Isolation and identification of histamine-forming bacteria in tuna sandwiches

Food Control 20 (2009) 1013–1017

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Isolation and identification of histamine-forming bacteria in tuna sandwiches Hsien-Feng Kung a, Tze-Ya Wang b, Yu-Ru Huang c, Chung-Saint Lin d, Wen-Sheng Wu a, Chia-Min Lin b, Yung-Hsiang Tsai b,* a

Department of Food Science and Technology, Tajen University, Pingtung, Taiwan, ROC Department of Seafood Science, National Kaohsiung Marine University, No. 142, Hai-Chuan Rd. Nan-Tzu, Kaohsiung City, 811 Taiwan, ROC c Department of Food Science, National Penghu University, Penghu, Taiwan, ROC d Department of Food Science, Yuanpei University, Hsin-Chu, Taiwan, ROC b

a r t i c l e

i n f o

Article history: Received 12 September 2008 Received in revised form 26 November 2008 Accepted 4 December 2008

Keywords: Histamine Biogenic amines Tuna sandwich Histamine-forming bacteria Raoultella ornithinolytica

a b s t r a c t Forty-three tuna sandwiches sold in diners and convenience store in southern Taiwan were purchased and tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH, water content, ash content, salt content, total volatile basic nitrogen (TVBN), aerobic plate count (APC), total coliform (TC), and Escherichia coli in all samples ranged from 4.5 to 6.9, 36.8% to 62.8%, 1.11% to 6.55%, 0.10% to 3.90%, 11.2 to 78.0 mg/100 g, <1.0 to 9.7 log CFU/g, <3 to 70,000 MPN/g and <3 to 1000 MPN/g, respectively. The average content of each of the eight biogenic amines in all samples was less than 3 mg/100 g, and only one tuna sandwich sample had histamine content (5.21 mg/100 g) greater than the 5.0 mg/100 g allowable limit suggested by the U.S. Food and Drug Administration. Five histamine-producing bacterial strains, capable of producing 42.1–595.4 ppm of histamine in trypticase soy broth supplemented with 1.0% L-histidine (TSBH), were identified as Hafnia alvei (one strain), Raoultella ornithinolytica (three strains) and Raoultella planticola (one strain), by 16S rDNA sequencing with PCR amplification. Ó 2009 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. In Taiwan, scombroid poisoning occurs occasionally (Chen & Malison, 1987; Chen et al., 2008; Tsai, Kung, et al., 2005), and the fish implicated in these outbreaks are tuna, mackerel, and black marlin. Recently, due to their popularity by Taiwanese people, sailfish, swordfish and marlin fillets have become the most frequently implicated fish species in scombroid outbreaks in Taiwan (Chang, Kung, Chen, Lin, & Tsai, 2008; Hwang, Chang, Shiau, & Chai, 1997; Hwang, Chang, Shiau, & Cheng, 1995; Tsai, Hsieh, et al., 2007).

* Corresponding author. Tel.: +886 7 3617141 3605; fax: +886 7 3640634. E-mail address: [email protected] (Y.-H. Tsai). 0956-7135/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2008.12.001

Biogenic amines are formed mainly through the decarboxylation of specific free amino acids by exogenous decarboxylases released by the microbial species associated with the seafood. Many different bacterial species are known to possess histidine decarboxylase and have the ability to produce histamine (An & Ben-Gigirey, 1998). Although only Morganella morganii, Klebsiella pneumoniae and Hafnia alvei have been isolated from the fish incriminated in scombroid poisoning (Taylor & Speckard, 1983), a variety of other bacterial species capable of producing histamine have been identified in fish (Middlebrooks, Toom, Douglas, Harrison, & McDowell, 1988; Taylor & Speckard, 1983; Yoshinaga & Frank, 1982). Among them are the enteric bacteria that include Proteus vulgaris, Proteus mirabilis, Enterobacter aerogenes, Enterobacter cloacae, Serratia fonticola, Serratia liquefaciens and Citrobacter freundii (Kim et al., 2003; Tsai, Lin, 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 (Middlebrooks et al., 1988; Okuzumi, Hiraishi, Kobayashi, & Fujii, 1994; Yatsunami & Echigo, 1991). Recently, we demonstrated the presence of histamine-forming Proteus, Enterobacter, Klebsiella, Rahnella and Acinetobacter in sailfish fillets in Taiwan, but failed to isolate any of the three above mentioned major histamine-formers – the H. alvei, M. morganii and K. pneumoniae (Tsai, Kung, Lee, Lin, & Hwang, 2004). In addition, the prolific histamine-forming bacteria Raoultella ornithinolytica

