Histamine Level and Histamine-Forming Bacteria in Dried ﬁsh Products Sold in Penghu Island of Taiwan

Food Control 21 (2010) 1234–1239

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Histamine level and histamine-forming bacteria in dried ﬁsh products sold in Penghu Island of Taiwan

Yu-Ru Huang a,*, Kuan-Ju Liu a, Hung-Sheng Hsieh b, Cheng-Hong Hsieh b, Deng-Fwu Hwang c, Yung-Hsiang Tsai d a Department of Food Science, National Penghu University, Penghu, Taiwan, ROC b Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan, ROC c Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan, ROC d Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung, Taiwan, ROC article info abstract

Article history: Forty-six dried fish products sold in retail markets in Penghu Islands, Taiwan were purchased and tested Received 25 July 2009 to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH, salt content, Received in revised form 2 February 2010 water content, water activity (Aw), total volatile basic nitrogen (TVBN), aerobic plate count (APC), Esch- Accepted 9 February 2010 erichia coli and total coliform (TC) in all samples ranged from 5.60 to 7.57, 1.8% to 27.1%, 19.32% to 61.90%, 0.63 to 0.92, 10.41 to 168.56 mg/100 g, 3.18 to 9.28 log CFU/g, <3 to 210 MPN/g and <3 to >1100 MPN/g, respectively. There had 30.4% of the tested dried fish products to contain histamine level more than 5 mg/ Keywords: 100 g of FDA guideline for scombroid fish and/or product. Among them, all of the nine samples of Selar- Histamine iodes leptolepis had the highest histamine content of 6.31–47.90 mg/100 g. Thirteen histamine-producing Hygienic quality Biogenic amines bacterial strains isolated from tested samples produced 8.7–531.2 ppm of histamine in trypticase soy Histamine-forming bacteria broth supplemented with 1.0% L-histidine (TSBH). Among these histamine-producing bacteria, Enterobac- ter aerogenes (one strain) isolated from S. leptolepis sample was proven to be a prolific histamine-former. Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.

1. Introduction content (5–25%). However, large amounts of histamine have often been detected in commercial fishery products of India, including Histamine is the causative agent of scombroid poisoning, a salt-dried products, which are not subjected to thermal treatment food-borne chemical hazard. Scombroid poisoning is usually a mild as the same as in Taiwan, could be the cause of some histamine illness with a variety of symptoms including rash, urticaria, nausea, outbreaks (Chakrabarti, 1991, 1993). Jeyasekaran and Jeyashakila vomiting, diarrhea, flushing, and tingling and itching of the skin (2003) reported histamine-forming bacteria were found to be high (Taylor, 1986). Severity of the symptoms can vary considerably in salted Indian ilisha (75%), salted tiger perch (70%), salted lethri- with the amount of histamine ingested and the individual’s sensi- nids (33%), and salted seer fish (24%) sold in India. Cadaverine tivity to histamine. Scombroid fish such as tuna, mackerel, bonito, formers were also high in salted seer fish (66%), dried anchovies and saury that contain high levels of free histidine in their muscle (48%), salted Indian ilisha (42%), salted tiger perch (40%) and salted are often implicated in scombroid poisoning incidents (Taylor, lethrinids (29%). Therefore, the biogenic amine amounts of salt- 1986). However, several species of nonscombroid fish such as dried fish products in Taiwan are expected to survey. mahi–mahi, bluefish, herring, and sardine have often been impli- Furthermore, an incident of histamine fish poisoning occurred cated in incidents of scombroid poisoning. Histamine is not the due to the consumption of dried sardine in Osaka, Japan (Kanki, only chemical responsible for toxicity, but its toxic effects are en- Yoda, Ishibashi, & Tsukamoto, 2004). Recently, an incident of hanced due to the presence of potentiating amines such as putres- food-borne poisoning causing illness in three victims due to inges- cine and cadaverine (Taylor & Sumner, 1986). tion of dried milkfish occurred in February, 2006, in southern Tai- Salt-drying is an ancient processing method of seafood in the wan. The suspected milkfish sample contained 616 ppm of world that involves several steps including back-cutting, degutting, histamine and Raoultella ornithinolytica was identified the major salting and sun-drying for several days. The dried product acquires histamine-producing bacterium responsible for the high content a hard consistency with low water activity (Aw, 0.75) and high salt of histamine in the implicated milkfish sample (Tsai, Kung, Chen, Chang, & Wei, 2007). * Corresponding author. Tel.: +886 6 9264115x3806; fax: +886 6 9260259. Biogenic amines are formed mainly through the decarboxylation E-mail address: [email protected] (Y.-R. Huang). of specific free amino acids by exogenous decarboxylases released

