillard reaction with pressure treatment (MPT-PV) via intragastric gavage
538 (3 mg/mouse, n=6) on days 21 and 28. Rectal temperatures were measured after the
539 challenge for 30 min on day 28. Blood was obtained on day 29.
540 B, The body temperature was measured at 30 min after intragastric gavage on day 28.
541 C-E, The levels of IgE, IgG1 and IgG2a were measured on day 29.
542 F, The release of histamine was measured in the serum.
544
545 Fig. 5. The influence of MPT products on B/T cells.
546 B/T cells were isolated from the spleens of mice on day 29 and stained for CD3 and CD19 cells.
547 The cells were cultured in the presence of 25 µg/mL anti-CD3-APC and 25 µg/mL
548 anti-CD19 for 30 min.
549
View Article Online
DOI: 10.1039/C8FO01167B
550 Table 1. Specific IgE levels in patients in relation to the fish.
Age Specific IgE

No. ID Sex Symptom
(years) level (kU/L)
1 839758 25 M 0.16 Cough

2 856386 38 F 0.10 Influenza
3 422384 5 M 0.47 Lung shadows

4 598870 45 M 0.70 Bronchial asthma
5 556766 21 M 1.10 Bronchial asthma
6 437753 35 F 1.10 Edema
7 568825 66 F 1.50 Bronchial asthma
8 307351 51 F 1.80 Allergic purpura
9 492982 4 M 2.20 Cough

Congenital
10 319293 3 M 2.20
Asthma
11 349416 75 F 2.80 Edema
12 413340 5 F 3.60 Bronchitis
13 483373 14 M 5.60 Edema
Cough variant
14 484581 1 M 7.60
asthma
15 494031 1 F 9.60 Edema
16 383250 2 M 10.00 Edema
17 480821 10 M 78.00 Allergic purpura
551 F: female, M: male, A serum with the specific IgE > 0.35 (kU/L) is defined as positive.
552
554
553
Figure 1
Food & Function
DOI: 10.1039/C8FO01167B
View Article Online

Page 28 of 33
Page 29 of 33
556
555
Figure 2
Food & Function
DOI: 10.1039/C8FO01167B
View Article Online

558
557
Figure 3
Food & Function
DOI: 10.1039/C8FO01167B
View Article Online

Page 30 of 33
Page 31 of 33
560
559
Figure 4
Food & Function
DOI: 10.1039/C8FO01167B
View Article Online

562
561
Figure 5
Food & Function
DOI: 10.1039/C8FO01167B
View Article Online

Page 32 of 33
Page 33 of 33
564
563
Graphical Abstract
Food & Function
DOI: 10.1039/C8FO01167B
View Article Online

Reduction of The Histamine Content and Immunoreactivity of Parvalbumin in by Maillarrd Reaction

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Reduction of The Histamine Content and Immunoreactivity of Parvalbumin in by Maillarrd Reaction

Uploaded by

Copyright:

Available Formats

View Article Online

acceptance, before technical editing, formatting and proof reading.

You can find more information about Accepted Manuscripts in the

Please note that technical editing may introduce minor changes

1 Reduction of the histamine content and immunoreactivity of parvalbumin in

2 Decapterus maruadsi by a Maillard reaction combined with pressure treatment

Food & Function Accepted Manuscript

10 Jimei University, 43 Yindou Road, Xiamen, 361021, Fujian, P.R. China.

12 Running title: Reduction of the content of histamine and immunoreactivity of parvalbumin

17 College of Food and Biological Engineering, Jimei University

24 of histamine and the immunoreactivity of parvalbumin in Decapterus maruadsi. As

25 demonstrated by reverse phase high performance liquid chromatography, no effect on

Food & Function Accepted Manuscript

27 ultrasonication, ultraviolet irradiation, pressure treatment (121 °C, 0.12 MPa).

29 was combined with pressure treatment (MPT). Further, the allergenicity of

35 the immunoreactivity of parvalbumin from Decapterus maruadsi.

37 Keywords: Decapterus maruadsi; Histamine; Maillard reaction combined with

38 pressure treatment; Parvalbumin; Reduction of allergenicity

40 Blue scad (Decapterus maruadsi) is a highly histamine-rich marine fish belonging to

41 the mackerel family. As an important economic seafood in China, its production

Food & Function Accepted Manuscript

45 Histamine fish poisoning, also known as ‘scombroid poisoning’, occurs commonly

46 worldwide. Histamine is a typical biogenic amine, and it is mainly formed by

47 decarboxylation of histidine or by amination and transamination of aldehydes and

48 ketones, as result of bacterial action during storage.4 A level of 50 mg/kg of histamine

52 it exerts many responses within the body, including anaphylaxis and

53 immunoreactions.6, 7 Histamine poisoning can lead to allergy-like food poisoning,

55 swelling and hypertension.8, 9

Food & Function Accepted Manuscript

68 share the same pathogenetic mechanism.14 Such histamine-induced reactions are

69 often misdiagnosed as IgE-mediated fish allergy. So it is necessary to prevent both

71 However, biogenic amines have been reported to be heat stable compounds.15

72 Most research has therefore focused on the prevention of histamine formation in

73 stored fish, by means such as temperature control, modified atmosphere packaging,

77 however the condition of canning is a very mature production technology that to

80 seems to be a more effective way of removing histamine. The glucose/histamine

83 of recombinant silver carp PV were partly weakened after the glucose/histamine

86 eliminate allergenicity during food processing and storage in industrial production.

Food & Function Accepted Manuscript

88 the Maillard reaction with pressure treatment was worth considering.

89 The following study compared the differences in reducing the content of

90 histamine and the immunoreactivity of parvalbumin upon boiling treatment (BT),

91 ultrasonication (US), ultraviolet irradiation (UV), PT and MR. In addition, a cell

98 low allergy foods.

100 2. Materials and Methods

101 2.1 Materials.

Food & Function Accepted Manuscript

119 2.2 Different processing methods.

121 contents of blue scad.

123 at 100 °C for 180 min.

127 with wavelength and power settings of 302 nm and 15 W, respectively.

129 0.12 MPa) for 20 min as the condition of canning.

130 Maillard reaction treatment: The reaction conditions of Maillard reaction

Food & Function Accepted Manuscript

133 30 min (pH 7.0).

134 Maillard reaction combined with pressure treatment: Samples and 4%

136 autoclave sterilizer (121 °C, 0.12 MPa) for 20 min.

137 2.3 Detection of the histamine content.

139 chromatography (RP-HPLC) after pre-column derivatization with Dns-Cl.20 A 5-g

Food & Function Accepted Manuscript

154 Samples were injected into an RP-HPLC to analyze the histamine

165 2.4 Detection of the immunoreactivity of parvalbumin.