Food Control 82 (2017) 136e144

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Histamine reduction by Maillard reaction with glucose

Wei Jiang a, Xiaoxia He b, Huicheng Yang c, Xingwei Xiang c, Shiwei Hu a, Shijie Li a,
Yu Liu a, *
a
Laboratory of Seafood Processing, Institute of Innovative and Application, Zhejiang Ocean University, Zhoushan, Zhejiang, 316022, China
b
Qingdao Entry-Exit Inspection and Quarantine Bureau, Qingdao, Shandong, 266002, China
c
Zhejiang Marine Development Research Institute, Zhoushan, Zhejiang, 316022, China

a r t i c l e i n f o a b s t r a c t

Article history: Histamine, well known as a toxic biogenic amine, is found in a variety of foods. Reducing its concen-
Received 15 April 2017 tration and toxicity is desirable. In this study, the glucose/histamine Maillard reaction was proposed as a
Received in revised form novel tool for histamine control. Effects of temperature, heating time, initial pH value, NaCl concentra-
19 June 2017
tion, initial histamine concentration and initial glucose concentration on percentage removal of hista-
Accepted 23 June 2017
Available online 24 June 2017
mine in the glucose/histamine Maillard reaction model were investigated. The results showed that
histamine reduction was affected by these variables, and could be almost eliminated under appropriate
conditions. Fluorescence intensity and ultravioletevisible spectroscopy analyses were used to charac-
Keywords:
Histamine
terize the glucose/histamine Maillard reaction. Cytotoxicity assay revealed that the glucose/histamine
Biogenic amine Maillard reaction significantly reduced the toxicity of histamine (P < 0.05). Furthermore, histamine
Glucose concentrations in canned tuna samples were significantly reduced by thermal treatment with glucose
Maillard reaction (P < 0.05). This study demonstrates that the glucose/histamine Maillard reaction is a promising method
Reduction for histamine control.
Cytotoxicity © 2017 Elsevier Ltd. All rights reserved.
Canned tuna

Chemical compounds studied in this article:

Histamine (PubChem CID: 774)
Glucose (PubChem CID: 5793)
Ampicillin (PubChem CID: 6249)
Methanol (PubChem CID: 887)
Dansyl chloride (PubChem CID: 11801)
Streptomycin (PubChem CID: 19649)
Sodium chloride (PubChem CID: 5234)
Dimethyl sulfoxide (PubChem CID: 679)

1. Introduction
Histamine, (2-(4-imidazolyl) ethylamine), is a biogenic amine
produced from the decarboxylation of amino acid histidine by
bacterial or tissue enzyme. As shown in Fig. 1, histamine is a het-
erocyclic monoamine chemical. High content of histamine is widely
found in various foods, especially in fermented foods (Koral et al.,
2013; Nei, Nakamura, Ishihara, Kimura, & Satomi, 2017; Pradenas,
Galarce-Bustos, Henríquez-Aedo, Mundaca-Uribe, & Aranda,
2016; Todoroki et al., 2014). Taking foods with high levels of his-
tamine can cause histamine poisoning, which is also known as
* Corresponding author.
E-mail address: liuyu1987@zjou.edu.cn (Y. Liu).
scombroid poisoning worldwide. It can not only cause a mild illness
with symptoms including dizziness, headache, hypotension, oral
burning and sweating, but also bring about life-threatening (Feng,
Teuber, & Gershwin, 2016).
The risk of histamine poisoning has attracted worldwide
attention. Reducing histamine concentration and toxicity in foods is
desirable. Briefly, histamine reduction can be achieved by two
means, namely preventing the formation of histamine and
consuming the existed histamine. For instance, high hydrostatic
pressure (K
rí

jkov
zek, Mate cha, & Dada
a, Va kova, 2014), irradiation
(Aflaki, Ghoulipour, Saemian, Shiebani, & Tahergorabi, 2015) and
modified atmosphere packing (Rodrigues et al., 2016) are employed
to inhibit the growth of microorganisms and thus suppress the
formation of histamine. However, these techniques are not appli-
cable for fermented foods where the growth of microorganisms is

http://dx.doi.org/10.1016/j.foodcont.2017.06.035
0956-7135/© 2017 Elsevier Ltd. All rights reserved.
 W. Jiang et al. / Food Control 82 (2017) 136e144 137

