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 Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan, ROC Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan, ROC d Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung, Taiwan, ROC b c

a r t i c l e

i n f o

Article history: Received 25 July 2009 Received in revised form 2 February 2010 Accepted 9 February 2010

Keywords: Histamine Hygienic quality Biogenic amines Histamine-forming bacteria

a b s t r a c t Forty-six dried fish products sold in retail markets in Penghu Islands, Taiwan were purchased and tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH, salt content, water content, water activity (Aw), total volatile basic nitrogen (TVBN), aerobic plate count (APC), Escherichia 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/ 100 g of FDA guideline for scombroid fish and/or product. Among them, all of the nine samples of Selariodes leptolepis had the highest histamine content of 6.31–47.90 mg/100 g. Thirteen histamine-producing bacterial strains isolated from tested samples produced 8.7–531.2 ppm of histamine in trypticase soy broth supplemented with 1.0% L-histidine (TSBH). Among these histamine-producing bacteria, Enterobacter 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 Histamine is the causative agent of scombroid poisoning, a food-borne chemical hazard. Scombroid poisoning is usually a mild illness with a variety of symptoms including rash, urticaria, nausea, vomiting, diarrhea, flushing, and tingling and itching of the skin (Taylor, 1986). Severity of the symptoms can vary considerably with the amount of histamine ingested and the individual’s sensitivity to histamine. Scombroid fish such as tuna, mackerel, bonito, and saury that contain high levels of free histidine in their muscle are often implicated in scombroid poisoning incidents (Taylor, 1986). However, several species of nonscombroid fish such as mahi–mahi, bluefish, herring, and sardine have often been implicated in incidents of scombroid poisoning. Histamine is not the only chemical responsible for toxicity, but its toxic effects are enhanced due to the presence of potentiating amines such as putrescine and cadaverine (Taylor & Sumner, 1986). Salt-drying is an ancient processing method of seafood in the world that involves several steps including back-cutting, degutting, salting and sun-drying for several days. The dried product acquires a hard consistency with low water activity (Aw, 0.75) and high salt * Corresponding author. Tel.: +886 6 9264115x3806; fax: +886 6 9260259. E-mail address: [email protected] (Y.-R. Huang).

content (5–25%). However, large amounts of histamine have often been detected in commercial fishery products of India, including salt-dried products, which are not subjected to thermal treatment as the same as in Taiwan, could be the cause of some histamine outbreaks (Chakrabarti, 1991, 1993). Jeyasekaran and Jeyashakila (2003) reported histamine-forming bacteria were found to be high in salted Indian ilisha (75%), salted tiger perch (70%), salted lethrinids (33%), and salted seer fish (24%) sold in India. Cadaverine formers were also high in salted seer fish (66%), dried anchovies (48%), salted Indian ilisha (42%), salted tiger perch (40%) and salted lethrinids (29%). Therefore, the biogenic amine amounts of saltdried fish products in Taiwan are expected to survey. Furthermore, an incident of histamine fish poisoning occurred due to the consumption of dried sardine in Osaka, Japan (Kanki, Yoda, Ishibashi, & Tsukamoto, 2004). Recently, an incident of food-borne poisoning causing illness in three victims due to ingestion of dried milkfish occurred in February, 2006, in southern Taiwan. The suspected milkfish sample contained 616 ppm of histamine and Raoultella ornithinolytica was identified the major histamine-producing bacterium responsible for the high content of histamine in the implicated milkfish sample (Tsai, Kung, Chen, Chang, & Wei, 2007). Biogenic amines are formed mainly through the decarboxylation of specific free amino acids by exogenous decarboxylases released

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Y.-R. Huang et al. / Food Control 21 (2010) 1234–1239