0956-7135/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2010.02.008 Y.-R. Huang et al. / Food Control 21 (2010) 1234–1239 1235 by the microbial species associated with the seafood. Many differ- slurry was then measured using a Corning 145 pH meter (Corning ent bacterial species of the Enterobacteriaceae family are known Glass Works, Medfield, MA, USA). The salt content in each sample to possess histidine decarboxylase and have the ability to produce was determined according to the AOAC procedure (1995) by histamine. The species included Morganella (Proteus) morganii, Kleb- homogenizing 2 g of dried fish sample with 18 ml of distilled siella pneumoniae, Hafnia alvei, Proteus vulgaris, Proteus mirabilis, water. The homogenate was titrated with 0.1 M AgNO3 using 10% Enterobacter aerogenes, Enterobacter cloacae, Serratia fonticola, Serra- w/v K2CrO4 solution as an indicator. The water content was con- tia liquefaciens, Raoultella (formerly Klebsiella) planticola, R. ornithin- ducted with the standard gravimetric method by drying 1–3 g of olytica, Providencia stuartii and Citrobacter freundii (Ababouch, Afila, a test sample at 102.0 ± 2.0 °C under atmospheric pressure for Rhafiri, & Busta, 1991; Kim et al., 2001a, 2001b, 2003; López-Sabat- 2 h. Consistency of mass was tested by additional drying steps of er, Rodrí guez-Jerez, Roig-sagués, & Mora-Ventura, 1994; Tsai et al., 1 h until the difference in mass did not exceed 0.5 mg. Water activ- 2005). In addition to the enteric bacteria, Clostridium spp., Vibrio ity was determined by an Electric Hygrometer (Hygrodynamics, alginolyticus, Acinetobacter lowffi, Plesiomonas shigelloides, Pseudo- Inc., Silver Spring, MD) at 27 °C. monas putida, Pseudomonas fluorescens, Aeromonas spp., and Photo- bacterium spp. have also been reported as histamine producers 2.3. Microbiological analysis and isolation of histamine-forming (Lopez-Sabater, Rodriguez-Jerez, Hernandez-Herrero, Roig-Sagues, bacteria & Mora-Ventura, 1996; Okuzumi, Hiraishi, Kobayashi, & Fujii, 1994; Yatsunami & Echigo, 1991). Yatsunami and Echigo (1991, A 25 g portion of the dried fish sample was homogenized at 1992, 1993) identified halotolerant Staphylococcus spp., Vibrio high speed for 2 min in a sterile blender with 225 ml sterile potas- spp., and Pseudomonas III/IV-NH as the histamine formers from fer- sium phosphate buffer (0.05 M, pH 7.0). The blender was sterilized mented salted sardine and fish products. Hernandez-Herrero, Roig- by autoclaving for 15 min at 121 °C. The homogenates was serially Sagues, Rodriguez-Jerez, and Mora-Ventura (1999) and Rodriguez- diluted with a sterile phosphate buffer, and 1.0 ml aliquot of the di- Jerez, Mora-Ventura, Lopez-Sabater, and Hernandez-Herrero lute was spread on aerobic plate count (APC) agar (Difco, Detroit, (1994) isolated histamine-producing Staphylococcus epidermidis, MI, USA) containing 0.5% NaCl. Bacterial colonies were counted Staphylococcus xylosus, Klebsiella oxytoca, E. cloacae, Pseudomonas after the plates were incubated at 35 °C for 48 h. Bacterial numbers cepaciae, and Bacillus spp. from salted Spanish anchovies. in the dried fish samples were expressed as log10 colony forming Dried fish products are important food item for consumption in units (CFU)/g. Penghu Island, Taiwan. There has been no report on the occurrence To isolate histamine-forming bacteria, 0.1 ml aliquot of the of biogenic amine, including histamine, histamine-forming bacte- sample dilute was spread on histamine-forming bacterium isola- ria, total coliform and Escherichia coli in dried fish products in Pen- tion agar (HBI agar) fortified with L-histidine (Niven, Jeffreg, & Cor- ghu Island. Therefore, 46 dried fish products sold in retail markets lett, 1981). Following incubation of the differential agar plates for 4 in Penghu Islands, Taiwan were collected and analyzed