NH2
water activity (Caballero, Finglas, & Toldr
a, 2016). It can be used as
an important tool for acquiring valuable Maillard reaction products
(MRPs) (Kanzler, Haase, Schestkowa, & Kroh, 2016; Lee et al., 2016).
Moreover, the Maillard reaction of glucose/fumonisin B1 was used
to the reduction of mycotoxin-fumonisin B1 (Lu et al., 2002),
implying that Maillard reaction might be applied to the reduction of
harmful chemicals with amino groups. Considering that histamine
has a free amino group (Fig. 1), it may be consumed by Maillard
reaction theoretically. Nevertheless, the Maillard reaction between
sugar and histamine has not been studied previously.
This study investigated the effects of temperature, initial pH
value, NaCl concentration, initial glucose concentration and initial
histamine concentration on histamine reduction in a glucose/his-
tamine Maillard reaction model. The intermediate and final stages
of this reaction were characterized by fluorescence intensity and
ultravioletevisible spectroscopy. Cytotoxicity of histamine, glucose,
glucose/histamine mixture, and glucose/histamine MRPs was
compared. Moreover, the reduction of histamine content in canned
tuna by the glucose/histamine Maillard reaction was assessed
preliminarily. The aim was to evaluate the possibility of applying

N
the glucose/histamine Maillard reaction to reduce histamine level
and detoxification of it.

2. Materials and methods

2.1. Chemicals

Histamine, dansyl chloride and dimethyl sulfoxide were pro-

vided by Sigma-Aldrich, Inc. (St. Louis, MO, USA). Glucose was
provided by Solarbio Inc. (Beijing, China). Methanol (HPLC grade)
was provided by Merck (Darmstadt, Germany). Ultrapure water
was prepared by Milli-Q (Millipore, Billerica, MA, USA). Roswell
Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum,

N
streptomycin sulphate, ampicillin, trypsin, 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) were provided by
Gibco (Invitrogen Co., Burlington, ON, Canada).

2.2. Glucose/histamine maillard reaction model

H Fig. 1. Structure of histamine.

