Assessing the risk of histamine from the Indonesian salted-boiled fish (pindang)

Indonesian salted-boiled fish, known as pindang, is the second largest traditional fish product in Indonesia and has important economic and social impacts for Indonesian societies, especially those who live in coastal areas. Pindang is made from Scombroid fish, such as tuna, mackerel and scad. Pindang processors are family- or neighbourhood-based industries equipped with basic or traditional processing equipment, with processing techniques passed from generation to generation. Salting and steaming (or boiling) are combined as methods of preservation in pindang processing. Some of the largest pindang processing centres are located in Pelabuhan Ratu-Sukabumi District (West Java Province), Juwana (Central Java Province) and Klungkung (Bali Province). As with many other Scombroid-based products, pindang has a high risk of being contaminated with histamine. Reports indicate that pindang has caused several histamine fish poisoning (HFP) outbreaks in Indonesia. HFP is one of the major problems in seafood industries worldwide. This foodborne intoxication is caused by the ingestion of fish or other foods containing high levels of histamine. The formation of histamine depends on the availability of free histidine in fish flesh and the presence of histamine-producing bacteria (HPB) that harbour the histidine decarboxylase (Hdc) enzyme and convert histidine into histamine. Histamine is a heat-stable amine and cannot be destroyed by common cooking practices such as boiling, steaming, heating or freezing. In Pelabuhan Ratu, Sukabumi District, West Java Province, Indonesia, pindang processing relies on the availability of fresh tuna from the local catchment. However, due to the limited number of fresh fish caught from the surrounding waters, pindang processors use frozen tuna as an alternative raw material. When frozen fish is used, a thawing step is introduced in the preparation phase of pindang processing. Improper thawing practices of raw material may lead to HPB growth and histamine formation. Therefore, this study aimed to provide a better understanding of the current processing of pindang in Pelabuhan Ratu and how the processing affects histamine accumulation in the final product. Field observations and laboratory experiments were combined to collect relevant data on pindang processing, to evaluate the behaviour of HPB isolated from pindang and to identify intervention strategies which can be applied to prevent histamine accumulation in the final product as well as to improve the safety and quality of the product. To identify HPB that are commonly found in pindang, fish samples were collected from several pindang processors in Pelabuhan Ratu. Fish from different processing stages (raw, thawed/washed, and cooked) were sampled. HPB were screened using modified Niven’s agar and a PCR-based assay and identified using the API system and 16S rRNA gene sequencing. The ability of the isolates to produce histamine was evaluated using histidine decarboxylase broth and an ELISA method. In total, fourteen different HPB genera were identified, i.e. Citrobacter, Enterobacter, Escherichia, Erwinia, Hafnia, Klebsiella, Morganella, Pantoea, Proteus, Providencia, Pseudomonas, Serratia, Shigella and Vibrio. In particular, Enterobacter sp., Klebsiella sp., Morganella morganii and Providencia were found on thawed fish. Further confirmation showed that an Enterobacter aerogenes isolate produced ≥ 4,000 μg/ml histamine in histidine-rich broth media after 24 h incubation at 30°C. A predictive modelling study was done to understand the growth of E. aerogenes isolated from pindang and its ability to produce histamine, at different conditions relevant to pindang production. The isolate was inoculated into histidine broth with different concentrations of salt (1.5, 6, 10 and 20% w/v) and in fish with 6% of salt concentration, then incubated at 10, 15, 20 and 30°C. Growth and histamine formation during the incubation were recorded. The growth parameters (growth rate-μ and lag time-λ) of E. aerogenes were modelled using the Roberts and Baranyi model as implemented in the DMFit version 3.5 Excel® add-in. A multiple linear regression and the extended Ratkowsky model were used to describe the effect of temperature and salt on the growth of E. aerogenes. Histamine formation during incubation was determined by combining a yield factor to the growth model. The results showed linear relationships among the bacterial growth rate, with temperature and salt. In broth experiment, the highest histamine level (>6,000 μg/ml) was produced at 30°C with 1.5% of salt. Although the isolate survived at 10% salt, the amount of histamine produced was very low (less than 10 μg/ml). To evaluate the growth and histamine formation of E. aerogenes under conditions that mimic the thawing process of pindang, a broth experiment was done by growing the isolate to different cell densities and freezing at -20°C for 72 h. Thawing of the frozen culture was done at 4, 18 and 25°C for 4 h. The bacterial growth and histamine formation during the thawing process were recorded. The inoculum was reduced from the initial counts after freezing, and no bacterial growth was observed during subsequent thawing at different temperatures for 4 h. For treatment with high initial bacterial counts, the histamine production during thawing was more pronounced than treatment with low initial counts. To describe the existing processing practices of pindang, field observations were done at several processors located in Pelabuhan Ratu. Two types of pindang processing were observed. The first processing type used fresh Skipjack tuna, while the second used frozen Little Eastern tuna. Pindang processing took four to five hours, depending on the type of raw materials used. When frozen fish were used as raw material, the processing required an additional 60 - 90 minutes of preparation, which predominantly was thawing. The histamine levels of fresh Skipjack and pindang made from this fish were very low (less than 50 μg/g). However, when frozen Little tuna was used as raw material, the histamine levels of some raw fish were higher than 100 μg/g. This greater level of histamine was also observed for cooked fish. Microbial community profiles for fish from different sources and at different processing steps, identified using the Automated Ribosomal Intergenic Spacer Analysis (ARISA), showed that the bacterial composition of pindang was affected by the type of fish used as raw material, fish processing (salt addition and heating), as well as post-processing contamination. Six critical control points (CCPs) were identified from pindang processing, i.e. receiving raw materials, thawing and washing, paper wrapping and arranging fish in the cooking container, salting, cooking and post-process handling. The first CCP in pindang processing is the choice of raw materials. To prevent high histamine levels in the final product, the histamine levels of raw fish should not exceed the allowable limit of fresh fish (100 mg/kg as regulated by the Indonesian Standardization Body). For processing units that use frozen fish as raw material, the second CCP is the thawing and washing step. Thawing becomes a critical step as temperature abuse and time delays likely occur during this step. These conditions allow HPB, in particular, to grow and to produce histamine. Since histamine is a heatstable amine, the subsequent processing steps of pindang, e.g., paper wrapping, salting and cooking, do not eliminate pre-formed histamine. In general, the histamine levels of raw tuna used in pindang processing will determine the histamine levels in the final product. When fresh tuna is not available, frozen tuna can be used as raw material. However, the temperature during the preparation step, especially during thawing and washing should be maintained below 18°C to avoid bacterial activity and histamine formation. Furthermore, the use of higher concentrations of salt (10%) is recommended to prevent the growth of halophilic HPB that may otherwise survive salting. Improved hygienic practices during processing are also necessary. For example, clean and circulated water should be used during thawing and washing to prevent contamination from the environment. Proper packaging combined with low storage temperature of the cooked product might also prevent post-processing contamination and increase the product shelf-life.