for the lev- dat35°C, colonies with blue or purple color on the plates were els of biogenic amine, total coliform, E. coli, total volatile basic picked and further streaked on trypticase soy agar (TSA) (Difco) nitrogen (TVBN) and histamine-forming bacteria. to obtain pure cultures. Their ability to produce biogenic amines was determined by inoculating the isolates in trypticase soy broth (TSB) (Difco) supplemented with 1% L-histidine (TSBH) and incu- 2. Materials and methods bated without shaking at 35 °C for 24 h. One milliliter of the culture broth were taken for quantization of biogenic amines. The 2.1. Samples method of biogenic amines analysis is described in Section 2.6. Analyses of total coliform and E. coli in these dried fish samples Forty-six dried fish products were purchased from retail mar- were conducted using the three-tube most probable number kets in Penghu Island, Taiwan. The species of dried fish included (MPN) method (FDA, 1992). Lauryl sulfate tryptose broth (LST bullet mackerel (Auxis tapeinosoma, three samples), round scad broth) and brilliant green lactose bile (2%) broth (BGLB broth) were (Decapterus maruadsi, four samples), smooth-tailed trevally (Selar- used for presumptive and confirmatory tests for total coliform, iodes leptolepis, nine samples), Pacific round herring (Etrumeus respectively. E. coli was determined by using the LST broth and teres, six samples), stout moray (Gymnothorax eurostus, three sam- EC broth. Cultures that showed positive production of gas in EC ples) and blue-spotted stingray (Dasyatis kuhlii, three samples). In broth were then confirmed by eosine methylene blue agar (EMBA) addition, the sample of single species collected are less than three and IMViC test. items, the data presents merged into others, including spotted tan- gingi (Scomberomorus guttatus, two samples), silver round herring 2.4. Identification of histamine-forming isolates (Spratelloides gracilis, two samples), silver rabbitfish (Siganus fuscescens, two samples), sand lizardfish (Synodus dermatogenys, The presumptive histamine-forming isolates were identified on two samples), Chinese emperor (Lethrinus haematopterus, two sam- the basis of morphology, Gram stain, endospore stain, catalase and ples), flatfish (Pseudorhombus cinnamomeus, two samples), rockfish oxidase reaction. Cell morphology was examined by phase-con- grouper (Epinephelus quoyanus, two sample), netted sweetlips trast microscopy. Gram reaction, the presence of oxidase and cata- (Plectorhinchus flavomaculatus, one sample), chicken grunt (Par- lase were determined as described by Smiberit and Krieg (1981). apristipoma trilineatum, one sample), red mullet goatfish (Upeneus The identity of histamine-forming isolates was further confirmed japonicus, one sample) and Japanese topeshark (Hemitriakis japon- by amplifying and sequencing approximately 1400 bp of the 16S ica, one sample). All collected samples were wrapped in aseptic ribosomal DNA (rDNA) for bacteria (Kuhnert, Capaul, Nicolet, & bags, placed in ice, and immediately transported to the laboratory Frey, 1996; Kuhnert, Heyberger-Meyer, Nicolet, & Frey, 2000). for use within 2 h. Amplification of histamine-forming bacteria was performed using the reported primers UNI-L (50-AGAGTTTGATCATGGCTCAG-30) 2.2. pH value, salt content, water content and water activity and UNI-R (50-GTGTGACGGGCGGTGTGTAC-30) for detecting 16S determination ribosomal DNA (rDNA) of bacteria (Kuhnert et al., 1996, 2000). Each presumptive histamine producer was cultured overnight in Dried fish samples (10 g) were homogenized in sterile blenders 2 ml of TSB at 35 °C and then centrifuged at 5000g for 10 min. with 10 ml of distilled water to make thick slurry. The pH of this The cell pellet was washed and resuspended in 0.5 ml of TE-buffer 1236 Y.-R. Huang et al. / Food Control 21 (2010) 1234–1239