by the microbial species associated with the seafood. Many different bacterial species of the Enterobacteriaceae family are known to possess histidine decarboxylase and have the ability to produce histamine. The species included Morganella (Proteus) morganii, Klebsiella pneumoniae, Hafnia alvei, Proteus vulgaris, Proteus mirabilis, Enterobacter aerogenes, Enterobacter cloacae, Serratia fonticola, Serratia liquefaciens, Raoultella (formerly Klebsiella) planticola, R. ornithinolytica, Providencia stuartii and Citrobacter freundii (Ababouch, Afila, Rhafiri, & Busta, 1991; Kim et al., 2001a, 2001b, 2003; López-Sabater, Rodrí guez-Jerez, Roig-sagués, & Mora-Ventura, 1994; Tsai et al., 2005). In addition to the enteric bacteria, Clostridium spp., Vibrio alginolyticus, Acinetobacter lowffi, Plesiomonas shigelloides, Pseudomonas putida, Pseudomonas fluorescens, Aeromonas spp., and Photobacterium spp. have also been reported as histamine producers (Lopez-Sabater, Rodriguez-Jerez, Hernandez-Herrero, Roig-Sagues, & Mora-Ventura, 1996; Okuzumi, Hiraishi, Kobayashi, & Fujii, 1994; Yatsunami & Echigo, 1991). Yatsunami and Echigo (1991, 1992, 1993) identified halotolerant Staphylococcus spp., Vibrio spp., and Pseudomonas III/IV-NH as the histamine formers from fermented salted sardine and fish products. Hernandez-Herrero, RoigSagues, Rodriguez-Jerez, and Mora-Ventura (1999) and RodriguezJerez, Mora-Ventura, Lopez-Sabater, and Hernandez-Herrero (1994) isolated histamine-producing Staphylococcus epidermidis, Staphylococcus xylosus, Klebsiella oxytoca, E. cloacae, Pseudomonas cepaciae, and Bacillus spp. from salted Spanish anchovies. Dried fish products are important food item for consumption in Penghu Island, Taiwan. There has been no report on the occurrence of biogenic amine, including histamine, histamine-forming bacteria, total coliform and Escherichia coli in dried fish products in Penghu Island. Therefore, 46 dried fish products sold in retail markets in Penghu Islands, Taiwan were collected and analyzed for the levels of biogenic amine, total coliform, E. coli, total volatile basic nitrogen (TVBN) and histamine-forming bacteria. 2. Materials and methods 2.1. Samples Forty-six dried fish products were purchased from retail markets in Penghu Island, Taiwan. The species of dried fish included bullet mackerel (Auxis tapeinosoma, three samples), round scad (Decapterus maruadsi, four samples), smooth-tailed trevally (Selariodes leptolepis, nine samples), Pacific round herring (Etrumeus teres, six samples), stout moray (Gymnothorax eurostus, three samples) and blue-spotted stingray (Dasyatis kuhlii, three samples). In addition, the sample of single species collected are less than three items, the data presents merged into others, including spotted tangingi (Scomberomorus guttatus, two samples), silver round herring (Spratelloides gracilis, two samples), silver rabbitfish (Siganus fuscescens, two samples), sand lizardfish (Synodus dermatogenys, two samples), Chinese emperor (Lethrinus haematopterus, two samples), flatfish (Pseudorhombus cinnamomeus, two samples), rockfish grouper (Epinephelus quoyanus, two sample), netted sweetlips (Plectorhinchus flavomaculatus, one sample), chicken grunt (Parapristipoma trilineatum, one sample), red mullet goatfish (Upeneus japonicus, one sample) and Japanese topeshark (Hemitriakis japonica, one sample). All collected samples were wrapped in aseptic bags, placed in ice, and immediately transported to the laboratory for use within 2 h. 2.2. pH value, salt content, water content and water activity determination Dried fish samples (10 g) were homogenized in sterile blenders with 10 ml of distilled water to make thick slurry. The pH of this

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slurry was then measured using a Corning 145 pH meter (Corning Glass Works, Medfield, MA, USA). The salt content in each sample was determined according to the AOAC procedure (1995) by homogenizing 2 g of dried fish sample with 18 ml of distilled water. The homogenate was titrated with 0.1 M AgNO3 using 10% w/v K2CrO4 solution as an indicator. The water content was conducted with the standard gravimetric method by drying 1–3 g of a test sample at 102.0 ± 2.0 °C under atmospheric pressure for 2 h. Consistency of mass was tested by additional drying steps of 1 h until the difference in mass did not exceed 0.5 mg. Water activity was determined by an Electric Hygrometer (Hygrodynamics, Inc., Silver Spring, MD) at 27 °C. 2.3. Microbiological analysis and isolation of histamine-forming bacteria A 25 g portion of the dried fish sample was homogenized at high speed for 2 min in a sterile blender with 225 ml sterile potassium phosphate buffer (0.05 M, pH 7.0). The blender was sterilized by autoclaving for 15 min at 121 °C. The homogenates was serially diluted with a sterile phosphate buffer, and 1.0 ml aliquot of the dilute was spread on aerobic plate count (APC) agar (Difco, Detroit, MI, USA) containing 0.5% NaCl. Bacterial colonies were counted after the plates were incubated at 35 °C for 48 h. Bacterial numbers in the dried fish samples were expressed as log10 colony forming units (CFU)/g. To isolate histamine-forming bacteria, 0.1 ml aliquot of the sample dilute was spread on histamine-forming bacterium isolation agar (HBI agar) fortified with L-histidine (Niven, Jeffreg, & Corlett, 1981). Following incubation of the differential agar plates for 4 d at 35 °C, colonies with blue or purple color on the plates were picked and further streaked on trypticase soy agar (TSA) (Difco) 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 incubated without shaking at 35 °C for 24 h. One milliliter of the culture broth were taken for quantization of biogenic amines. The method of biogenic amines analysis is described in Section 2.6. Analyses of total coliform and E. coli in these dried fish samples were conducted using the three-tube most probable number (MPN) method (FDA, 1992). Lauryl sulfate tryptose broth (LST broth) and brilliant green lactose bile (2%) broth (BGLB broth) were used for presumptive and confirmatory tests for total coliform, respectively. E. coli was determined by using the LST broth and EC broth. Cultures that showed positive production of gas in EC broth were then confirmed by eosine methylene blue agar (EMBA) and IMViC test. 2.4. Identification of histamine-forming isolates The presumptive histamine-forming isolates were identified on the basis of morphology, Gram stain, endospore stain, catalase and oxidase reaction. Cell morphology was examined by phase-contrast microscopy. Gram reaction, the presence of oxidase and catalase were determined as described by Smiberit and Krieg (1981). The identity of histamine-forming isolates was further confirmed by amplifying and sequencing approximately 1400 bp of the 16S ribosomal DNA (rDNA) for bacteria (Kuhnert, Capaul, Nicolet, & Frey, 1996; Kuhnert, Heyberger-Meyer, Nicolet, & Frey, 2000). Amplification of histamine-forming bacteria was performed using the reported primers UNI-L (50 -AGAGTTTGATCATGGCTCAG-30 ) and UNI-R (50 -GTGTGACGGGCGGTGTGTAC-30 ) for detecting 16S ribosomal DNA (rDNA) of bacteria (Kuhnert et al., 1996, 2000). Each presumptive histamine producer was cultured overnight in 2 ml of TSB at 35 °C and then centrifuged at 5000g for 10 min. The cell pellet was washed and resuspended in 0.5 ml of TE-buffer