Glucose/histamine Maillard reaction was conducted in
16 mm
155 mm glass screw tubes capped with tight screw-caps.
Unless otherwise stated, the incubations contained 50 mM potas-
sium phosphate, pH 8.0, 40 mM glucose, and 2.5 mM histamine in a
total volume of 5.0 mL without the addition of NaCl. The tubes were
incubated at designed temperatures in a Digital Dry Bath (Jinxin,
very important. Furthermore, the existing histamine cannot be
JX100-4, Shanghai, China). To ensure that the reaction was stopped
consumed by these methods. Thus, some other researchers isolated
at the appropriate time point, the tubes were cooled in a mixture of
histamine degrading strains as functional starter cultures (Xu, Liu,
ice and water for 30 min.
Xu, Wang, & Jiang, 2016) or applied amine oxidase (Naila et al.,
2012) for the degradation of histamine. However, the amine oxi-
2.3. Factors affecting percentage removal of histamine in the
dase is expensive at present, and professional equipment/staff are
glucose/histamine maillard reaction model
required for the application of histamine degrading strains. Hence,
a more feasible method is required for histamine control in foods.
Effect of temperatures (60, 70, 80, 90, 100 and 110  C) on per-
Maillard reaction, also known as non-enzymatic browning re-
centage removal of histamine was investigated by the reaction of
action, is a complex chemical reaction between carbonyl groups of
40 mM glucose and 2.5 mM histamine at pH 8.0 for 12 h without
reducing sugars and free amino groups of amino acids, peptides,
the addition of NaCl. Samples were taken at 0, 0.5, 1, 1.5, 2, 2.5, 3, 4,
proteins or some other nitrogen containing compounds. Maillard
6, 8, 10 and 12 h. Effects of initial pH values (5.0, 6.0, 7.0, 8.0, 9.0,
reaction plays an important role in food industry and occurs during
10.0, 11.0 and 12.0), NaCl concentrations (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0,
thermal processing or food storage. According to the compounds
4.0, 6.0, 8.0, 10.0 and 12.0%, w/v), initial glucose concentrations (5,
generated during Maillard reaction, the reaction process is divided
10, 20, 40, 60, 80, 160 and 320 mM) and initial histamine concen-
into early, intermediate and final stages (Nursten, 2005). Among
trations (0.5, 1.0, 2.5, 5.0, 10.0 and 20.0 mM) on percentage removal
them, the intermediate and final stages can be characterized by
of histamine were investigated by changing one parameter at a
fluorescence intensity and ultravioletevisible absorption. Maillard
time in the reaction of 40 mM glucose and 2.5 mM histamine at
reaction is affected by a variety of variables, such as temperature,
100  C and pH 8.0 for 5 h without the addition of NaCl. Samples
reaction time, pH, substrate concentration, substrate type, and
were taken at 0.5, 1, 3 and 5 h.
138 W. Jiang et al. / Food Control 82 (2017) 136e144
2.4. Histamine concentration determination
Histamine concentration was determined using HPLC method
by pre-column derivatization with dansyl chloride (Jiang, Xu, Li,
Dong, & Wang, 2014). Derivatization process was performed as
previously reported (Dad akova, K
rí
nova
zek, & Pelika , 2009) with
some modifications. One milliliter of sample was mixed with 1.5 mL
of carbonate buffer (pH 11) in a 10 mL centrifuge tube. The car-
bonate buffer was prepared according to a previously reported
method (Dada kova et al., 2009). After mixed, 1.0 mL of dansyl
chloride solution (dansyl chloride in acetone, 10 mg/mL) was
added. The centrifuge tube was shaken at 40  C in a water bath for
1 h in darkness. To consuming the remaining dansyl chloride,
0.2 mL of proline solution (proline in ultrapure water, 100 mg/mL)
was added and the mixture was shaken for another 1 h in darkness.
Then 3 mL of heptane was added and the mixture was shaken for
30 min. After stratification, 1.0 mL of the heptane part was dried at
40  C using a gas blowing concentrator (Jinxin, JXDC-400, Shanghai,
China). The dried residue was dissolved in 1 mL of methanol.
Samples were filtered through a 0.22 mm filter (Millipore, Bedford,
MA, USA) prior to analysis. The HPLC system consist of a Waters
2695 series (Milford, USA) equipped with a degasser, quaternary
pump, column oven, ultraviolet detector and automatic sampler. A
reversed-phase chromatographic column (Capcell PAK 5 mm C18
MG, 150
4.6 mm) was provided by Shiseido Co. (Tokyo, Japan).
The chromatographic column was equilibrated at 30  C with the
mobile phase of 55% methanol and 45% ultrapure water. The
derivatized histamine was detected at 254 nm. The injection vol-
ume was 10 mL. Calibration curves were constructed of peak area
versus standard histamine concentration (0.01e2.5 mM).
2.5. Fluorescence intensity
Fluorescence intensities of samples were determined by a
fluorescence spectrophotometer (Hitachi, F-4500, Kyoto, Japan) as
nez-Pe
the method described previously (Morales & Jime rez, 2001).
Samples were diluted 10-fold with ultrapure water. The fluores-
cence intensities were measured at an excitation wavelength of
350 nm and an emission wavelength of 420 nm.
2.6. Ultravioletevisible spectroscopy
Ultravioletevisible spectra of samples were determined by an
ultravioletevisible spectrophotometer (Shimadzu, UV-2600, Kyoto,