(10 mM Tris–HCl, 1 mM EDTA; pH 8.0), and then lysed by 200 llof tonitrile:water at a flow rate of 1.0 ml/min for 19 min, followed by 20% sodium dodecyl sulfate (SDS). After the solution was boiled for a linear increase to 90:10 acetonitrile/water (1.0 ml/min) for 1 min, 20 min and the cellular debris was discarded following centrifuga- and then the ratio of acetonitrile/water decreased to 50:50 (1.0 ml/ tion at 13,000g for 3 min, the total DNA in the supernatant was min) for 10 min. precipitated with 70% ethanol and used as template DNA for PCR. PCR amplification was performed in 20 ll reaction mixture con- 2.7. Statistical analysis taining 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2,20 qmol of each primer, 0.2 mM concentration for each of four deox- All statistical analyses were performed using the Statistical ynucleotide triphosphates, 0.5 U of Taq DNA polymerase (Applied Package for Social Sciences, SPSS Version 9.0 for windows (SPSS Biosystems, Foster City, CA, USA), and template DNA (10 ng). Inc., Chicago, IL, USA). Value of P < 0.05 was used to indicate signif- Amplifications were carried out for 35 cycles (94 °C for 30 s, icant deviation. 55 °C for 30 s, and 72 °C for 60 s) in a GeneAmp PCR 2400 Thermal Cycler (Applied Biosystems) with an initial denaturation at 94 °C for 4 min and a final extension at 72 °C for 7 min (Kuhnert et al., 3. Results and discussion 1996, 2000). Amplicons were detected by electrophoresis on a 1.5% agarose gel and then stained with ethidium bromide. Ampli- Values of the pH, water content, water activity (Aw), salt con- cons were purified using a QIAquick PCR Purification Kit (Qiagen, tent, total volatile basic nitrogen (TVBN), aerobic plate count Valencia, CA, USA) and eluted in Tris–HCl (10 mM, pH 8.5) prior (APC), E. coli, and total coliform of the 46 dried fish samples from to sequencing. The amplified DNA was directly sequenced with Penghu Island are presented in Table 1. The levels of pH, water con- the ABI TaqDye Deoxy Terminator Cycle sequencing kit and ABI tent, water activity, salt content, APC, TVBN, E. coli, and total coli- Model 377 automated DNA sequencer (Applied Biosystems). The form in all samples ranged from 5.60 to 7.57, 19.32–61.90%, sequences were analyzed with the BLAST (NCBI) for identification 0.63–0.92, 1.8–27.1%, 10.41–168.56 mg/100 g, 3.18–9.28 log CFU/ of histamine-forming bacteria. g, <3 to 210 MPN/g and <3 to >1100 MPN/g, respectively. The rates of unacceptable dried fish samples were 56.5% (26/46) for TVBN, 2.5. Determination of total volatile base nitrogen (TVBN) based on the decomposition limit level of 30 mg/100 g for fish quality determination. The unacceptable rate of TVBN in individual The TVBN content of the dried fish sample was measured by the dried fish sample was 66.7% (2/3) for A. tapeinosoma, 75.0% (3/4) method of Conway’s dish (Cobb, Aoaniz, & Thompson, 1973). The for D. maruadsi, 77.8% (7/9) for S. leptolepis, 33.3% (2/6) for E. teres, TVBN extract of the fish sample in 6% trichloroacetic acid (TCA, Sig- 33% (1/3) for G. eurostus, 66.7% (2/3) for D. kuhlii, 50.0% (9/18) for ma, St. Louis, Mo., USA) was absorbed by boric acid and then ti- other species. Among them, the highest unacceptable rate for TVBN trated with 0.02 N HCl. The TVBN content was expressed in mg/ was 77.8% (7/9) for S. leptolepis samples. Besides, TVBN values on E. 100 g fish. quoyanus (two samples), D. kuhlii (one sample) and S. fuscescens (one sample) exceeded the 100 mg/100 g. TVBN is mainly com- 2.6. Biogenic amine analysis posed of ammonia and primary, secondary and tertiary amines. Its increase is related to the activity of spoilage bacteria and endog- Each dried fish sample was ground in a Waring Blender for enous enzymes. 3 min. The ground samples (5 g) were transferred to 50 ml centri- The rates of unacceptable dried fish samples were 34.8% (16/46) fuge tubes and homogenized with 20 ml of 6% trichloroacetic acid for APC, based on the Taiwanese regulatory standard of 6.47 log (TCA) for 3 min. The homogenates were centrifuged (10,000 g, CFU/g. The unacceptable rate of APC in individual dried fish sample 10 min, 4 °C) and filtered through Whatman No. 2 filter paper was 33.3% (1/3) for A. tapeinosoma, 100.0% (4/4) for D. maruadsi, (Whatman, Maidstone, England). The filtrates were then placed 11.1% (1/9) for S. leptolepis, 33.3% (2/6) for E. teres, 0% (0/3) for G. in volumetric flasks, and TCA was added to bring to a final volume eurostus, 100% (3/3) for D. kuhlii, and 22.2% (4/18) for other kinds. of 50 ml. Samples of standard biogenic amine solutions and 1 ml According to report of Kalaimani, Gopakumar, and Nair (1988), the aliquots of the dried fish extracts were derivatized with dansyl APC of 103 CFU/g is normal in salted and dried fishery products. chloride. The dansyl derivatives of amines were prepared accord- However, Shakila, Lakshmanan, and Jeyasekaran (2002) have re- ing to the previously described method (Chen et al., 2010). To ported that the APC in the salted fish ranged from 104 to 105 1 ml of mixed amines solution containing 0–20 lg of each amine CFU/g, while dried fish product contained about 104 CFU/g. or 1 ml of dried fish extract, 0.2 ml of 2 M sodium hydroxide and Although the average water content (30.88%) of G. eurostus samples 0.3 ml of saturated sodium bicarbonate were mixed with. The solu- was not significantly different from those in the other samples, but tion was added 2 ml of 1% dansyl chloride solution dissolved in the average Aw (0.68) and APC (4.5 log CFU/g) in this dried fish acetone, and allowed to stand at 40 °C for 45 min. After the reac- samples were the lowest (Table 1). The dried salted fish product tion, 100 ll of ammonia was added and allowed to stand for with less than 40% moisture showed the lower APC, indicating that 30 min. Acetonitrile was added to a final volume of 5 ml and the low moisture could inhibit the growth of aerobic bacteria. In addi- solution was centrifuged (10,000 g, 5 min, 4 °C). The supernatant tion, two S. leptolepis samples and one A. tapeinosoma sample con- was filtered through a 0.45-lm membrane, and then used for tained more than 50 MPN/g of E. coli, the allowable limit of the HPLC. One milliliter of each presumptive histamine-forming bacte- Taiwanese regulatory standard. These poor microbiological quali- rial culture broth was also dansylated using the same procedures ties showed that the dried fishery products in Taiwan have been for dried fish extracts to ensure that each bacterium was hista- unhygienically handled or processed. The dried fishery samples mine-former. were made by sun-drying for several days and kept at room tem- Biogenic amines were determined with a high performance li- perature, resulting that flies may easily contaminate. Concerning quid chromatograph (Young Lin, Anyang, Korea) consisting of a the hygienic quality, Hsu et al. (2009) also reported that the tested Model 9100 pump, a Rheodyne Model 7125 syringe loading sample dried milkfish products, with 12.5% (4/32) samples contained injector and a Model 9160 photodiode array detector (set at greater than 50 MPN/g of E. coli. 254 nm). A HiQsil C18 column (5 lm, 150 4.6 mm, i.d., KYA Table 2 summarized the contents of biogenic amines in the Technology, Yokohama, Japan) was used for chromatographic sep- tested dried fish products. Nine samples of S. leptolepis had the aration. The gradient elution program began with 50:50 (v/v) ace- highest average histamine content of 21.07 mg/100 g. Table 3 Y.-R. Huang et al. / Food Control 21 (2010) 1234–1239 1237