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isolated from dried milkfish implicated in food poisoning has been studied (Tsai, Kung, et al., 2007). Quality loss and histamine accumulation often occur after frozen tuna fish are thawed and kept for long periods of time at room temperature before further processing. Since histamine is heat resistant, it can remain intact in canned tuna products (Lopez-Sabater, Rodriguez-Jerez, Roig-Sagues, & Mora-Ventura, 1994). The use of poor quality tuna fish as raw material for canning, or defective handling techniques of high quality tuna fish during processing results in the presence of toxic levels of histamine in canned tuna products (Price & Melvin, 1994). Tuna sandwich is an important and popular ready to eat (RTE) food processed from canned tuna. The 18 tuna sandwich samples sold in Hong Kong had histamine contents ranging from 0.6 to 20 ppm, while it was found that the histamine formation in opened canned tuna could be rapidly increased if stored at 33 °C for 6 h (Anonymous, 2005). In Taiwan, there exists no report on the occurrence of biogenic amines, including histamine, histamine-forming bacteria and related bacteria in tuna sandwiches. Therefore, this research was undertaken by testing 43 tuna sandwich samples sold in diners and convenience stores in southern Taiwan so that a better understanding of the safety quality of the products can be accomplished to better protect the consumers. 2. Materials and methods 2.1. Samples Forty-three samples of tuna sandwich were purchased from diner chain-A stores (10 samples), diner chain-B stores (eight samples), convenience stores (three samples), and other individual diner stores (22 samples) in southern Taiwan from August and September, 2007. Main ingredients of sandwich tested in this study were bread, mayonnaise, ham, egg, tuna meat, and vegetables such as cucumber slices. Usually, pickled cucumbers and cheese are not used for making tuna sandwich in Taiwan. The tuna sandwiches were all packed in plastic bags. Except for convenience store samples stored at 18 °C, the diner samples were stored at room temperature (27–32 °C) on shelf. Shelf life of sandwich purchased from diner chain and convenient stores was 1 and 2 days, respectively. The canned tuna meats (600 g) were purchased from supermarket of Kaohsiung city for recovery analysis. After purchase, all samples were kept at 4 °C and immediately transported to the laboratory for analysis. Tuna meat taken from the sandwiches purchased from the same store purchased at the same time was collected for following tests. 2.2. pH value, water content, ash content, and salt content determination The tuna meat sample (5 g) was homogenized in sterile blenders with 10 ml of distilled water to make thick slurry. The pH of this slurry was then measured using a Corning 145 pH meter (Corning Glass Works, Medfield, MA, USA). 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 is tested by additional drying steps of 1 h until the difference in mass does not exceed 0.5 mg. The ash and salt contents in each sample was determined according to the AOAC procedures (1995). 2.3. Determination of total volatile basic nitrogen (TVBN) The TVBN content of each tuna meat sample was measured by the method of Conway’s dish (Cobb, Aoaniz, & Thompson, 1973).