Japan) as the method described previously (Zhou et al., 2013) with
some modifications. Samples were diluted 50-fold with ultrapure
water. Ultravioletevisible spectra were recorded from 220 to
800 nm with an interval of 0.5 nm. Meanwhile, the absorbance at
294 and 420 nm was measured.
2.7. Cell culture
The normal human liver HL-7702 cell line (BNCC 100966) was
provided by BeNa Culture Collection Co. Ltd. (Beijing, China). Cells
were maintained in RPMI 1640 medium containing 10% (v/v) fetal
bovine serum, 0.01% (w/v) ampicillin and 0.05% (w/v) streptomycin
at 37  C under a humidified atmosphere containing 5% CO2.
2.8. Cytotoxicity assay
Cytotoxicity of histamine, glucose, glucose/histamine mixture,
and glucose/histamine MRPs were evaluated by the MTT assay
using HL-7702 cells as a previously reported method (Xia et al.,
2014) with some modifications. Briefly, cells were seeded into a
96-well plate at density of 5
103 cells per well with 100 mL of
RPMI 1640 medium containing 10% (v/v) fetal bovine serum, 0.01%
(w/v) ampicillin and 0.05% (w/v) streptomycin. After incubated at
37  C for 24 h under a humidified atmosphere containing 5% CO2,
100 mL of culture medium containing different test substances (as
shown in Table 1) were added to the wells of experiment groups.
Meanwhile, 100 mL of culture medium without test substances were
added to the wells of the negative control group. The cells were
subsequently cultured for 24 h at 37  C under a humidified atmo-
sphere containing 5% CO2. Then 10 mL of MTT solution (5 mg/mL)
was added to each well and the plate was incubated for another 4 h.
After carefully removal of the supernatant, 100 mL of dimethyl
sulfoxide was added to each well and the plate was shaken at room
temperature for 20 min. The absorbance at 490 nm was measured
in a microplate reader (PULANG, DNM-9606, Beijing, China). The
cell growth inhibition rate was calculated as follow: Cell growth
inhibition rate (%) ¼ (Absorbance value of control - Absorbance
value of sample)/Absorbance value of control
100.
2.9. Thermal treatment of canned tuna with glucose
To evaluate the histamine reduction ability of Maillard reaction
in foods, two commercial canned tuna samples were obtained from
a market in Zhoushan, Zhejiang, China. The salt concentrations of
sample A and sample B were measured as 2.03± 0.21% (w/v) and
1.85± 0.37% (w/v), respectively. The pH values of sample A and
sample B were measured as 6.68 ± 0.07 and 6.82 ± 0.16, respec-
tively. The obtained canned tuna was opened and its net weight
was measured. Then 2% (w/w) of glucose (equal to 125 mM of
glucose) was added and mixed gently. The mixture was transferred
to a sealable glass container. After that, the container was incubated
in a water bath at 95  C. Samples were taken at the heating time
points of 0, 1, 2 and 3 h.
2.10. Histamine extraction from canned tuna
Histamine was extracted from canned tuna by a reported
method (Evangelista et al., 2016) with some modifications. The
samples were chopped and ground using a glass tissue grinder.
After homogenization, 2.0 g of samples were placed into centrifuge
tubes containing 3 mL of 5% (w/v) trichloroacetic acid. The tubes
were mixed for 70 s by a vortex mixer (IKA, MS3, Staufen, German)
and centrifuged at 11250 g and 4  C for 3 min by a refrigerated
centrifuge (Zhongke, HC-3018R, Anhui, China). The supernatant
was filtered through qualitative paper. The acid extraction step was
repeated twice again. The filtrates were all transferred into a cali-
brated volumetric flask and diluted with 5% trichloroacetic acid to
10 mL. The diluted solution was used for the determination of
histamine concentration in canned tuna.
Table 1
Experiment design for cytotoxicity assay of histamine, glucose, glucose/histamine
mixture and glucose/histamine Maillard reaction products (MRPs).
Levels Concentration (mM)
Histamine Glucose Mixturea MRPs-1b MRPs-5b
1 2 32 34 34 34
2 4 64 68 68 68
3 6 96 102 102 102
4 8 128 136 136 136
5 10 160 170 170 170
a
It was prepared by freeze-drying of the mixture of 2.5 mM histamine and 40 mM
glucose in 50 mM PBS (pH 8.0).
b
They were prepared by freeze-drying of the MRPs of 2.5 mM histamine and
40 mM glucose after heating at 100  C for 1 h (MRPs-1) or 5 h (MRPs-5) in 50 mM
PBS (pH 8.0).
 W. Jiang et al. / Food Control 82 (2017) 136e144
2.11. Statistical analyses
Statistical analyses were performed using the software IBM SPSS
Statistics 22.0 (IBM, USA). Data were analyzed by one-way analysis
of variance (ANOVA) with the Tukey test. The experiments were
performed in triplicate and the data were expressed as
average ± standard deviation.
3. Results and discussion
3.1. Effects of temperature and heating time on percentage removal
of histamine
Changes of percentage removal of histamine during thermal
treatment with or without glucose are shown in Fig. 2. It was
observed that histamine concentration remained essentially un-
changed when heated alone at 110  C for up to 12 h, indicating that
histamine was thermally stable at this temperature. Nevertheless,
histamine was consumed in the glucose/histamine Maillard reac-
tion model, and the percentage removal was influenced by tem-
perature and heating time.
It can be seen in Fig. 2 that percentage removal of histamine
increased as the reaction temperature increased from 60 to 110  C.
