Rapid detection of histamine in foods
Kikkoman Biochemifa Company
1 Introduction
Histamine poisoning is one of the most common forms of food
intoxication and is caused by the ingestion of foods containing
high level of histamine. Histamine poisoning is characterized by
a variety of symptoms similar to allergic reactions and include hy-
potension, flushing, headache, puffy eyes, and skin rash (Taylor,
1986). In Japan, instances of histamine poisoning occur every year
(Table 1).
Histamine is a biogenic amine produced through the decar-
boxylation of free histidine by histamine-forming bacteria (Lade-
ro et al., 2010). This ability has been described in different genera,
species, and strains of bacteria. Histamine is rarely found in fresh
fish but its level increases with the progress of decomposition.
Freezing, cooking, or canning can inhibit the histamine-forming
reactions, but histamine formation cannot be eliminated because
histamine is heat stable. In addition to fish, histamine can also be
found in aged or fermented foods such as soy sauce, fish sauce,
miso, wine and cheese, in which histamine-forming bacteria can
be found (Okamoto et al., 1997). Histamine poisoning caused by
Japanese seasonings such as soy sauce and miso have rarely oc-
curred and most cases of poisoning are caused by raw and pro-
cessed seafood. It is likely that the small intake of seasonings does
not contain sufficient amounts of histamine causing poisoning
(Guidi and Gloria, 2012).
2 Regulations in histamine
Fish handling practices are critical with regard to histamine pro-
duction. For the purposes of consumer protection, fish importing
countries have regulations and varying limits for histamine in fish
and fishery products. For example, according to Commission Reg-
ulation EC No 2073/2005 (Regulation 2073/2005/EC) maximum
levels for histamine were established in fishery products from fish
species associated with high histidine amounts, value raising from
100 to 200 mg/kg, while the products which have undergone en-
zyme maturation treatment in brine, the aforementioned limits
raised to 200 and 400 mg/kg. The last Regulation amended Annex
I to Regulation EC No 2073/2005 added a maximum value for fish
sauce produced by fermentation of fishery products, equal to 400
mg/kg. According to the FDA guidelines, the toxicity and defect
action levels of histamine established for fish is equal to or greater
than 50 ppm. (FDA 2011).
3 Analytical methods for histamine detection
A number and variety of methods exist for determination of hista-
mine levels in fish, fishery products, and fermented foods includ-
ing the well-accepted Association of Official Analytical Chemists
(AOAC) fluorometric method (AOAC 977.13), enzyme-linked im-
munosorbent assay (ELISA) methods (Pessatti et al., 2004, Kose
et al., 2011), the colorimetric enzyme test (Sato et al., 2005) and
high-performance liquid chromatography (HPLC) methods that
can measure multiple biogenic amines (Malle et al., 1996; Brillantes
and Samosorn, 2001). While each method has strengths and limi-
tations, and they vary in terms of related cost, operator expertise,
time to obtain a result, portability, etc. (Table 2), most methods
provide good agreement and are capable of reliably measuring
histamine in seafood at levels of interest.
Table 1: Histamine poisoning outbreaks in Japan
Number of cases Number of patients
2011 7 206
2012 9 113
2013 7 190
2014 7 61
2015 13 405
2016 15 283
2017 8 74
1
 Table 2: Comparison of the test methods for determination of histamine levels (modified from the FAO Meeting report, 2012)

Method Enzymatic test Fluorescent assay HPLC ELISA Lateral flow

(AOAC 977.13)
Product Histamine Test Veratox (Neogen) Reveal (Neogen)
1-2h
Testing time 1h (column preparation 1-2h 1h 0.5h
time is not included)
Equipment Spectrophotometer Fluorometer HPLC Micro plate reader None
Limit of quantification 10 ppm 1-5 ppm 1.5-5 ppm 2.5 ppm 50 ppm
Complicated,use of
Procedure Easy Complicated Easy Easy
hazardous solvents
Easy, fast, low cost, Quantification of Easy, fast, multiple
General Advantages accuracy, Repeatable, accuracy all biogenic amines, tests simultaneously, Easy, fast
simple calibration accuracy accuracy