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(10 mM Tris–HCl, 1 mM EDTA; pH 8.0), and then lysed by 200 ll of 20% sodium dodecyl sulfate (SDS). After the solution was boiled for 20 min and the cellular debris was discarded following centrifugation at 13,000g for 3 min, the total DNA in the supernatant was precipitated with 70% ethanol and used as template DNA for PCR. PCR amplification was performed in 20 ll reaction mixture containing 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 deoxynucleotide triphosphates, 0.5 U of Taq DNA polymerase (Applied Biosystems, Foster City, CA, USA), and template DNA (10 ng). Amplifications were carried out for 35 cycles (94 °C for 30 s, 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., 1996, 2000). Amplicons were detected by electrophoresis on a 1.5% agarose gel and then stained with ethidium bromide. Amplicons were purified using a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and eluted in Tris–HCl (10 mM, pH 8.5) prior to sequencing. The amplified DNA was directly sequenced with the ABI TaqDye Deoxy Terminator Cycle sequencing kit and ABI Model 377 automated DNA sequencer (Applied Biosystems). The sequences were analyzed with the BLAST (NCBI) for identification of histamine-forming bacteria. 2.5. Determination of total volatile base nitrogen (TVBN) The TVBN content of the dried fish sample was measured by the method of Conway’s dish (Cobb, Aoaniz, & Thompson, 1973). The TVBN extract of the fish sample in 6% trichloroacetic acid (TCA, Sigma, St. Louis, Mo., USA) was absorbed by boric acid and then titrated with 0.02 N HCl. The TVBN content was expressed in mg/ 100 g fish. 2.6. Biogenic amine analysis Each dried fish sample was ground in a Waring Blender for 3 min. The ground samples (5 g) were transferred to 50 ml centrifuge tubes and homogenized with 20 ml of 6% trichloroacetic acid (TCA) for 3 min. The homogenates were centrifuged (10,000 g, 10 min, 4 °C) and filtered through Whatman No. 2 filter paper (Whatman, Maidstone, England). The filtrates were then placed in volumetric flasks, and TCA was added to bring to a final volume of 50 ml. Samples of standard biogenic amine solutions and 1 ml aliquots of the dried fish extracts were derivatized with dansyl chloride. The dansyl derivatives of amines were prepared according to the previously described method (Chen et al., 2010). To 1 ml of mixed amines solution containing 0–20 lg of each amine or 1 ml of dried fish extract, 0.2 ml of 2 M sodium hydroxide and 0.3 ml of saturated sodium bicarbonate were mixed with. The solution was added 2 ml of 1% dansyl chloride solution dissolved in acetone, and allowed to stand at 40 °C for 45 min. After the reaction, 100 ll of ammonia was added and allowed to stand for 30 min. Acetonitrile was added to a final volume of 5 ml and the solution was centrifuged (10,000 g, 5 min, 4 °C). The supernatant was filtered through a 0.45-lm membrane, and then used for HPLC. One milliliter of each presumptive histamine-forming bacterial culture broth was also dansylated using the same procedures for dried fish extracts to ensure that each bacterium was histamine-former. Biogenic amines were determined with a high performance liquid chromatograph (Young Lin, Anyang, Korea) consisting of a Model 9100 pump, a Rheodyne Model 7125 syringe loading sample injector and a Model 9160 photodiode array detector (set at 254 nm). A HiQsil C18 column (5 lm, 150  4.6 mm, i.d., KYA Technology, Yokohama, Japan) was used for chromatographic separation. The gradient elution program began with 50:50 (v/v) ace-