Table 1 Values of the pH, water content, salt content, total volatile basic nitrogen (TVBN), aerobic plate count (APC), E. coli and total coliform (TC) in 46 dried ﬁsh products.

Scientiﬁc name No. of pH Water Aw Salt TVBN APC E. coli TC samples content (%) content (%) (mg/100 g) (log CFU/g) (MPN/g) (MPN/g) Auxis tapeinosoma 5.92–6.78 44.40–48.41 0.84–0.91 2.2–5.2 24.64–40.04 6.30–8.20 <3–75 <3–>1100 3 (6.25 ± 0.46)aA (47.00 ± 2.26)A (0.87 ± 0.04)D (4.07 ± 1.63)A (33.60 ± 8.00)A (7.38 ± 0.98)BC Decapterus maruadsi 6.14–6.54 26.24–41.98 0.81–0.91 2.8–19.8 29.00–63.56 7.16–9.28 <3 <3–>1100 4 (6.27 ± 0.19)A (33.88 ± 6.50)AB (0.85 ± 0.04)CD (7.3 ± 8.3)AB (45.40 ± 15.09)A (8.04 ± 0.91)C Selariodes leptolepis 5.98–6.46 21.77–54.48 0.65–0.78 1.9–20.2 10.79–68.70 3.28–6.88 <3–210 <3–1100 9 (6.16 ± 0.16)A (31.09 ± 12.57)A (0.70 ± 0.04)AB (5.3 ± 5.8)A (44.68 ± 18.17)A (5.67 ± 1.05)AB Etrumeus teres 5.60–6.57 23.61–59.40 0.69–0.92 3.0–16.7 18.34–57.90 3.61–8.71 <3 <3–110 6 (6.24 ± 0.35)A (43.85 ± 16.73)A (0.84 ± 0.08)CD (7.78 ± 6.35)AB (30.91 ± 13.98)A (5.75 ± 2.06)AB Gymnothorax eurostus 6.16–6.40 19.32–42.75 0.64–0.73 3.3–14.6 14.56–33.19 3.60–6.03 <3 <3 3 (6.24 ± 0.14)A (30.88 ± 11.72)A (0.68 ± 0.05)A (7.2 ± 6.4)A (21.04 ± 10.53)A (4.50 ± 1.33)A Dasyatis kuhlii 6.53–7.57 25.71–46.10 0.69–0.91 2.3–27.1 10.42–156.30 6.56–8.04 <3 <3–20 3 (7.07 ± 0.52)B (35.30 ± 10.25)AB (0.79 ± 0.11)BCD (16.8 ± 12.9)B (69.81 ± 76.62)A (7.23 ± 0.75)B Othersb 6.00–7.54 24.11–61.90 0.63–0.89 1.8–19.6 10.41–168.56 3.18–8.79 <3 <3–>1100 18 (6.49 ± 0.38)A (37.43 ± 11.32)A (0.77 ± 0.07)ABC (7.96 ± 6.36)AB (53.17 ± 46.70)A (5.49 ± 1.52)AB

a Mean ± SD values in the same column with different letters are statistically different (P < 0.05). b Scomberomorus guttatus (two samples), Spratelloides gracilis (two samples), Siganus fuscescens (two samples), Synodus dermatogenys (two samples), Lethrinus haematopterus (two samples), Pseudorhombus cinnamomeus (two samples), Epinephelus quoyanus (two samples), Plectorhinchus ﬂavomaculatus (one sample), Parapristipoma trilineatum (one sample), Upeneus japonicus (one sample) and Hemitriakis japonica (one sample).

Table 2 Contents of biogenic amines in 46 dried ﬁsh products.

