artikel about Histamin Evaluation

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DOI 10.18551/rjoas.2021-12.23
HISTAMIN EVALUATION ON FROZEN TUNA LOINS (THUNNUS SP.) AS AN EXPORT
REQUIREMENT STANDARD IN DENPASAR LABORATORY, BALI, INDONESIA
Budiadnyani I Gusti Ayu1
*, Astiana Ika1
, Samanta Pinky Natalia1
,
Handayani Tri Rahayu2
, Sasabila Vita Ayu1
1
Politeknik Kelautan dan Perikanan Jembrana, Indonesia
2
Politeknik Kelautan dan Perikanan Sidoarjo, Indonesia
*E-mail: igustiayu678@gmail.com
ABSTRACT
Tuna is one of the essential export commodities in Indonesia. Histamine in this species can
cause a food safety hazard. Therefore, a maximum standard of histamine content must be
applied to frozen loin tuna products to reduce food safety risks. This study aims to identify
the histamine content of frozen tuna loins. Samples were 121 frozen tuna loins from nine
different companies. Histamine testing was done using the ELISA method based on AOAC-
RI 070703. Test results on 121 frozen tuna loins from nine companies confirmed that one
sample, coded EP2/H/1-6, had a histamine level of >50 ppm. This sample had a histamine
level of 83 ppm. Since only one sample had a histamine level of >50 ppm, it showed that
these exporting companies had applied good handling to prevent high histamine content in
the products they exported.
KEY WORDS
ELISA, export, loin tuna, histamine.
Tuna is one of the most economically important fish species globally and is the third-
largest fishery commodity in Indonesia after shrimp and bottom fish. The Indonesian tuna
fishery business has been attractive to foreign countries and is attracting many world fish
businesspeople. The high demand for tuna in the global market and the high economic value
of the global tuna trade make the tuna industry increases rapidly (DJPDS, 2019). The
increase in Indonesian tuna exports is also strongly influenced by increased tuna production.
The growth of tuna export value increased by 3.8% from 2000 to 2009 (Suwarno et al., 2012).
However, tuna exports face various food safety barriers, including the issue of histamine, CO,
heavy metals (mercury and cadmium), inadequate sanitation handling, and a poorly
maintained distribution process. Histamine ranks first as the cause of rejection of tuna
exports. Data from 2011 to 2017 showed 27 rejections of tuna exports to the European Union,
of which 13 cases were caused by the inability of the export tuna products to meet the
standard of histamine levels (Irawati et al., 2019).
The government of Indonesia has issued the Indonesian National Standard (Standar
Nasional Indonesia – SNI) on frozen tuna products coded as SNI 01-2710:2006 (BSN, 2006).
This standard requires companies exporting frozen tuna products to comply with a histamine
content of a maximum of 100 ppm or the standards required by the destination country.
These companies need to carry out good tuna handling so that these export requirements
can be met. DJPDS data (2019) shows that frozen tuna loins rank as the third-largest
Indonesian tuna export commodity. The export destination countries for Indonesian frozen
tuna loins in 2018 were dominated by the United States, followed by Japan and Vietnam, and
other countries including France, Australia, Hong Kong, Mexico, Canada, Singapore, and the
Philippines. The Food and Drug Administration sets the histamine content standard on frozen
tuna products for the United States of America at the maximum of 50 ppm (FDA, 2020).
Histamine is a biogenic compound in fish due to the breakdown of free histidine, a type
of essential amino acid. Histamine is formed from the decarboxylation of free histidine in the
fish body, especially fish species from the Scombridae family (Radjawane et al., 2016).
Histamine is formed by decarboxylation of histidine catalyzed by histidine decarboxylase
enzyme present in the fish body or carried out by bacteria such as Morganella morganii.

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Histamine is toxic if found in large quantities in the body (Maulayani et al., 2016). Histamine
poisoning can occur after consuming fish that contain high histamine. The effects of
histamine poisoning include allergy symptoms, fever, vomiting, nausea, diarrhoea, dizziness,
and palpitations. Therefore, it is vital to evaluate histamine levels in frozen loin tuna to be
exported. The purpose of this study was to evaluate the histamine level of frozen tuna loins
as an export requirement.