tonitrile:water at a flow rate of 1.0 ml/min for 19 min, followed by a linear increase to 90:10 acetonitrile/water (1.0 ml/min) for 1 min, and then the ratio of acetonitrile/water decreased to 50:50 (1.0 ml/ min) for 10 min. 2.7. Statistical analysis All statistical analyses were performed using the Statistical Package for Social Sciences, SPSS Version 9.0 for windows (SPSS Inc., Chicago, IL, USA). Value of P < 0.05 was used to indicate significant deviation. 3. Results and discussion Values of the pH, water content, water activity (Aw), salt content, total volatile basic nitrogen (TVBN), aerobic plate count (APC), E. coli, and total coliform of the 46 dried fish samples from Penghu Island are presented in Table 1. The levels of pH, water content, water activity, salt content, APC, TVBN, E. coli, and total coliform in all samples ranged from 5.60 to 7.57, 19.32–61.90%, 0.63–0.92, 1.8–27.1%, 10.41–168.56 mg/100 g, 3.18–9.28 log CFU/ 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, based on the decomposition limit level of 30 mg/100 g for fish quality determination. The unacceptable rate of TVBN in individual dried fish sample was 66.7% (2/3) for A. tapeinosoma, 75.0% (3/4) for D. maruadsi, 77.8% (7/9) for S. leptolepis, 33.3% (2/6) for E. teres, 33% (1/3) for G. eurostus, 66.7% (2/3) for D. kuhlii, 50.0% (9/18) for other species. Among them, the highest unacceptable rate for TVBN was 77.8% (7/9) for S. leptolepis samples. Besides, TVBN values on E. quoyanus (two samples), D. kuhlii (one sample) and S. fuscescens (one sample) exceeded the 100 mg/100 g. TVBN is mainly composed of ammonia and primary, secondary and tertiary amines. Its increase is related to the activity of spoilage bacteria and endogenous enzymes. The rates of unacceptable dried fish samples were 34.8% (16/46) for APC, based on the Taiwanese regulatory standard of 6.47 log CFU/g. The unacceptable rate of APC in individual dried fish sample was 33.3% (1/3) for A. tapeinosoma, 100.0% (4/4) for D. maruadsi, 11.1% (1/9) for S. leptolepis, 33.3% (2/6) for E. teres, 0% (0/3) for G. eurostus, 100% (3/3) for D. kuhlii, and 22.2% (4/18) for other kinds. According to report of Kalaimani, Gopakumar, and Nair (1988), the APC of 103 CFU/g is normal in salted and dried fishery products. However, Shakila, Lakshmanan, and Jeyasekaran (2002) have reported that the APC in the salted fish ranged from 104 to 105 CFU/g, while dried fish product contained about 104 CFU/g. Although the average water content (30.88%) of G. eurostus samples was not significantly different from those in the other samples, but the average Aw (0.68) and APC (4.5 log CFU/g) in this dried fish samples were the lowest (Table 1). The dried salted fish product with less than 40% moisture showed the lower APC, indicating that low moisture could inhibit the growth of aerobic bacteria. In addition, two S. leptolepis samples and one A. tapeinosoma sample contained more than 50 MPN/g of E. coli, the allowable limit of the Taiwanese regulatory standard. These poor microbiological qualities showed that the dried fishery products in Taiwan have been unhygienically handled or processed. The dried fishery samples were made by sun-drying for several days and kept at room temperature, resulting that flies may easily contaminate. Concerning the hygienic quality, Hsu et al. (2009) also reported that the tested dried milkfish products, with 12.5% (4/32) samples contained greater than 50 MPN/g of E. coli. Table 2 summarized the contents of biogenic amines in the tested dried fish products. Nine samples of S. leptolepis had the highest average histamine content of 21.07 mg/100 g. Table 3

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Y.-R. Huang et al. / Food Control 21 (2010) 1234–1239 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 samples

Auxis tapeinosoma 3 Decapterus maruadsi 4 Selariodes leptolepis 9 Etrumeus teres 6 Gymnothorax eurostus 3 Dasyatis kuhlii 3 Othersb 18

pH

Water content (%)

Aw

Salt content (%)

TVBN (mg/100 g)

APC (log CFU/g)

E. coli (MPN/g)

TC (MPN/g)

5.92–6.78 (6.25 ± 0.46)aA 6.14–6.54 (6.27 ± 0.19)A 5.98–6.46 (6.16 ± 0.16)A 5.60–6.57 (6.24 ± 0.35)A 6.16–6.40 (6.24 ± 0.14)A 6.53–7.57 (7.07 ± 0.52)B 6.00–7.54 (6.49 ± 0.38)A