Scientiﬁc name No. of Contents of biogenic amine (mg/100 g) samples Puta Cad Try Phe Spd Spm His Tyr Auxis 3NDb-1.56 ND–0.33 ND–0.35 ND ND–75.96 8.57–30.21 ND–11.20 ND–3.44 tapeinosoma (0.82 ± 0.78)c (0.11 ± 0.19) (0.12 ± 0.20) (25.62 ± 43.60) (16.26 ± 12.10) (3.93 ± 6.30) (1.15 ± 1.99) Decapterus 4 ND–15.30 ND–3.00 ND ND–6.80 ND–8.80 7.89–73.80 ND–12.73 ND–19.53 maruadsi (4.13 ± 7.47) (1.33 ± 1.56) (1.70 ± 3.40) (2.20 ± 4.40) (25.80 ± 32.13) (3.18 ± 6.37) (4.88 ± 9.77) Selariodes 9 ND–31.80 4.13–65.50 ND–12.27 ND–38.10 ND ND–10.32 6.31–47.90 ND–15.05 leptolepis (6.33 ± 9.79) (14.50 ± 19.31) (1.36 ± 4.09) (4.23 ± 12.70) (1.15 ± 3.44) (21.07 ± 13.12) (5.92 ± 6.38) ND–4.50 ND–16.5 ND–0.35 ND–4.50 ND–51.5 ND–67.30 ND–4.50 ND Etrumeus teres 6 (1.14 ± 1.89) (3.02 ± 6.63) (0.06 ± 0.14) (0.75 ± 1.84) (8.58 ± 21.02) (25.83 ± 29.75) (0.91 ± 1.80) ND Gymnothorax 3 ND–3.49 ND–6.32 ND ND ND–8.07 ND–8.98 ND–2.62 ND–1.34 eurostus (1.61 ± 1.76) (2.21 ± 3.56) (2.69 ± 4.66) (2.99 ± 5.18) (0.99 ± 1.42) (0.45 ± 0.77) Dasyatis kuhlii 3 ND–2.04 2.06–15.30 ND ND ND–16.9 ND–63.30 ND–0.48 ND (0.68 ± 1.18) (8.22 ± 6.67) (5.91 ± 9.53) (25.89 ± 33.19) (0.16 ± 0.28) Othersd 18 ND–43.60 ND–292.80 ND–8.85 ND–24.40 ND–34.1 ND–64.40 ND–15.66 ND–58.90 (3.63 ± 10.34) (34.14 ± 75..34) (0.49 ± 2.09) (1.65 ± 5.75) (2.34 ± 8.04) (14.56 ± 21.33) (3.30 ± 5.24) (5.20 ± 14.05)

a Put: putrescine; Cad: cadaverine; Try: tryptamine; Phe: 2-phenylethylamine; Spd: spermidine; Spm: spermine; His: histamine; and Tyr: tyramine. b ND: not detected (amine level less than 0.05 mg/100 g). c Mean ± SD. d Scomberomorus guttatus (two samples), Spratelloides gracilis (two samples), Siganus fuscescens (two samples), Synodus dermatogenys (two samples), Lethrinus haematopterus (two samples), Pseudorhombus cinnamomeus (two samples), Epinephelus quoyanus (two samples), Plectorhinchus ﬂavomaculatus (one sample), Parapristipoma trilineatum (one sample), Upeneus japonicus (one sample) and Hemitriakis japonica (one sample).

Table 3 Distribution of the histamine contents in 46 dried fish products. tion level because histamine is not uniformly distributed in a decomposed fish. Therefore, if 5 mg/100 g is found in one section, Content of histamine Dried fish products there is a possibility that other units may exceed 50 mg/100 g (mg/100 g) No. of samples % of samples (Lehane & Olley, 2000). Several countries have set legal limits of <4.9 32 69.6 histamine concentrations that are regarded as safe for human con- 5.0–19.9 10 21.7 sumption: Australia, 20 mg/100 g (Australian Food Standards Code, 20.0–49.9 4 8.7 2001), Europe, 10 mg/100 g (EC, 2003) and South Africa, 10 mg/ Total 46 100 100 g (South African Bureau of Standards, 2001). In this study, we apply the stricter FDA level of 5 mg/100 g is an indicator of shows the distribution of histamine contents in tested dried fish decomposition. products, with 30.4% (14/46) samples containing greater than In our present study, all of nine samples of S. leptolepis had the 5 mg/100 g of histamine, the allowable limit of the US Food and highest histamine content of 6.31–47.90 mg/100 g. However, no Drug Administration (FDA) for scombroid fish and/or products. one with greater than 50 mg/100 g of histamine as the toxicity le- Based on an analysis of poisoning episodes, Shalaby (1996) sug- vel based on FDA. But the four samples of S. leptolepis with greater gested the following guideline levels for histamine content of fish: than 20 mg/100 g of histamine may be sufficient to cause the (i) <5 mg/100 g (safe for consumption); (ii) 5–20 mg/100 g (possi- symptoms of scombroid poisoning (CDC, 2000; EEC, 1991). bly toxic), (iii) 20–100 mg (probably toxic), and (iv) >100 mg/ Although the tested P. cinnamomeus and H. japonica samples of 100 g (toxic and unsafe for human consumption). In addition, the others did not contain high levels of histamine, they contained FDA guidelines (1995) for tuna, mahi–mahi and related fish specify 292.8 and 166.4 mg/100 g (data not shown) of cadaverine, respec- 50 mg/100 g as the toxicity level and 5 mg/100 g as the defect ac- tively (Table 2). The presence of cadaverine and putrescine may 1238 Y.-R. Huang et al. / Food Control 21 (2010) 1234–1239