MATERIALS AND METHODS OF RESEARCH
Samples of frozen tuna loins for histamine evaluation came from nine companies
exporting frozen tuna loin products. The evaluation was done in a laboratory in Denpasar,
Bali, accredited by the National Accreditation Committee of Indonesia (Komite Akreditasi
Nasional – KAN) as a testing laboratory (Laboratorium Penguji coded as LP-670-IDN). The
laboratory has fulfilled the standard of SNI ISO/IEC 17024-2008 in Bali as an export
requirement. Samples came in a frozen state in ready-to-export packages, such as a tuna
Saku, steak, half, cube, or ground meat. Samples were checked for the temperature, and it
should not exceed 14o
C because a temperature higher than 14o
C will affect the histamine
content. Samples tested for histamine levels were coded in a unique way to maintain their
confidentiality. The sample code consisted of the name of the company and the time of the
test/type of test/number of samples tested in one test.
The histamine evaluation involved the Veratox histamine testing kit. The histamine
evaluation was done using a wash buffer solution containing 10mM PBS-Tween Veratox, a
saline phosphate buffer solution of pH 7.4, distilled water, histamine tuna reagent (Veratox
blue, green, and red reagent), and 70% alcohol. The tools used were plastic conical tubes,
white and red wells, 100µl yellow tip, 100µl finned pipettes, tube racks, well racks, large trays,
PE (polyethene) plastic, plastic bags, stickers, pens, stat fax reader, ice (ice pack gel), a
digital scale, scissors, filter tubes, filters, plastic cups, a stopwatch, reagent containers,
rubber, and washing bottles.
The evaluation employed the ELISA method using an automatic ELISA reader within
10 minutes with a wavelength of 450 ± 10 nm and a reference filter with a wavelength of 630
nm. Samples were tested using histamine level control of 0, 2.5, 10, 20, and 50 ppm.
Frozen tuna loin samples were thawed first and then weighed as much as 50g.
Samples were divided into two: 10 grams for individual testing and 3 grams for composite
testing. The sample was then given 90 ml of distilled water and homogenized for 15-20
seconds—the process was repeated every 5 minutes for 3 times. Extracted and
homogenized samples were then filtered to get the filtrate. Filtering was done using a filter
with cotton, and a filter tube was put under the filter to collect the filtered result. The collected
filtrate samples were transferred to a conical tube containing 10 mL of phosphate buffer
saline (PBS) using a yellow tip micropipette of 100 µL. The centrifuge tube with the filtrate
sample was homogenized manually by raising and lowering the tube for 6 seconds. Then,
100 µL of the filtrate was transferred to a red well that had been given 100 µL of blue reagent
(conjugate solution), which was then homogenized by raising and lowering the yellow tip
micropipette 3 times. After that, 100 µL of the filtrate was taken and transferred into a white
well coated with an antibody at the bottom of the well to bind histamine. After the filtrate was
placed in the white well, it was allowed to stand for 10 minutes at room temperature of 18 to
30°C so that the filtrate was bound to the antibody in the well.
When the binding process for the filtrate and antibody was completed, the wells were
then washed using a wash buffer solution containing 10 mM PBS-Tween with a pH of 7.4 to
remove unbound compounds. This solution was used by mixing it with 1 liter of distilled water.
The well washing was carried out 3 times. The green reagent (substrate solution) was added
after washing. The green reagent addition was done by taking 100 µL of the liquid using a
yellow tip micropipette and then putting it into a well that had been washed. After the addition
was complete, the filtrate was let to stand for 10 minutes at a temperature of 18 -to30°C.