44.40–48.41 (47.00 ± 2.26)A 26.24–41.98 (33.88 ± 6.50)AB 21.77–54.48 (31.09 ± 12.57)A 23.61–59.40 (43.85 ± 16.73)A 19.32–42.75 (30.88 ± 11.72)A 25.71–46.10 (35.30 ± 10.25)AB 24.11–61.90 (37.43 ± 11.32)A

0.84–0.91 (0.87 ± 0.04)D 0.81–0.91 (0.85 ± 0.04)CD 0.65–0.78 (0.70 ± 0.04)AB 0.69–0.92 (0.84 ± 0.08)CD 0.64–0.73 (0.68 ± 0.05)A 0.69–0.91 (0.79 ± 0.11)BCD 0.63–0.89 (0.77 ± 0.07)ABC

2.2–5.2 (4.07 ± 1.63)A 2.8–19.8 (7.3 ± 8.3)AB 1.9–20.2 (5.3 ± 5.8)A 3.0–16.7 (7.78 ± 6.35)AB 3.3–14.6 (7.2 ± 6.4)A 2.3–27.1 (16.8 ± 12.9)B 1.8–19.6 (7.96 ± 6.36)AB

24.64–40.04 (33.60 ± 8.00)A 29.00–63.56 (45.40 ± 15.09)A 10.79–68.70 (44.68 ± 18.17)A 18.34–57.90 (30.91 ± 13.98)A 14.56–33.19 (21.04 ± 10.53)A 10.42–156.30 (69.81 ± 76.62)A 10.41–168.56 (53.17 ± 46.70)A

6.30–8.20 (7.38 ± 0.98)BC 7.16–9.28 (8.04 ± 0.91)C 3.28–6.88 (5.67 ± 1.05)AB 3.61–8.71 (5.75 ± 2.06)AB 3.60–6.03 (4.50 ± 1.33)A 6.56–8.04 (7.23 ± 0.75)B 3.18–8.79 (5.49 ± 1.52)AB

<3–75

<3–>1100

<3

<3–>1100

<3–210

<3–1100

<3

<3–110

<3

<3

<3

<3–20

<3

<3–>1100

a

Mean ± SD values in the same column with different letters are statistically different (P < 0.05). 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). b

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

Auxis tapeinosoma Decapterus maruadsi Selariodes leptolepis Etrumeus teres Gymnothorax eurostus Dasyatis kuhlii Othersd

No. of samples 3 4 9

6 3 3 18

Contents of biogenic amine (mg/100 g) Puta

Cad

Try

Phe

Spd

Spm

His

Tyr

NDb-1.56 (0.82 ± 0.78)c ND–15.30 (4.13 ± 7.47) ND–31.80 (6.33 ± 9.79) ND–4.50 (1.14 ± 1.89) ND–3.49 (1.61 ± 1.76) ND–2.04 (0.68 ± 1.18) ND–43.60 (3.63 ± 10.34)

ND–0.33 (0.11 ± 0.19) ND–3.00 (1.33 ± 1.56) 4.13–65.50 (14.50 ± 19.31) ND–16.5 (3.02 ± 6.63) ND–6.32 (2.21 ± 3.56) 2.06–15.30 (8.22 ± 6.67) ND–292.80 (34.14 ± 75..34)

ND–0.35 (0.12 ± 0.20) ND

ND

ND–75.96 (25.62 ± 43.60) ND–8.80 (2.20 ± 4.40) ND

8.57–30.21 (16.26 ± 12.10) 7.89–73.80 (25.80 ± 32.13) ND–10.32 (1.15 ± 3.44) ND–67.30 (25.83 ± 29.75) ND–8.98 (2.99 ± 5.18) ND–63.30 (25.89 ± 33.19) ND–64.40 (14.56 ± 21.33)

ND–11.20 (3.93 ± 6.30) ND–12.73 (3.18 ± 6.37) 6.31–47.90 (21.07 ± 13.12) ND–4.50 (0.91 ± 1.80) ND–2.62 (0.99 ± 1.42) ND–0.48 (0.16 ± 0.28) ND–15.66 (3.30 ± 5.24)

ND–3.44 (1.15 ± 1.99) ND–19.53 (4.88 ± 9.77) ND–15.05 (5.92 ± 6.38) ND ND ND–1.34 (0.45 ± 0.77) ND

ND–12.27 (1.36 ± 4.09) ND–0.35 (0.06 ± 0.14) ND

ND–6.80 (1.70 ± 3.40) ND–38.10 (4.23 ± 12.70) ND–4.50 (0.75 ± 1.84) ND

ND

ND

ND–8.85 (0.49 ± 2.09)

ND–24.40 (1.65 ± 5.75)

ND–51.5 (8.58 ± 21.02) ND–8.07 (2.69 ± 4.66) ND–16.9 (5.91 ± 9.53) ND–34.1 (2.34 ± 8.04)