[Fluorescent assay (AOAC 977.13)] 4 Rapid Enzymatic assay

Fluorescent measurement (AOAC 977.13) has been established
and is recognized as the most suitable method for the determi-
nation of histamine contained in fish and fermented foods. In this
method, product is extracted with 75% (v/v) methanol. Extract
is passed through ion exchange column. o-Phthaldialdehyde as
a fluorescent reagent is added to elute to form fluorescent his-
tamine derivatives. The intensity of the fluorophore is measured
using photofluorometer and histamine is quantified using exter-
nal standards. In this method, the procedure is not simple and
the samples need to be extracted with hot methanol. In order to
obtain derivatives from this fluorophore and histamine, impurities
in the sample must be removed by column. Thus, well-trained lab-
oratory labor must be employed and time required to carry out
“cleanup” procedures are unavoidable.
[High-performance liquid chromatography (HPLC)]
HPLC is suitable method of histamine assay. In addition to his-
tamine, biogenic amines such as putrescine and cadaverine can
be simultaneously and quantitatively determined by HPLC. In
this method, however HPLC analysis is not run simultaneously, so
sequential analysis of multiple samples can take time. The HPLC
equipment needed in this assay is also expensive.
Kikkoman Histamine Test (code 61341)
Histamine test kits using rapid enzymatic method with easy
extraction are commercially available (Kikkoman Histamine Test
Code 61341). This product has been issued the AOAC Performance
Tested Methods (PTM) certificate (License number 041802).
4.1 Assay principal
A colorimetric enzyme assay for quantitative analysis of his-
tamine in foods has been developed using histamine dehydro-
genase (Sato et al., 2005). Histamine dehydrogenase specifically
catalyzes the oxidation of histamine. In the test kit of Kikkoman
Biochemifa Company, the principle of this photometric assay is as
follows;
Histamine dehydrogenase catalyzes oxidative deamination of his-
tamine in the presence of 1-methoxy PMS (electron carrier), which
Histamine dehydrogenase

Histamine 1-methoxy PMS WST-8 formazan

[Enzyme-linked Immune Sorbent Assay (ELISA)] (color development)
ELISA assay kits are commercially available. ELISA kits pro-
Imidazole 1-methoxy PMS WST-8
vide good reproducibility, however, the kits are expensive for Acetaldehyde (reduced form)
routine testing of numerous samples on a quality-assurance
Figure 1: Determination of histamine using enzyme
or preventive testing basis.

2
converts WST-8 (tetrazolium salt) to a formazan (Figure 1). Thus, one 1,2
y = 0,3403x +0,0127
molecule of formazan is formed by one molecule of histamine. This （1.9）
1,0 R2 = 0,99989

product is measured in the visible range at 460-470 nm.

0,8

OD460
0,6
The correlation between histamine level and absorbance is excel- （1.7）

lent when assay is performed using histamine standard in terms of 0,4

linearity passing the origin of coordinate axes (Figure 2). Therefore, 0,2
（2.3）
the histamine in a tested sample can be determined with good （2.5） （CV: %）
0,0
precision from absorbance using only one point of histamine con- 0 1 2 3

centration as a standard. In addition, the cost of this enzymatic Histamine concentration in solution (ppm)

method is very reasonable as compared to other method such as Figure 2: Determination of histamine standard
Fluorescent assay, HPLC, and ELISA method.

4.2 Analytical protocol of raw and canned tuna is not needed.

Histamine level in raw and canned tuna can be determined as be-
low (Figure 3);
[Preparation of sample solution]
1. Approximately 10 g of the sample was weighed and homoge-
nized.
2. Weigh out precisely 1 g of homogenized sample and transfer
into a heat resistant plastic tube with cap.
3. Precisely 24 ml of 0.1 M EDTA•2Na solution (pH8.0) is added and
mixed vigorously. In this case, the sample is diluted 25-fold.
4. The sample tube is boiled for 20 min and then cooled on ice. In a
case of heated sample such as canned tuna, this boiling process
5. The sample is mixed vigorously and the supernatant is collected
by filtering through folded filter paper or centrifugation (10,000g
for 5 min).
[Determination of histamine amount]
1. To set the absorbance of the spectrophotometer to zero, dis-
tilled water should be used as reference according to its instruc-
tion manual.
2. To assay N samples, prepare (2N + 2) plastic tubes for duplicated
assay.
3. To carry out sample assay, add 0.5 ml of the extracted sample
solution. Then add 0.5 ml each of the colorimetric reagent and