When the reagent is added, the well will be light blue if the sample is negative, and the well
will be transparent if the sample is positive. The addition of the red reagent (red stop-solution)

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was carried out after the addition of the green reagent was completed—the red reagent was
to stop the staining when the green reagent was added. The red reagent was added by
taking 100 µL of the liquid using a yellow tip micropipette and then putting it into the well
where the color change reaction happened. After that, the samples were tested using an
automatic ELISA reader within 10 minutes with a wavelength of 450 ± 10 nm and a reference
filter of 630 nm.
RESULTS AND DISCUSSION
Tuna belongs to the Scromboidae family that can produce scrombotoxin, which causes
histamine poisoning. In addition to come from the Scromboidae family, histamine poisoning
may also happen due to the low quality of fish during processing, such as not being fresh
(Maulayani et al., 2016). The lowest the quality of the fish (the most damaged it has), the
more histamine is formed in the fish. The process of damage to tuna occurs rapidly in the
tropics (Wodi et al., 2014) due to hot temperatures (>27°C) and high humidity. This condition
is ideal for microbial growth and enzyme activity in the fish body.
Table 1 – Histamine Evaluation Results on Frozen Loin Tuna
Sample Grup Sample Code Sample Number Sample Types Histamine Evaluation Results
1
BG1/H/10
AS1/H/6
EP1/H/6
10
6
6
Frozen tuna steak 0.0
0.0
0.0
2 BM1/H/10 10 Tuna Saku 1 sample has a histamine level of 2.6
3 BG2/H/10 10 Frozen tuna steak 0.0
4
BG3/H/8
EP2/H/6
DA1/H/4
RM1/H/8
8
6
4
8
Frozen tuna steak
0.4-1
2 sample has a histamine level of 24 and 83
0.2-0.6
0.0
5 BG4/H/10 10 Frozen tuna steak 0.0
6
BG5/H/6
EP3/H/7
6
7 Frozen tuna steak
0.0
1 sample has a histamine level of 0.7
7 HM1/H/6 6 Frozen tuna steak 0.0
8
EP4/H/6
AS2/H/4
6
4
Frozen tuna steak
1 sample has a histamine level of 0.7
2 sample has a histamine level of 0.7 and 5.9
9 BG6/H/6 6 Frozen tuna steak 0.0
10 CK1/H/4 4 Frozen tuna steak 0.0
11 CD1/H/6 6 Frozen tuna steak 0.2-0.4
Table 2 – Histamine Evaluation Results on Frozen Tuna Loins with a Value of > 0.0 ppm
No
Company Code
BG AS BM EP DA CD
1 0.7 5.9 2.6 24 0.4 0.3
2 0.4 0.7 83 0.6 0.2
3 0.5 0.7 0.5 0.2
4 0.4 0.7 0.2 0.4
5 0.6 0.4
6 0.7 0.2
7 1
8 0.6
Notes
8 samples from 50
tested samples
2 samples from 10
tested samples
1 sample from 10
tested samples
4 samples from 25
tested samples
4 samples from 4
tested samples
6 samples from 6
tested samples
Our evaluation of the frozen tuna loins involved 121 samples from nine different
companies. These nine companies exported their tuna. Our test results confirmed that most
of the samples did not contain histamine (0.0 ppm). There were samples with histamine
levels ranged from 0.2 ppm to 83 ppm. The requirements for the histamine content of export
tuna vary from country to country, depending on the requirement set by the responsible
agency, such as the FDA (Food and Drug Administration), EC (European Commission),
Codex, and SNI. The FDA requires a maximum histamine content of 50 ppm (FDA, 2020),
EC requires a maximum histamine content of 100 ppm (EC 2005), and SNI requires a
maximum histamine content of 100 ppm (BSN, 2006). The minimum standard of histamine is
50 ppm, which the FDA requires for companies sending frozen loin tuna to the United States.
There is a large gap between the SNI standard and the FDA. This difference in requirements
can affect competitiveness, especially the quality of export products (Resnia et al., 2020).
Thus, the recommended histamine content limit is < 50 ppm to avoid the tuna products being

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219
returned by buyers. When fish products are detected to have a histamine level of 50 ppm,
the possibility for other fish body parts to also have that histamine level is also high.