ND–58.90 (5.20 ± 14.05)

a

Put: putrescine; Cad: cadaverine; Try: tryptamine; Phe: 2-phenylethylamine; Spd: spermidine; Spm: spermine; His: histamine; and Tyr: tyramine. 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). b

Table 3 Distribution of the histamine contents in 46 dried fish products. Content of histamine (mg/100 g)

Dried fish products No. of samples

% of samples

<4.9 5.0–19.9 20.0–49.9 Total

32 10 4 46

69.6 21.7 8.7 100

shows the distribution of histamine contents in tested dried fish products, with 30.4% (14/46) samples containing greater than 5 mg/100 g of histamine, the allowable limit of the US Food and Drug Administration (FDA) for scombroid fish and/or products. Based on an analysis of poisoning episodes, Shalaby (1996) suggested the following guideline levels for histamine content of fish: (i) <5 mg/100 g (safe for consumption); (ii) 5–20 mg/100 g (possibly toxic), (iii) 20–100 mg (probably toxic), and (iv) >100 mg/ 100 g (toxic and unsafe for human consumption). In addition, FDA guidelines (1995) for tuna, mahi–mahi and related fish specify 50 mg/100 g as the toxicity level and 5 mg/100 g as the defect ac-

tion level because histamine is not uniformly distributed in a decomposed fish. Therefore, if 5 mg/100 g is found in one section, there is a possibility that other units may exceed 50 mg/100 g (Lehane & Olley, 2000). Several countries have set legal limits of histamine concentrations that are regarded as safe for human consumption: Australia, 20 mg/100 g (Australian Food Standards Code, 2001), Europe, 10 mg/100 g (EC, 2003) and South Africa, 10 mg/ 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 decomposition. In our present study, all of nine samples of S. leptolepis had the highest histamine content of 6.31–47.90 mg/100 g. However, no one with greater than 50 mg/100 g of histamine as the toxicity level based on FDA. But the four samples of S. leptolepis with greater than 20 mg/100 g of histamine may be sufficient to cause the symptoms of scombroid poisoning (CDC, 2000; EEC, 1991). Although the tested P. cinnamomeus and H. japonica samples of the others did not contain high levels of histamine, they contained 292.8 and 166.4 mg/100 g (data not shown) of cadaverine, respectively (Table 2). The presence of cadaverine and putrescine may

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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

PHF PHF PHF PHF PHF PHF PHF PHF PHF PHF PHF PHF PHF a b

1-1 1-3 1-5 2-1 19-1 24-1 24-1 34-1 37-1 23-1 35-2 35-4 45-2

Strain source

Siganus fuscescens Siganus fuscescens Siganus fuscescens Synodus dermatogenys Hemitriakis japanica Pseudorhombus cinnamomeus Pseudorhombus cinnamomeus Dasyatis kuhlii Etrumeus teres Selaroides leptolepis Selariodes leptolepis Selariodes leptolepis Selariodes leptolepis

Organism identified

Citrobacter freundii C. freundii C. freundii Staphylococcus saprophyticus S. saprophyticus Pantoea agglomerans P. agglomerans Salmonella typhi Sal. typhi C. freundii Salmonella enteritidis S. typhi Enterobacter aerogenes

Percentage identity (%)

Gene bank accession number

100 100 100 100 100 100 100 100 100 100 100 100 100

AB244451.1 AB244451.1 AB244451.1 EU419944.1 EU419944.1 EU879089.1 EU879089.1 EU118115.1 EU118115.1 AB244451.1 EU118102.1 EU118114.1 AB244456.1

Levels of biogenic amine (ppm) Trya

Put

Cad

His

Tyr

NDb 13.6 17.2 20.7 7.9 8.7 ND 0.4 ND ND ND ND ND

ND 10.2 ND 1.7 ND 18.7 ND 37.8 30.0 6.1 ND 18.0 ND

ND 3.9 2.5 2.7 0.6 4.5 ND 411.8 331.8 16.1 236.1 224.5 388.3

27.9 49.4 34.5 20.7 12.0 9.6 11.3 14.3 21.5 57.1 15.1 8.7 531.2

16.5 17.7 17.8 18.0 ND ND ND ND ND ND 1.0 ND 7.8

Try: tryptamine; Put: putrescine; Cad: cadaverine; His: histamine; Tyr: tyramine. ND: not detected (amine level less than 0.05 ppm).