① Homogenize ② Add 1g of the ③ Boil for 20 minutes ④ Cool down ⑤ Filtrate

the sample sample to 24ml of
sample treatment
buffer ※You can skip ③,④ for fish meal,
canned fish and fish sauce.

⑥ Add reagents ⑦ Add sample ⑧ Incubate for ⑨ Measure the absorbance

to test tubes solution and 15 minutes at 470nm
enzyme
※Protect from light ※Use plastic lab wares

Figure 3: Determination of histamine in raw fish using enzymatic method

3
 the enzyme solution. Mix well and incubate at 37°C for 15 Table 3: Spike and recovery test for raw and canned tuna
min. The sample should be protected from light, if possible,
Spiked histamine Recovered histamine (ppm) *N=3
especially irradiate strong light and, in particular sunlight, dur- (ppm) Canned tuna in oil Canned tuna in water Raw tuna
ing the operations. Measure the absorbance at 470 nm. 10 11.3 10.8 10.6
4. To carry out sample blank assay, add 0.5 ml of the buffer solu- 20 21.6 21.2 20.0
50 51.0 49.5 50.9
tion instead of the enzyme solution. Carry on the same oper-
75 72.0 73.5 77.6
ation as in (3). Measure the absorbance at 470 nm. Recovery rate 96-113% 98-108% 100-106%
5. To carry out histamine standard assay, use 0.5 ml of histamine
standard solution instead of extracted sample solution. Carry
Table 4: Comparison of results from four histamine test methods
on the same operations as in (3). Measure the absorbance at in raw and canned tuna (unit:ppm)
470 nm.
6. To carry out reagent blank assay, add 0.5 ml of distilled water Raw tuna
(stored at 20℃ for 2 days) Canned tuna
instead of the extracted sample solution and add 0.5 ml of Rapid Enzymatic test 2,886 537
buffer instead of enzyme solution. Carry on the same opera- HPLC 2,545 566
AOAC 977.13 2,646 530
tion as in (3). Measure the absorbance at 470 nm.
EIA method 2,728 574

The histamine concentration in the sample can be calculat-

Table 5: Comparison of results from two histamine test methods
ed by the ratio of the absorbance of the sample and standard in fish sauce produced in Thailand (unit: ppm)
solution with the subtractions of each blank. In this method, no
harmful reagents are used and each operation is not difficult. Colorimetric enzymatic
Sample HPLC
method
All procedures including extraction can be completed within Brand A 42 47
1 hour. Brand B 148 148
Brand C 290 288
Table 3 shows recovery of spiked sample in raw and canned
Brand D 290 309
tuna. Good recovery was obtained at the concentration from
10 ppm through 75 ppm. When commercial raw tuna is stored
at 20°C for 2 days, histamine levels can be increase. As shown
Histamine dehydrogenase
in Table 4, high concentration of histamine is detected, and
the result of this method showed good correlation with HPLC, Histamine 1-methoxy PMS WST-8 formazan
(color development)
AOAC fluorescent assay, and EIA methods. Histamine concen-
tration in a commercial canned tuna sample, in which a high Imidazole 1-methoxy PMS WST-8
Acetaldehyde (reduced form)
concentration of histamine was present, was also measured by
four different methods. The result using this enzymatic method Interference by oxidation and
reduction substrates
was consistent with the other methods. In conclusion, this sim-
Color development could be interfered or enhanced by
ple and rapid enzymatic histamine test is as reliable as the other oxidation and reduction substrates
conventional methods tested.
Figure 4: Interference by oxidation and reduction substrates