The TVBN extract of tuna meat samples 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 calculated and expressed in mg/100 g sample. 2.4. Microbiological analysis and isolation of histamine-forming bacteria A 25-g portion of the tuna meat sample of sandwich was homogenized at high speed for 2 min in a sterile blender with 225 ml of sterile potassium phosphate buffer (0.05 M, pH 7.0). The sterile blender was prepared by autoclaving for 15 min at 121 °C. The homogenates were serially diluted with a sterile phosphate buffer, and 1.0 ml aliquots of the dilutes were inoculated into 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. The bacterial numbers in the tuna sandwich samples were expressed as log10 colony forming units (CFU)/g. To isolate histamine-forming bacteria, a 0.1 ml aliquot of the diluted sample 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 days 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 milliliters of the culture broth were taken for quantitation of biogenic amines. Analyses of total coliform and E. coli in tuna meat samples were conducted using the three tubes most probable number (MPN) methods (FDA, 1992). Lauryl sulfate tryptose broth (LST broth) and brilliant green lactose bile (2%) broth (BGLB broth) were used for presumptive and confirmed tests for total coliform, respectively. E. coli was determined by using the LST broth and EC broth. Cultures that showed positive production of gas were then confirmed by eosine methylene blue agar (EMBA) and IMViC test. 2.5. 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. 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 universal primers UNI-L (50 -AGAGTTTGATCATGGCTCAG-30 ) and UNI-R (50 -GTGTGACGGGCGGTGTGTAC-30 ) (Kuhnert et al., 1996; Kuhnert et al., 2000). Bacterial cells were cultured overnight in 2 ml of TSB at 35 °C and then centrifuged at 8000 rpm for 10 min. The cell pellet was washed and resuspended in 0.5 ml of TE-buffer (10 mM Tris–HCl, 1 mM EDTA; pH 8.0), and then lysed by 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 pmol of each primer, a 0.2 mM concentration for each of the 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

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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; Kuhnert et al., 2000). Amplicons were detected by electrophoresis on a 1.5% agarose gel staining with ethidium bromide. Amplicons were purified using a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) 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. 2.6. Biogenic amines analysis Each tuna meat of sandwich 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 (10,000g, 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 tuna meat extracts were derivatized with dansyl chloride according to the previously described method (Eerola, Hinkkanen, Lindfors, & Hirvi, 1993). One milliliter of each bacterial culture broth were derivatized with dansyl chloride using the same procedure as for the dansylation of standard amine solution. The 20 ll aliquots of dansylated derivatives were used for HPLC injection. Biogenic amines were determined with a Hitachi liquid chromatograph (Hitachi, Tokyo, Japan) consisting of a Model L-7100 pump, a Rheodyne Model 7125 syringe loading sample injector, a Model L-4000 UV–Vis detector (set at 254 nm), and a Model D-2500 Chromato-integrator. A LiChrospher 100 RP-18 reversedphase column (5 lm, 125  4.6 mm, E. Merck, Damstadt, Germany) was used for chromatographic separation. The gradient elution program began with 50:50 (v/v) acetonitrile:water at a flow rate of 1.0 ml/min for the 19 min, followed by a linear increase to 90:10 acetonitrile:water (1.0 ml/min) during the next 1.0 min. The acetonitrile:water mix decreased to 50:50 (1.0 ml/min) for 10 min. 2.7. Recovery of amines spiked into canned tuna meats The recovery of biogenic amines was determined by fortifying homogenized canned tuna meats with 1.0, 5.0, 10 mg/100 g of each amine. Each spiked amount was extracted and derivatized with dansyl chloride using the same procedure as for tuna sandwich sample in triplicate including a blank test to evaluate the average recovery.

2.8. Statistical analysis Pearson correlation was carried out to determine relationships between pH, water content, ash content, salt content, TVBN, APC, total coliform, E. coli and histamine contents in the 43 samples collected from southern Taiwan. 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, ash content, salt content, aerobic plate count (APC), total coliform (TC), and E. coli in the tuna sandwich products are presented in Table 1. The levels of pH, water content, ash content, salt content, total volatile basic nitrogen (TVBN), APC, TC, and E. coli in all samples ranged from 4.5 to 6.9, 36.8% to 62.8%, 1.11% to 6.55%, 0.10% to 3.90%, 11.2 to 78.0 mg/ 100 g, <1.0 to 9.7 log CFU/g, <3 to 70,000 MPN/g and <3 to 1000 MPN/g, respectively. The average content for each of the eight analysis items in all samples were not significantly different (p > 0.05). The rates of unacceptable diner chain-A, diner chain-B and other diners samples were 20% (2/10), 75% (6/8), and 41% (9/22), respectively, based on the ‘‘General foods served without preparing treatments” regulatory level of Taiwan for total coliform (1000 MPN/g). Based on Taiwanese regulatory standard of negative for E. coli, 10% (1/10) of the samples obtained in diner chain-A were unacceptable as compared to 13.6% (3/22) of other diners samples. Common practice for making tuna sandwich in Taiwan leaves opened tuna cans, which usually are at commercial size (more than 1 kg), at room temperature during process. In addition, the sandwiches contained several non-sterile ingredients such as slices of fresh cucumbers. Therefore, microorganisms in tuna should be from other ingredients of sandwiches or utensils. In general, no correlation existed among the pH values, water content, ash content, salt content, TVBN, APC, TC, E. coli, and histamine contents in the tested 43 samples. Biogenic amines were not detected in blank canned tuna meats (detection limit was 0.05 ppm). The average recoveries of the amines spiked into canned tuna meats were 82.4% for tryptamine, 104.1% for 2-phenylethylamine, 99.8% for putrescine, 113.6% for cadaverine, 106.8% for histamine, 107.7% for tyramine, 97.4% for spermidine, and 89.2% for spermine (Table 2). Except for tryptamine at low amount addition (1.0 mg/100 g), the coefficients of variation (CV, %) of other seven amines were lower than 9.4%. This result indicated the HPLC analytical method with dansylation was quite accurate for the determination of biogenic amines in canned tuna meats. None of the 43 tested tuna sandwich samples contained tryptamine (Table 3). Although the average content for each of the remaining seven biogenic amines in all samples was lower than 3.0 mg/100 g, one of the diner chain-A sample had histamine