synergistically enhance histamine toxicity by inhibiting histamine metabolizing enzymes such as diamine oxidase and histamine methyl transferase (Antoine et al., 2002). Recently, we demonstrated that most of tested dried milkfish products (78.1%) sold in Taiwan had histamine levels greater than the FDA guideline of 5 mg/100 g for scombroid fish and/or product (Hsu et al., 2009). Although high unacceptable rate was detected in dried milkfish and S. leptolepis products tested in previous and this studies, respectively, very few cases of food-borne histamine intoxication have been reported due to consumption of dried fish products. Symptoms of histamine poisoning are not particularly definitive. Therefore, histamine intoxication is frequently misdiagnosed as an allergic reaction. Biogenic amines are formed mainly through the decarboxylation of specific free amino acids by exogenous decarboxylases released by microbial species associated with seafood. Many different bacterial species are known to possess histidine decarboxylase and have the ability to produce histamine (Rawles, Flick, & Martin, 1996). Table 4 lists the identity of 13 histamine-forming bacteria isolated from the dried fish products as determined by 16S rDNA sequences using NCBI database analysis. Thirteen histamineproducing bacterial strains, capable of producing 8.7–531.2 ppm of histamine in trypticase soy broth supplemented with 1.0% L-histidine (TSBH), were identified as C. freundii (four strains), Pantoea agglomerans (two strains), Staphylococcus saprophyticus (two strains), Salmonella typhi (three strains), Salmonella enteritidis (one strain) for weak histamine formers (557.1 ppm), and E. aerogenes (one strain) for prolific histamine formers (531.2 ppm). Some of them also produced different amounts of other biogenic amines through the action of their respective decarboxylase enzymes on various amino acids that also existed in the culture medium (Table 4). Recently, we reported that 30 histamine-producing bacterial strains were isolated from dried milkfish (Chanos chanos) products, capable of producing 5.4–562 ppm of histamine in TSBH broth, and identified as E. aerogenes (seven strains) and Citrobacter sp. (one strain) for prolific histamine formers (=553 ppm), and S. xylosus (10 strains), S. sciuri (one strain), Bacillus thuringiensis (two strains), C. freundii (five strains), K. pneumoniae (one strain) and E. cloacae (three strains) for weak histamine formers (551.4 ppm) (Hsu et al., 2009). Enterobacter spp. and Citrobacter spp. were most frequently reported prolific histamine formers in various species of scombroid fish such as tuna, albacore and sailfish (Kim et al., 2001a, 2001b; Lopez-Sabater et al., 1996; Tsai, Kung, Lee, Lin, & Hwang, 2004). The E. aerogenes isolate from the sailfish fillets was found to be a prolific histamine-former capable of producing more than 1000 ppm of histamine in the culture broth (Tsai et al., 2004). Sim-

ilarly, E. aerogenes isolated from S. leptolepis sample was a potent histamine-forming bacteria capable of producing 531.2 ppm of histamine and 388.3 ppm of cadaverine in TSBH. C. freundii isolated from S. leptolepis and S. fuscescens samples was a weak histamine-former, producing only 27.9–57.1 ppm histamine in TSBH broth (Table 4). Potential histamine-forming bacteria, including halo-tolerant bacteria, have been isolated from salted fish samples. Staphylococcus spp., Vibrio spp. and Pseudomonas III/IV-NH were isolated from fermented salted sardine products (Yatsunami & Echigo, 1991, 1992), while S. epidermidis, S. xylosus, K. oxytoca, E. cloacae, P. cepaciae and Bacillus spp. were isolated from salted Spanish anchovies (Hernandez-Herrero et al., 1999; Rodriguez-Jerez et al., 1994). However, only one of these histamine-forming species, Staphylococcus saprophyticus was isolated in this study from salt-dried fish samples. Staphylococcus spp. were the most frequently reported histamine-formers in fermented salted fish, accounting for nearly 50% of histamine-forming microorganisms. They were usually shown to have powerful histamine-forming activity (Yatsunami & Echigo, 1991, 1992). For example, S. epidermidis and S. capitis, isolated from salted Spanish anchovies, produced more than 1000 ppm and 400 ppm of histamine, respectively, in TSBH broth (Hernandez-Herrero et al., 1999). The S. capitis recently isolated from mustard pickle products in Taiwan was a potent histamineformer, capable of producing more than 1000 ppm of histamine in TSBH broth (Kung et al., 2006). However, the recently isolated S. pasteuri from miso products in Taiwan was a weak histamineformer, producing only 28.1 ppm histamine in TSBH broth (Kung, Tsai, & Wei, 2007). Similarly, the S. saprophyticus isolated from the samples of Selaroides dermatogenys and Hemitriakis japanica in this study was also a weak histamine-former capable of producing only 20.7 and 12.0 ppm of histamine in TSBH broth, respectively (Table 4). Since Staphylococci are the major microbial groups that inhabit human skin, it is reasonable to expect that they would be transferred to food products through considerable human contact during preparation and processing. Lakshmanan, Jeya-Shakila, and Jeyasekaran (2002) pointed out that the incidence of halophytic amine forming bacteria appeared the fluctuation in the sardine processing. It was around 20% in fresh sardines, reached a maximum level of 84%, decreased during the drying process, and finally was not found in salt-dried sardines after final drying. However, Moori, Cann, and Taylor (1988) observed that the penetration of histamine-forming bacteria of the gut into the inner muscle of whole fish is possible during the sun-drying process by rupture of the belly walls. Furthermore, contamination may happen when the fish is eviscerated during manual processing, cut with a dirty knife, and mixed in the same