Table 4 Identiﬁcation of histamine-forming bacteria isolated from the dried ﬁsh products by 16S rDNA analysis, and their production of biogenic amines (ppm) in culture broth.

Strain Strain source Organism identiﬁed Percentage Gene bank accession number Levels of biogenic amine (ppm) identity (%) Trya Put Cad His Tyr PHF 1-1 Siganus fuscescens Citrobacter freundii 100 AB244451.1 NDb ND ND 27.9 16.5 PHF 1-3 Siganus fuscescens C. freundii 100 AB244451.1 13.6 10.2 3.9 49.4 17.7 PHF 1-5 Siganus fuscescens C. freundii 100 AB244451.1 17.2 ND 2.5 34.5 17.8 PHF 2-1 Synodus dermatogenys Staphylococcus saprophyticus 100 EU419944.1 20.7 1.7 2.7 20.7 18.0 PHF 19-1 Hemitriakis japanica S. saprophyticus 100 EU419944.1 7.9 ND 0.6 12.0 ND PHF 24-1 Pseudorhombus cinnamomeus Pantoea agglomerans 100 EU879089.1 8.7 18.7 4.5 9.6 ND PHF 24-1 Pseudorhombus cinnamomeus P. agglomerans 100 EU879089.1 ND ND ND 11.3 ND PHF 34-1 Dasyatis kuhlii Salmonella typhi 100 EU118115.1 0.4 37.8 411.8 14.3 ND PHF 37-1 Etrumeus teres Sal. typhi 100 EU118115.1 ND 30.0 331.8 21.5 ND PHF 23-1 Selaroides leptolepis C. freundii 100 AB244451.1 ND 6.1 16.1 57.1 ND PHF 35-2 Selariodes leptolepis Salmonella enteritidis 100 EU118102.1 ND ND 236.1 15.1 1.0 PHF 35-4 Selariodes leptolepis S. typhi 100 EU118114.1 ND 18.0 224.5 8.7 ND PHF 45-2 Selariodes leptolepis Enterobacter aerogenes 100 AB244456.1 ND ND 388.3 531.2 7.8