Temperature plays an important role in Maillard reaction, and this
reaction can occur during the storage, processing and sterilization
of foods. Recently, different research groups reported the sugar/
whey protein Maillard reactions at 37  C and 123  C (Leiva, Naranjo,
& Malec, 2017; Ruiz et al., 2016). As Maillard reaction is an endo-
thermic reaction, the degree of this reaction is always enhanced at
higher temperature (Lu et al., 2002).
Heating time is another important factor in Maillard reaction. As
shown in Fig. 2, percentage removal of histamine increased with
the prolonging of heating time. Histamine was almost eliminated
within 1.5, 2.5, and 6 h when heated at 110, 100, and 90  C,
respectively. The equal degree of Maillard reaction can be achieved
by a high-temperature short-time treatment or a low-temperature
long-time treatment (Caballero et al., 2016).
3.2. Effect of initial pH value on percentage removal of histamine
Results of percentage removal of histamine in the glucose/
100
Percentage removal (%)
80
60
40
20
0 As indicated in Fig. 3C and D, percentage removal of histamine in
0 1 2 3 4 5 6 7 8 9 10 11
Heating time (h)
Fig. 2. Changes of percentage removal of histamine in the glucose/histamine Maillard
reaction model during heating at different temperatures (60  C, ; 70  C, ; 80  C,
; 90  C, ; 100  C, ; 110  C, ) for up to 12 h. The reaction was performed with
2.5 mM histamine and 40 mM glucose at pH 8.0. Histamine heated alone at 100  C and
pH 8.0 was also studied ( ).
139
histamine Maillard reaction model with different initial pH values
are shown in Fig. 3A. Percentage removal of histamine significantly
increased as the initial pH value increased from 5.0 to 12.0
(P < 0.05). Specifically, if the initial pH value was no less than 7.0,
histamine was almost eliminated within 5 h. The promotion of
reactants consumption at high initial pH value was reported in
various Maillard reactions, such as glucose/casein (Ajandouz,
Desseaux, Tazi, & Puigserver, 2008), glucose/proline (Blank,
Devaud, Matthey-Doret, & Robert, 2003), and fructose/lysine
(Ajandouz & Tchiakpe, 2001). The inhibition of glucose/histamine
Maillard reaction in acidic solution might be attributed to the
protonation of histamine (Liu, Yang, Chen, Chen, & Chen, 2011).
Under the condition of low pH value, the nitrogen atom in the
amino group of histamine would be protonated and transformed
into an ammonium ion. This phenomenon resulted in suppressing
the condensation of amino group with carbonyl group and thus
reducing the consumption of histamine. Moreover, reaction rate
could be slowed down by the fact that the pH value always de-
creases in the process of Maillard reaction, which was attributed to
the consumption of the basic amino group and the formation of
acidic MRPs (Cai et al., 2016). Furthermore, it was reported that the
activation energy value decreased as the system pH increased in
Maillard reactions, indicating that Maillard action occurred easier
at higher pH value (Ajandouz et al., 2008).
3.3. Effect of NaCl concentration on percentage removal of
histamine
Since histamine was always found in fermented foods with high
salt content (Jiang et al., 2014), we investigated the effect of salt
concentration on percentage removal of histamine in the glucose/
histamine Maillard reaction model. As shown in Fig. 3B, the addi-
tion of NaCl decreased percentage removal of histamine in the
glucose/histamine Maillard reaction model. It was worth noting
that histamine was almost eliminated within 5 h if no more than
8.0% of NaCl was added. The results were consistent with a previous
study in which the addition of 0.5e2.5% NaCl inhibited the fructose/
surimi wash water Maillard reaction at 95  C and pH 9.0 for 12 h
(Thongraung & Kangsanan, 2010).
3.4. Effects of initial glucose concentration and initial histamine
concentration on percentage removal of histamine
Results of percentage removal of histamine in the glucose/his-
tamine Maillard reaction model at 100  C and pH 8.0 with 2.5 mM
histamine and different concentrations of glucose (5e320 mM) are
showed in Fig. 3C. Percentage removal of histamine increased with
increasing glucose concentration. Histamine was almost eliminated
after 1, 3, and 5 h with 2.5 mM histamine and no less than 320, 20,
and 10 mM of glucose, respectively. Results of percentage removal
of histamine in the glucose/histamine Maillard reaction model at
100  C and pH 8.0 with 40 mM glucose and different concentrations
of histamine (0.5e20 mM) are showed in Fig. 3D. Percentage
removal of histamine decreased as histamine concentration
increased. Histamine was almost eliminated after 3 and 5 h with
40 mM glucose and no more than 5 and 10 mM of histamine,
respectively.
12 the glucose/histamine Maillard reaction model positively corre-
lated with initial glucose concentration and negatively correlated
with initial histamine concentration, suggesting that it might be
determined by the concentration ratio between glucose and his-
tamine. When the reaction was proceeded with the concentration
ratio of glucose to histamine as four to one, percentage removal of
histamine in Fig. 3C by 10 mM glucose and 2.5 mM histamine
140 W. Jiang et al. / Food Control 82 (2017) 136e144