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tank with salting process for a long time. These may be the reasons that E. coli, TC and histamine-forming bacteria strains occurred in the fishery products. 4. Conclusion This study, to determine the safety of 46 dried fish products sold in Penghu Island, Taiwan, showed that the TVBN content in 26 samples (56.5%) exceeded the 30 mg/100 g decomposition limit. The tested dried fish products (30.4%) had histamine levels greater than the FDA guideline of 5 mg/100 g for scombroid fish and/or product. Among them, all of the nine samples of S. leptolepis had the highest histamine content of 6.31–47.90 mg/100 g. Consumption of these products could lead to scombroid poisoning in consumers. The weak histamine-producing bacteria (557.1 ppm) have been isolated and included C. freundii (four strains), Pantoea agglomerans (two strains), Staphylococcus saprophyticus (two strains), Salmonella typhi (three strains) and Salmonella enteritidis (one strain), but a prolific histamine-former (531.2 ppm) E. aerogenes (one strain) was also isolated. Therefore, not only histamine but also hygienic quality of dried fish products in Penghu Island need to be improved. Acknowledgement The study was supported by the National Science Council, ROC (Contract No. NSC 97-2313-B-346-001-MY3). References Ababouch, L., Afila, M. E., Rhafiri, S., & Busta, F. F. (1991). Identification of histamineproducing bacteria isolated from sardine (Sardina pilchardus) stored in ice and at ambient temperature (25 °C). Food Microbiological, 8, 127–136. Antoine, F. R., Wei, C. I., Otwell, W. S., Sims, C. A., Littell, R. C., Hogle, A. D., et al. (2002). Analysis of biogenic amines and their precursor free amino acids in mahi–mahi (Coryphaena hippurus). Journal of Food Biochemistry, 26, 131–152. AOAC (1995). Official methods of analysis of AOAC international (16th ed.). Arlington, VA: AOAC International. Australian Food Standards Code (2001). Part D: Fish and fish products. Standards D1 and D2. Version 18. . CDC (Centers for Disease Control and Prevention) (2000). Scombroid fish poisoningPennsylvania, 1998. MMWR, 49, 398–400. Chakrabarti, R. (1991). Histamine content in dried fish products from kakinada coast. Fishery Technology, 28, 59–62. Chakrabarti, R. (1993). Processing of Penaeus indicus, Decapterus sp. and Stolephorus sp. to dried product with low histamine and their storage characteristics. Fishery Technology, 30, 130–133. Chen, H. C., Huang, Y. R., Hsu, H. H., Lin, C. S., Chen, W. C., Lin, C. M., et al. (2010). Determination of histamine and biogenic amines in fish cubes (Tetrapturus angustirostris) implicated in a food borne poisoning. Food Control, 21, 13–18. Cobb, B. F., Aoaniz, I., & Thompson, C. A. (1973). Biochemical and microbial studies on shrimp: Volatile nitrogen and amino nitrogen analysis. Journal of Food Science, 38, 431–435. EC (2003). Commission recommendation of 10 January 2003 concerning a coordinated programme for the official control of foodstuffs for 2003 (2003/ 10/EC). Official Journal of the European Commission, 7, 76–81. EEC (European Economic Community) (1991). Laying down the health conditions for the production and the placing on the market of fishery products. Council Directive (EEC) 91/493/EEC. FDA (Food, and Drug Administration) (1992). Bacteriological analytical manual. Arlington, VA: AOAC International. FDA (1995). Decomposition and histamine – Raw, frozen tuna and mahi–mahi, canned tuna, and related species, revised compliance guide, availability. Federal Registration, 149, 39754–39756. Hernandez-Herrero, M. M., Roig-Sagues, A. X., Rodriguez-Jerez, J. J., & Mora-Ventura, M. T. (1999). Halotolerant and halophilic histamine-forming bacteria isolated during the ripening of salted anchovies. Journal of Food Protection, 62, 509– 514. Hsu, H. H., Chuang, T. C., Lin, H. C., Huang, Y. R., Lin, C. M., Kung, H. F., et al. (2009). Histamine content and histamine-forming bacteria in dried milkfish (Chanos chanos) products. Food Chemistry, 114, 933–938. Jeyasekaran, G., & Jeyashakila, R. (2003). Occurrence of biogenic amine forming bacteria in cured fishery products of Thoothukkudi region of Tamil Nadu, India. Asian Fisheries Science, 16, 195–202. Kalaimani, N., Gopakumar, K., & Nair, T. S. U. (1988). Quality characteristics of cured fish of commerce. Fishery Technology, 25, 54–56.

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