4.3 Interference by oxidation and reduction substrates in

some foods
In this method, the tetrazolium salt develops color due to the
electron transfer, which is caused by histamine decomposition
by enzyme. If any oxidation or reduction substrate is present
in the reaction, interference or enhancement of color develop-
ment is possible with the result that histamine level is not de-
termined properly. Such substrates are known to be present in
fermented foods such as miso, soy sauce, and fish sauce.
Sample dilution can eliminate these interferences when signif-

4
icant concentrations of histamine are present in food. In fact,
Dilution Washing Elution
we found that the histamine could be determined in fish sauce
when sample is diluted at least 200 times. Four commercial fish
sauces were tested in two methods - 1) the rapid enzymatic test Column

as described above and 2) HPLC. As shown in Table 5, the rap-

id enzymatic test produced results in very close agreement to Histamine
Testing
the results in HPLC method. Thus, the rapid enzymatic method Interfering substance

could produce in accurate and consistent results for fish sauce.

① Soy sauce is diluted 100-folds by buffer
In the case of soy sauce, because histamine levels are not typ-
② Diluted solution is applied to column
ically as high as in fish sauce, dilution is not applicable. In this    Histamine adheres to column

case, pre-treatment of sample with column could be applied ③ Column is washed by buffer
   Interfering substances are washed out
(Figure 5). The procedure of column-treatment in soy sauce is
④ Histamine is eluted by buffer
shown below.
Figure 5: Eliminating interference by column-treatment

1. 0.1 ml of soy sauce is diluted in 10 ml of 20 mM phosphate

buffer (pH6.0).
900
2. This solution is applied to column (Sep-Pak Plus Accell CM,
Waters). 800

3. Column is washed by 10 mL of 20 mM phosphate buffer (pH 700

Correlation coefficient＝0.998
6.0). 600
Enzymatic method

4. Histamine is eluted by 10 ml of 175 mM NaCl in 20 mM phos- 500

phate buffer (pH7.0). The eluent was applied to histamine test. 400
As shown in Figure 6, various concentration of histamine in 300
soy sauce were determined by enzymatic method and HPLC 200
method. Both results were consistent and their correlation co- 100
efficient was 0.998.
0
0 100 200 300 400 500 600 700 800 900

5 Conclusion HPLC method

Figure 6: Correlation of Enzymatic method and HLPC method

(Sample: Soy sauce)
The rapid enzymatic method for the measurement of histamine
in food has been developed and Histamine Test (AOAC-PTM
041802) is commercially available. A high correlation was ob-
served between the results obtained by this method and those
obtained by conventional methods. This method has a lot of
advantages; extraction procedure is simple, calibration curve
of histamine can be easily drawn, and the histamine amount
can be rapidly measured. In the FAO Meeting report (2012), this
method was cited as reliable analysis as same as AOAC fluores-
cent assay, HPLC, and ELISA methods. Thus, this method would
be a useful tool for assessing food spoilage and preventing his-
tamine poisoning.

5
 6 References

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FAO/WHO (Food and Agriculture Organization of the United

Nations/World Health Organization) (2012) Public health risks of
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Food and Drug Administration (FDA). (2011). Fish and fishery

products hazards and controls guidance, 4th edn. Washington
DC: department of health and human services, Food and Drug
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Kose, S., Kaklikkaya, N., Koral, S., Tufan, B., Buruk, D. K., and Alydin,
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Pessatti, T. L., Fontana, J. D., and Pessatti, M. L. (2004). Spectro-

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Regulation 2073/2005/EC. Commission Regulation EC No

2073/2005. Microbiological criteria for foodstuffs. OJ. L 338, 1-26. 2-1-1, Nishi-shinbashi, Minato-ku, Tokyo 105-0003, Japan
Phone +81-3-5521-5481 Fax +81-3-5521-5498
Sato, T., Horiuchi, T., and Nishimura, I. (2005). Simple and rapid E-mail biochemifa@mail.kikkoman.co.jp
determination of histamine in food using a new histamine de- URL https://biochemifa.kikkoman.co.jp/e/
http://www.kikkomana3.com/
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This article is excerpted and modified from "A technical journal on food chemistry & chemicals,” September 2016, pp. 86-90. ©2018 Kikkoman Corp..(H-002-2Y200401)