(a) g
(b) f
c dc dc f dc f dc g f dc f f f f ff f f
100 e 100
b
e
Percentage removal (%)

Percentage removal (%)

e d
c
80 de
80 d cd c
e g

f
60 b 60 b
e
de d
c d f cd
a ef abb
40 40 de c
a
c d d b a
a b ab
cd
c
a
20 a 20 b
b ab
a a

0 0
5 6 7 8 9 10 11 12 0 1 3 5 8 11 14 19 24 29
Initial pH NaCl concentration (%)

(c) (d) c b
b cb cb cb c b cb hcb e c b c b c b b
100 g 100
e

Percentage removal (%)

h
Percentage removal (%)

b
80 f 80 f
g d b
d e
a e a

60 c ef f 60 c
a
a
d d
b b
40 40
c
c
a
a
20 b 20 b
a a

0 0
5 10 20 40 60 80 160 320 0.5 1 2.5 5 10 20
Glucose concentration (mM) Histamine concentration (mM)
Fig. 3. Effects of (a) initial pH value, (b) NaCl concentration, (c) initial glucose concentration and (d) initial histamine concentration on percentage removal of histamine in the
glucose/histamine Maillard reaction model system at different reaction time (0.5 h, ; 1 h, ; 3 h, ; 5 h, ). Reaction Conditions: (a) 2.5 mM histamine and 40 mM
glucose at 100  C and different initial pH values; (b) 2.5 mM histamine and 40 mM glucose at 100  C and pH 8.0 with different concentration of NaCl; (c) 2.5 mM histamine and
different concentration of glucose at 100  C and pH 8.0; (d) different concentration of histamine and 40 mM glucose at 100  C and pH 8.0. Bars of the same color (at the same
reaction time) with different letters are significantly different (P < 0.05).

(16.52% at 0.5 h, 36.74% at 1 h, 74.91% at 3 h, and 100% at 5 h) were Fluorescence intensity of ribose/a-lactoglobulin MRPs reached the
similar to those in Fig. 3D by 40 mM glucose and 10 mM histamine maximum within 2 h, followed by a rapid decline (Jiang &
(15.69% at 0.5 h, 35.61% at 1 h, 70.91% at 3 h and 100% at 5 h). Effect Brodkorb, 2012). Nevertheless, fluorescence intensity of xylan/
of the reactant ratio on Maillard reactions has been also reported in chitosan MRPs increased to a plateau phase in 2 h (Wu et al., 2014).
previous studies (Li, Luo, & Feng, 2011; Yang et al., 2015). In a binary Fluorescence intensity of MRPs is related to the generation and
Maillard reaction system, increasing of the concentration of one consumption of fluorescent substances, which is determined by
reactant always results in inhibiting its own reduction rate, but both the types of reactants and the extent of reaction. The results
enhancing the consumption of the other reactant. demonstrate that some fluorescent MRPs form during the glucose/
histamine Maillard reaction, and are converted into non-
fluorescent substances as the reaction proceeds.
3.5. Characterization on the glucose/histamine maillard reaction Ultravioletevisible spectrum, a useful tool for characterizing the
process of Maillard reaction, has been studied in a variety of models
Fluorescence substances can be detected in the intermediate (Hrynets, Ndagijimana, & Betti, 2015; Shrestha & De Meulenaer,
stage as the precursors of the brown products in the final stage 2014). Besides, the ultraviolet absorbance at 294 nm always rep-
(Jiang, Wang, Wu, & Wang, 2013). As shown in Fig. 4, during ther- resents the MRPs of the intermediate stage, and the visible absor-
mal treatment at 100  C and pH 8.0 for 4 h, fluorescence intensity of bance at 420 nm is associated with the browning development in
40 mM glucose alone or 2.5 mM histamine alone did not changed the final stage (Morales & Jime nez-Perez, 2001). Fig. 5 shows the
significantly. However, when the mixture of 40 mM glucose and ultravioletevisible spectra of the glucose/histamine MRPs, during
2.5 mM histamine were heated under the same conditions, the heating at 100  C and pH 8.0 with 40 mM glucose and 2.5 mM
fluorescence intensity of the glucose/histamine MRPs increased histamine for up to 4 h. The mixture of glucose and histamine
and reached the maximum value at 1.5 h. Thereafter, it decreased without thermal treatment (heating time ¼ 0 h) did not show
slightly as the heating time prolonged to 4 h. Previous studies detectable absorbance between 220 nm and 800 nm. Nevertheless,
suggested that fluorescence intensity of different MRPs changed in ultravioletevisible absorbance of the glucose/histamine MRPs
different modes. Fluorescence intensity of galactose/bovine casein increased obviously with the prolonging of heating time (0e4 h),
peptide MRPs was also found to quickly reach a maximum at 1 h implying that new substances generated in this process. As shown
and decreased dramatically thereafter (Jiang et al., 2013).
 W. Jiang et al. / Food Control 82 (2017) 136e144 141

500

Fluorescence intensity (A.U.) 400

300

200

100

0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
Heating time (h)
Fig. 4. Change of fluorescence intensity of 40 mM glucose ( ), 2.5 mM histamine ( ) and the MRPs of 40 mM glucose and 2.5 mM histamine ( ), during heating at 100  C and pH
8.0 for up to 4 h. Samples were diluted 10-fold with ultrapure water prior to analysis.