a Try: tryptamine; Put: putrescine; Cad: cadaverine; His: histamine; Tyr: tyramine. b ND: not detected (amine level less than 0.05 ppm). synergistically enhance histamine toxicity by inhibiting histamine ilarly, E. aerogenes isolated from S. leptolepis sample was a potent metabolizing enzymes such as diamine oxidase and histamine histamine-forming bacteria capable of producing 531.2 ppm of his- methyl transferase (Antoine et al., 2002). Recently, we demon- tamine and 388.3 ppm of cadaverine in TSBH. C. freundii isolated strated that most of tested dried milkfish products (78.1%) sold from S. leptolepis and S. fuscescens samples was a weak hista- in Taiwan had histamine levels greater than the FDA guideline of mine-former, producing only 27.9–57.1 ppm histamine in TSBH 5 mg/100 g for scombroid fish and/or product (Hsu et al., 2009). broth (Table 4). Although high unacceptable rate was detected in dried milkfish Potential histamine-forming bacteria, including halo-tolerant and S. leptolepis products tested in previous and this studies, bacteria, have been isolated from salted fish samples. Staphylococ- respectively, very few cases of food-borne histamine intoxication cus spp., Vibrio spp. and Pseudomonas III/IV-NH were isolated from have been reported due to consumption of dried fish products. fermented salted sardine products (Yatsunami & Echigo, 1991, Symptoms of histamine poisoning are not particularly definitive. 1992), while S. epidermidis, S. xylosus, K. oxytoca, E. cloacae, P. cepa- Therefore, histamine intoxication is frequently misdiagnosed as ciae and Bacillus spp. were isolated from salted Spanish anchovies an allergic reaction. (Hernandez-Herrero et al., 1999; Rodriguez-Jerez et al., 1994). Biogenic amines are formed mainly through the decarboxyl- However, only one of these histamine-forming species, Staphylo- ation of specific free amino acids by exogenous decarboxylases re- coccus saprophyticus was isolated in this study from salt-dried fish leased by microbial species associated with seafood. Many samples. Staphylococcus spp. were the most frequently reported different bacterial species are known to possess histidine decar- histamine-formers in fermented salted fish, accounting for nearly boxylase and have the ability to produce histamine (Rawles, Flick, 50% of histamine-forming microorganisms. They were usually & Martin, 1996). Table 4 lists the identity of 13 histamine-forming shown to have powerful histamine-forming activity (Yatsunami bacteria isolated from the dried fish products as determined by 16S & Echigo, 1991, 1992). For example, S. epidermidis and S. capitis, iso- rDNA sequences using NCBI database analysis. Thirteen histamine- lated from salted Spanish anchovies, produced more than producing bacterial strains, capable of producing 8.7–531.2 ppm of 1000 ppm and 400 ppm of histamine, respectively, in TSBH broth histamine in trypticase soy broth supplemented with 1.0% L-histi- (Hernandez-Herrero et al., 1999). The S. capitis recently isolated dine (TSBH), were identified as C. freundii (four strains), Pantoea from mustard pickle products in Taiwan was a potent histamine- agglomerans (two strains), Staphylococcus saprophyticus (two former, capable of producing more than 1000 ppm of histamine strains), Salmonella typhi (three strains), Salmonella enteritidis in TSBH broth (Kung et al., 2006). However, the recently isolated (one strain) for weak histamine formers (557.1 ppm), and E. aerog- S. pasteuri from miso products in Taiwan was a weak histamine- enes (one strain) for prolific histamine formers (531.2 ppm). Some former, producing only 28.1 ppm histamine in TSBH broth (Kung, of them also produced different amounts of other biogenic amines Tsai, & Wei, 2007). Similarly, the S. saprophyticus isolated from through the action of their respective decarboxylase enzymes on the samples of Selaroides dermatogenys and Hemitriakis japanica various amino acids that also existed in the culture medium (Table in this study was also a weak histamine-former capable of 4). Recently, we reported that 30 histamine-producing bacterial producing only 20.7 and 12.0 ppm of histamine in TSBH broth, strains were isolated from dried milkfish (Chanos chanos) products, respectively (Table 4). Since Staphylococci are the major microbial capable of producing 5.4–562 ppm of histamine in TSBH broth, and groups that inhabit human skin, it is reasonable to expect that they identified as E. aerogenes (seven strains) and Citrobacter sp. (one would be transferred to food products through considerable hu- strain) for prolific histamine formers (=553 ppm), and S. xylosus man contact during preparation and processing. (10 strains), S. sciuri (one strain), Bacillus thuringiensis (two strains), Lakshmanan, Jeya-Shakila, and Jeyasekaran (2002) pointed out C. freundii (five strains), K. pneumoniae (one strain) and E. cloacae that the incidence of halophytic amine forming bacteria appeared (three strains) for weak histamine formers (551.4 ppm) (Hsu the fluctuation in the sardine processing. It was around 20% in et al., 2009). fresh sardines, reached a maximum level of 84%, decreased during Enterobacter spp. and Citrobacter spp. were most frequently re- the drying process, and finally was not found in salt-dried sardines ported prolific histamine formers in various species of scombroid after final drying. However, Moori, Cann, and Taylor (1988) ob- fish such as tuna, albacore and sailfish (Kim et al., 2001a, 2001b; served that the penetration of histamine-forming bacteria of the Lopez-Sabater et al., 1996; Tsai, Kung, Lee, Lin, & Hwang, 2004). gut into the inner muscle of whole fish is possible during the The E. aerogenes isolate from the sailfish fillets was found to be a sun-drying process by rupture of the belly walls. Furthermore, con- prolific histamine-former capable of producing more than tamination may happen when the fish is eviscerated during man- 1000 ppm of histamine in the culture broth (Tsai et al., 2004). Sim- ual processing, cut with a dirty knife, and mixed in the same Y.-R. Huang et al. / Food Control 21 (2010) 1234–1239 1239 tank with salting process for a long time. These may be the reasons Kanki, M., Yoda, T., Ishibashi, M., & Tsukamoto, T. (2004). Photobacteriu that E. coli, TC and histamine-forming bacteria strains occurred in phosphoreum caused a histamine fish poisoning incident. International Journal of Food Microbiology, 92, 79–87. the fishery products. Kim, S. H., Barros-Velazquez, J., Ben-Gigirey, B., Eun, J. B., Jun, S. H., Wei, C. I., et al. (2003). Identification of the main bacteria contributing to histamine formation in seafood to ensure product safety. Food Science and Biotechnology, 12, 4. Conclusion 451–460. Kim, S. H., Field, K. G., Morrissey, M. T., Price, R. J., Wei, C. I., & An, H. (2001a). Source and identification of histamine-producing bacteria from fresh and temperature This study, to determine the safety of 46 dried fish products sold abused albacore. Journal of Food Protection, 64, 1035–1044. in Penghu Island, Taiwan, showed that the TVBN content in 26 Kim, S. H., Field, K. G., Morrissey, M. T., Price, R. J., Wei, C. I., & An, H. (2001b). 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