CC BY
1
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
1Politeknik Kelautan dan Perikanan Jembrana, Indonesia 2Politeknik 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.
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 14oC because a temperature higher than 14oC 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, 100^l yellow tip, 100^l 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 ^L. The centrifuge tube with the filtrate sample was homogenized manually by raising and lowering the tube for 6 seconds. Then, 100 ^L of the filtrate was transferred to a red well that had been given 100 ^L of blue reagent (conjugate solution), which was then homogenized by raising and lowering the yellow tip micropipette 3 times. After that, 100 ^L 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 ^L 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)
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 ^L 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
BG1/H/10 10 Frozen tuna steak 0.0
1 AS1/H/6 6 0.0
EP1/H/6 6 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
BG3/H/8 8 0.4-1
4 EP2/H/6 DA1/H/4 6 4 Frozen tuna steak 2 sample has a histamine level of 24 and 83 0.2-0.6
RM1/H/8 8 0.0
5 BG4/H/10 10 Frozen tuna steak 0.0
6 BG5/H/6 6 0.0
EP3/H/7 7 Frozen tuna steak 1 sample has a histamine level of 0.7
7 HM1/H/6 6 Frozen tuna steak 0.0
8 EP4/H/6 6 Frozen tuna steak 1 sample has a histamine level of 0.7
AS2/H/4 4 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
Notes
Table 2 - Histamine Evaluation Results on Frozen Tuna Loins with a Value of > 0.0 ppm
_Company Code_
No -
_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
8 samples from 50 2 samples from 10 1 sample from 10 4 samples from 25 4 samples from 4 6 samples from 6 tested samples_tested samples_tested samples_tested samples_tested samples_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
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 0.20.6 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 4.4oC (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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