Material and methods

Pindang was prepared using Longtail tuna (Thunnus sp.) and Eastern Little tuna (Euthynnus sp.) as raw materials. Fish (raw, washed, thawed and cooked) (Table 2-1) were collected from traditional fish processors in Pelabuhan Ratu, Sukabumi District, West Java Province, Indonesia and kept cold or frozen during transportation. Longtail tuna were caught on a single day of fishing in local waters and were processed while fresh, while the Eastern Little tuna were obtained from fish

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Table 2-1. Sampling point and sample description

Fish source Sampling points Sample terms

Fresh Longtail tuna Raw fish, fresh upon arrival Raw-local Fish after washing Washed-local Fish after salting and boiling Cooked-local Frozen Eastern little tuna Raw fish, frozen upon arrival Raw-imported

Fish after thawing and washing Thawed-imported

Fish after salting and boiling Cooked-imported

2.2.2.Screening of histamine-producing bacteria

Bacterial isolation followed the procedures described by Torido et al. (2014) with modification of psychrotrophic incubation temperature, i.e., due to the instrument unavailability, the incubation of psychrotrophic isolates was done at 17°C. Prior to isolation, 5 g fish tissue was incubated in 45 ml of histidine broth medium containing 1% bacteriological peptone (Oxoid, UK), 0.3% yeast extract (Oxoid, UK), 1.5% sodium chloride (Merck, USA) and enriched with 0.5% of L-histidine (Sigma, USA). Bacteria were incubated at 30°C for 24 h for mesophilic and at 17°C for 72 h for psychrotrophic HPB species. One ml of enriched culture was then transferred into 9 ml of fresh histidine broth and incubated under the same conditions.

After incubation, 100 µl of the broth culture was streaked onto Niven agar (Niven et al., 1981) containing 0.5% tryptone (Oxoid, UK), 0.5% yeast extract (Oxoid, UK), 0.5% sodium chloride (Merck, USA), 2.7% L-histidine (Sigma, USA), 2% agar, 0.006% bromcresol purple with pH adjusted to 5.5 – 5.7. Plates were incubated at 30°C for 24 h for mesophilic and at 17°C for 72 h for psychrotrophic HPB. Typical HPB colonies that were a purple-greyish colour with or without media colour change were streaked onto tryptone soy agar (TSA) (Oxoid, UK) containing 2% (w/v) of sodium chloride (Merck, USA), and incubated at the same time and temperature conditions as above to isolate single colonies of HPB for DNA extraction.

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Qualitative assays to identify HPB from isolates positive in Niven agar were done by growing the isolates in 96-well plates of histidine decarboxylase broth (HD broth) incubated at 30°C for 24 h and at 17°C for 72 h. HD broth contained (per litre): 2 g peptone (Oxoid, UK), 1 g Lab-Lemco powder (Oxoid, UK), 5 g NaCl (Merck, Germany), 10 g L-histidine (Sigma, USA), 10 ml of 0.1% bromcresol green solution, 10 ml of 0.2% of chlorophenol red solution and had pH adjusted to 5.3 (Yamani & Untermann, 1985). Presumptive histidine decarboxylase - positive bacteria were identified by colour change from light green to violet.

Isolates that returned positive results in both the Niven agar and HD broth tests were further examined by screening for the hdc gene. Bacterial DNA was extracted using the Isolate II Genomic DNA Kit (Bioline, AU) according to the manufacture’s protocol. hdc-F (5’-TCH ATY ARY AAC TGY GGT GAC TGG RG-3’) and hdc-R (5’-CCC ACA KCA TBA RWG GDG TRT GRC C-3’) primer pairs which target a 709-bp fragment were used in the PCR reaction (Takahashi et al., 2003). The reaction was performed in 50 µl reaction mixtures containing 25 µl of MyTaq™ HS Mix (Bioline, AU), 20 pmoles of each primer (Geneworks, AU), 10 ng of template DNA and nuclease-free water using the following conditions: initial denaturation at 94°C for 4 min, followed by 35 cycles of amplification (94°C for 1 min, 58°C for 1 min, 72°C for 1 min) and final extension at 72°C for 4 min. PCR products were separated in 1.5% agarose gel (Promega, AU) in 1x Tris-borate-EDTA (TBE) buffer at 90V for 50 min and visualized with a Gel Doc™ XR Imaging System (BIO-RAD, AU).