Several test samples were detected to contain histamine, including (a) one sample of
tuna Saku coded BM1/H/1-10 of 2.6 ppm, (b) two samples of frozen tuna steak coded
EP2/H/1-6 of 24 and 83 ppm, (c) four samples of frozen tuna steak coded DA1/H/1-4 of 0.2-
0.6 ppm, (d) one sample of frozen tuna steak coded EP3/H/1-7 of 0.7 ppm, (e) one sample of
frozen tuna steak coded EP4/H/1-6 of 0.7 ppm, (f) two samples of frozen tuna steak coded
AS2/H/4 of 0.7 and 5.9 ppm, and (g) six samples of frozen tuna steak coded CD1/H/1-6 of
0.2-0.4 ppm. Based on these results, only one sample was found with a histamine level
exceeding 50 ppm, namely 83 ppm for the sample code EP2/H/6 from the EP company. The
results were submitted to the company, and it was recommended not to be exported to
destination countries with histamine standards < 50 ppm, such as the United States.
However, the company can export its tuna if the buyer requires histamine content of <100
ppm as in European Union countries. Indonesian processed tuna products in the European
Union market has an above-the-average competitiveness level from 2003 to 2018
(Rahmansyah et al., 2021). This is influenced by import duty rates, export product prices, the
exchange rate of the rupiah against the US dollar, and export volume.
Tuna exports to European Union countries with a histamine level of 83 ppm can also
be at risk of being rejected because there is the potential for an increase in the histamine
level if a loss of temperature happens on the way to the destination countries. Histamine
formation can be inhibited by keeping tuna in frozen storage, while the growth of histamine-
forming bacteria can be controlled by cold storage below 4.4o
C (Lee et al., 2012). The
formation of histamine is not only caused by decarboxylation bacteria that convert histidine
into histamine but is also caused by the histidine decarboxylase enzyme on the fish itself.
There were 25 samples included in four groups of frozen tuna Saku tested for
histamine levels from the EP company, namely EP1/H/1-6, EP2/H/1-6, EP3/H/1-7, and
EP4/H/1-6. The EP2/H/1-6 group had high histamine levels compared to the other test
sample groups. Of these six samples, only one was detected with a ppm content of > 50 ppm,
namely 83 ppm. This indicates that the EP company has implemented good handling of
frozen tuna steak, but there might have been an error when handling frozen tuna steak
coded EP2/H/6. The company can perform traceability on tuna samples that have high
histamine levels. The company should separate products with the same production code with
samples having high histamine levels from other products with histamine levels that still meet
export requirements. Next, the company must explore the causes of the high histamine
content in these tuna products. This high histamine content can be caused by a loss of
control of the cold chain and post-harvest handling. An increase in temperature and the
presence of stomach contents in tuna during storage can increase histamine concentrations
(Mahusain et al., 2017). Histamine control in the exported frozen tuna products must have
been implemented since the fish was caught (Santoso et al., 2020). Tuna fishing vessels
must already have a Good Fish Handling (Cara Penanganan Ikan yang Baik – CPIB)
certificate and a Fish Processing Unit (Unit Pengolahan Ikan – UPI) applying the HACCP
(Hazard Analysis Critical Control Point) throughout the process chain. The high histamine in
export tuna products may be due to the poor handling of tuna in primary production (Irawati
et al., 2019). Primary production includes the handling of fish at the time of capture.
Inadequate knowledge regarding sanitation and hygiene and the poorly managed cold chain
from handling fish onboard to UPI will increase fish histamine levels.
CONCLUSION
Evaluation results confirmed that from 121 test samples from nine different companies,
only 1 sample with a histamine content < 50 ppm, namely 83 ppm. Thus, it can be concluded
that tuna exporting companies have implemented good handling of frozen tuna loin products
to prevent high histamine levels. Frozen export tuna products sent without meeting the
required product quality will cause losses to the exporting company because there is a risk
that the product will be returned.

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