0.250
0.10
Absorbance at 294 nm
0.09 Absorbance at 420 nm

0.08
0.200 0.07

0.06
Absorbance

0.05

0.04
0.150
Absorbance

0.03

0.02

0.01
4h
0.00
0.100 3.5 h 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
Heating time (h)
3h heating time
2.5 h

2h
0.050
1.5 h
1h
0.5 h
0.25 h
0.000 0h

220 250 300 350 400 450 500 550 600 650 700 750 800
nm
Fig. 5. Ultravioletevisible absorbance spectra of the MRPs of 40 mM glucose and 2.5 mM histamine during heating at 100  C and pH 8.0 for up to 4 h. Samples were diluted 50-fold
with ultrapure water prior to analysis. The inserted figure showed the absorbance changes at 294 nm and 420 nm.

in the figure inserted in Fig. 5, the absorbance at 294 nm increased
dramatically as the heating time went on. It was also observed that
the absorbance increasing speed at 294 nm was lower in the first
1.5 h (increase from 0.003 to 0.024) than that between 1.5 h and 3 h
(increase from 0.024 to 0.074). The results indicate that the glucose/
histamine MRPs of the intermediate stage form immediately within
0.25 h. However, most of these MRPs generate from 1.5 to 3 h. The
inserted figure in Fig. 5 also shows that the absorbance at 420 nm
does not increase in the first 0.5 h, but increases dramatically
thereafter (from 0.001 at 0.5 h to 0.017 at 4 h) with the extended
heating time. The results indicate that browning pigments, gener-
ating in the final stage of Maillard reaction, do not form
142 W. Jiang et al. / Food Control 82 (2017) 136e144

Table 2
Cytotoxicity assay of histamine, glucose, glucose/histamine mixture and glucose/histamine Maillard reaction products (MRPs).

Levelsa Cell growth inhibition rate (%)

Histamine Glucose Mixtureb MRPs-1c MRPs-5c

1 14.29 ± 2.09a 1.87 ± 0.56**a 13.21 ± 1.97a 7.86 ± 0.61**a 1.82 ± 0.43**a
2 18.49 ± 0.68ab 1.34 ± 0.76**b 19.64 ± 0.81b 13.14 ± 0.88**b 2.59 ± 0.54**a
3 23.70 ± 1.33bc 1.35 ± 0.12**b 22.21 ± 1.12b 19.57 ± 1.35**c 2.22 ± 0.33**a
4 28.32 ± 2.12c 1.14 ± 0.94**b 28.58 ± 0.99c 20.51 ± 1.42**c 4.61 ± 2.90**ab
5 47.25 ± 2.82d 1.29 ± 0.81**b 50.21 ± 2.00d 26.72 ± 2.80**d 5.79 ± 1.65**b

Data in the same row with ** are significantly different vs Histamine group (P < 0.01).
Data in the same column with different letters are significantly different (P < 0.05).
a
The levels of different groups were shown in Table 1.
b
It was prepared by freeze-drying of the mixture of 2.5 mM histamine and 40 mM glucose in 50 mM PBS (pH 8.0).
c
They were prepared by freeze-drying of the MRPs of 2.5 mM histamine and 40 mM glucose after heating at 100  C for 1 h (MRPs-1) or 5 h (MRPs-5) in 50 mM PBS (pH 8.0).