2.2.3.Histamine measurement

Approximately half of the isolates positive in hdc gene screening were chosen at random and tested for their ability to produce histamine using the quantitative HistaSure™ ELISA Fast Track kit (LDN Labor Diagnostika Nord GmbH & Co. KG, Germany), which applies indirect competitive ELISA allowing binding competition between the bound (histamine) and unbound (acylated-histamine) antigen with the histamine antiserum conjugate. Mesophilic and psychrotrophc isolates were grown in histidine broth at 30°C for 24 h and 15°C for 72 h, respectively. Morganella morganii (32) and

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Morganella psychrotolerans (FE 3_12) from the Tasmanian Institute of Agriculture (TIA) culture collection were used as reference isolates for mesophilic and psychrotrophic incubation conditions, respectively. Prior to histamine analysis, a 1.5 ml aliquot of culture was transferred into micro centrifuge tubes and centrifuged at 10,000 g for 2 min at room temperature. Up to 1 ml of the supernatant was then filtered using 0.2 µm sterile filters (Corning, Germany) and placed into new and clean microtubes for histamine analysis. Samples were acylated prior to the quantification. The ELISA test was performed according to the manufacture’s protocol. Six histamine standard solutions (0, 0.12, 0.4, 1.2, 4 and 12 ppm) with the absorbance value of each measured at 450 nm, were used to derive a calibration curve. The 4-parameter logistic (4PL) non-linear regression model (Cox et al., 2004) of the calibration curve was used to calculate the measured histamine concentration.

Histamine was also tested from representative samples of raw and cooked fish using HPLC (SNI 2354.10:2009) . Fish flesh (50 g) was ground with a blender and treated with 100 ml of 10% trichloroacetid acid (C2HCl3O2) and centrifuged at 3,000 rpm for 10 min to remove any suspended particulates. Pre-column derivatization was done using o-phtalaldehyde (OPA). Separation was performed using a C18 column in gradient elution with acetonitrile:sodium dihydrogen sulphate (30:70) as the mobile phase. The amount of histamine was quantified by fluorescense at 350 nm (excitation) and 450 nm (emission). Significant differences (P<0.01) between the histamine concentration were analysed using analysis of variance (ANOVA) in Real Statistic Excel add-ins (http://www.real-statistics.com/).

2.2.4.Identification of histamine-producing bacteria

Histamine-producing isolates were randomly selected for identification using the API® 20E biochemical detection system and sequencing of the 16S rRNA gene. Identification with the API® 20E detection system (Biomereux, France) was performed according to the manufacture’s protocol to confirm the identification of Enterobacteriaceae with single colonies of HPB grown overnight in TSA. Catalase and oxidase tests were performed to complete organism identification. Identification was

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based on the numerical profile of each test determined using the Analytical Profile Index databases (V4.1).

Isolates able to produce histamine were further identified by sequencing of the 16S rRNA gene using the universal primers 27F (5’-AGA GTT TGA TCC TGG CTC AG-3’) and 1492R (5’-TAC GGY TAC CTT GTT ACG ACT T-3’) (Weisburg et al., 1991). Forty microliters of each reaction mixture contained 20 µL of MyTaq™ HS Mix (Bioline, AU), 8 pmoles of each primer (Genework, AU) and 2.4 µL of template DNA. The PCR amplification was started with initial denaturation at 95°C for 2 min, followed by 30 cycles of 95°C for 1.5 min, 55°C for 1.5 min, and 72°C for 1.5 min and final extension at 72°C for 1.5 min (Bjornsdottir-Butler et al., 2011b). PCR products were cleaned using the Ultra Clean PCR clean up kit (MOBIO™, AU) and sent to the Ramaciotti Centre for Genomics (University of New South Wales, Sydney, AU) for sequencing.

Sequences were corrected using the BioEdit Sequence Alignment Editor (Hall, 1999) in any noisy areas or areas of poor resolution. Appropriate IUB codes were inserted for double traces. The SEQMATCH tool of the Ribosomal Database Project (http://rdp.cme.msu.edu/) combined with the nucleotide database of NCBI (http://www.ncbi.nlm.nih.gov/nucleotide/) were used to find similar sequences, type strains and outgroup taxa. ClustalW sequence alignment and phylogenetic analyses were done using the Neighbour Joining on pairwise distance (p-distance) method with 1000

bootstrap replications in the Molecular Evolutionary Genetics Analysis (MEGA) version 6.0 software (Tamura et al., 2013).