immediately at the beginning of the glucose/histamine Maillard
reaction.
3.6. Cytotoxicity comparison
Various toxic MRPs, such as 5-hydroxymethlofurfural, dicar-
bonyls, advanced glycation end products and heterocyclic amines,
have caused wide concern over the recent years (Zhang, Tao, Wang,
Chen, & Wang, 2015). Hence, although histamine concentration
decreased in the glucose/histamine Maillard reaction, the unknown
toxicity of the MRPs was still a problem. Hence, we assayed cyto-
toxicity of histamine, glucose, glucose/histamine mixture and
glucose/histamine MRPs using normal human liver HL-7702 cells
by MTT method. As shown in Table 2, histamine showed significant
cytotoxicity to HL-7702 cells in a dose-dependent manner
(P < 0.05). The cell growth inhibition rate of the histamine group
increased significantly from 14.29% with 2 mM histamine to 47.25%
with 10 mM histamine (P < 0.05). However, the cell growth inhi-
bition rates of the glucose group were all less than 2%, indicating no
observable toxicity. The mixture of histamine and glucose without
thermal treatment showed similar cytotoxicity to that of the his-
tamine group. However, after 40 mM glucose and 2.5 mM hista-
mine reacted at 100  C and pH 8.0 for 1 h, the histamine
concentration was reduced by 60.66% (Fig. 2), and the cytotoxicity
of MRPs-1 was significantly lower than that of histamine or the
mixture of glucose and histamine (P < 0.01). Furthermore, when
the reaction time was 5 h, the cell growth inhibition rates of MRPs-
5 were all lower than 10%, exhibiting even lower cytotoxicity than
MRPs-1. The results show that the glucose/histamine Maillard re-
action does not only reduce the histamine content, but also lowered
its cytotoxicity.
3.7. Preliminary application of glucose/histamine maillard reaction
in canned tuna
In order to evaluate the potential application of the glucose/
histamine Maillard reaction, we tested its performance in com-
mercial canned tuna samples which always contained high levels of
histamine (Akbari-Adergani, Hosseini, Shekarchi, & Pirali-
Hamedani, 2012). The salt concentration, pH value, and histamine

0.9
c
Histamine concentration (mM)

0.8

0.7
b
0.6
b
0.5
ab a
0.4 a
0.3 a a
0.2

0.1

0.0
0 1 2 3
Heating time (h)
Fig. 6. Histamine reduction in two canned tuna samples (sample A, ; sample B, ) by the glucose/histamine Maillard reaction at different incubation time (0, 1, 2, 3 h). The
incubation was performed with 125 mM glucose at 95  C. Bars of the same sample with different letters are significantly different (P < 0.05).
 W. Jiang et al. / Food Control 82 (2017) 136e144
concentration of sample A were measured as 2.03± 0.21% (w/v),
6.68 ± 0.07, and 0.75 ± 0.09 mM, while those of sample B were
1.85± 0.37% (w/v), 6.82 ± 0.16, and 0.48 ± 0.06 mM. As shown in
Fig. 6, histamine concentrations of both samples decreased signif-
icantly by thermal treatment with 125 mM glucose at 95  C for up
to 3 h (P < 0.05). The histamine concentration of sample A was
reduced significantly by 25.89, 46.87, and 53.57% after 1, 2, and 3 h,
respectively (P < 0.05). A similar trend was also observed in sample
B. The histamine concentration of sample B was reduced signifi-
cantly by 33.57, 54.55, and 61.54% after 1, 2, and 3 h, respectively
(P < 0.05). Higher percentage removal of histamine in sample B may
be attributed to its lower salt concentration, higher pH value and
lower initial histamine concentration, which could be explained by
the results in Fig. 3. It was obvious that percentage removal of
histamine were lower in canned tuna samples than in the glucose/
histamine Maillard reaction model. It might be concluded that
glucose reacted with not only histamine, but also with other
compounds, including amino acids, peptides, and proteins.
Furthermore, the existence of minerals in canned tuna may inhibit
the glucose/histamine Maillard reaction (Rizzi, 2017). In the next
step, the effect of Maillard reaction on the physicochemical and
nutritional properties of canned tuna should be investigated.
4. Conclusions
In conclusion, histamine was thermal stable when heated alone,
but could be reduced in the glucose/histamine Maillard reaction
model. Percentage removal of histamine was affected by temper-
ature, heating time, initial pH value, NaCl concentration, initial
glucose concentration and initial histamine concentration. Notice-
ably, histamine could be almost eliminated under appropriate
conditions in the glucose/histamine Maillard reaction model. The
intermediate and final stages of the glucose/histamine Maillard
reaction were characterized by fluorescence intensity and ultra-
violetevisible spectroscopy. The results showed that MRPs of the
intermediate stage formed within 0.25 h, but the browning pig-
ments of the final stage formed after 0.5 h under the conditions
analyzed in this study. Cytotoxicity assay indicated that the toxicity
of glucose/histamine MRPs was significantly lower than that of
histamine (P < 0.01). Furthermore, the glucose/histamine Maillard
reaction significantly reduced histamine concentration in two
commercial canned tuna samples (P < 0.05). Taken together, this
study demonstrates that the glucose/histamine Maillard reaction
has bright application prospect for histamine control in foods.
Acknowledgments
The work was supported by the Zhejiang Provincial Natural
Science Foundation of China [grant number LQ15C200008], the
Natural Science Foundation of China [grant number 31501573], and
the Research Start-up Funding of Zhejiang Ocean University [grant
numbers Q1442, Q1443].
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