Table 1 Values of the pH, water content, ash content, salt content, total volatile basic nitrogen (TVBN), aerobic plate count (APC), total coliform (TC), and E. coli in tested tuna sandwiches. Sample sources

No. of samples

pH

Water content (%)

Ash content (%)

Salt content (%)

TVBN (mg/100 g)

APC (log CFU/g)

TC (MPN/g)

E. coli (MPN/g)

Diner chain-A

10

Diner chain-B

8 3

36.8–50.6 (45.2 ± 5.1)A 45.7–60.3 (54.1 ± 6.6)A 55.7–60.6 (58.3 ± 2.5)A 42.4–62.8 (54.7 ± 5.9)A

1.35–1.76 (1.56 ± 0.16)A 1.11–1.88 (1.52 ± 0.29)A 2.06–6.55 (4.12 ± 2.27)A 1.49–2.24 (1.77 ± 0.26)A

0.46–0.97 (0.75 ± 0.21)A 0.55–0.82 (0.71 ± 0.10)A 0.10–0.57 (0.41 ± 0.27)A 0.82–3.90 (1.17 ± 0.88)A

13.7–31.7 (20.1 ± 9.0)A 11.2–78.0 (28.6 ± 28.2)A 14.6–17.7 (16.4 ± 1.6)A 12.4–35.8 (24.1 ± 7.3)A

1.7–8.2 (5.3 ± 1.9)A 4.5–9.7 (6.6 ± 1.7)A 1.9–9.1 (5.3 ± 1.8)A <1–4.7 (3.1 ± 2.7)A

<3–30000 (6003 ± 12133)A <3–70000 (12535 ± 23718)A <3

<3–1000 (100 ± 316)A <3

Convenience store Other diners

4.7–6.4 (5.8 ± 0.5)aA 4.5–6.9 (5.8 ± 0.7)A 5.2–5.8 (5.4 ± 0.3)A 5.0–6.2 (5.9 ± 0.3)A

<3–49000 (6498 ± 14251)A

<3–100 (6.4 ± 21)A

a

22

Means ± SD. Value in the same column with same letters are not statistically different (p > 0.05).

<3

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Table 2 Recoveries of biogenic amines spiked into canned tuna meats. Analyte

Recoverya of spiked amine (%) 1 mg/100 g

5 mg/100 g

10 mg/100 g

Average

Tryptamine 2-Phenylethylamine Putrescine Cadaverine Histamine Tyramine Spermidine Spermine

72.4 (17.1)b 98.8 (1.8) 98.8 (2.0) 103.9 (4.8) 105.4 (2.8) 89.2 (6.8) 97.4 (6.0) 88.9 (2.9)

81.5 (1.4) 104.9 (3.9) 100.3 (2.5) 117.2 (1.7) 107.9 (0.5) 118.2 (4.1) 98.3 (3.0) 92.5 (4.2)

93.4 (4.2) 108.6 (3.5) 100.3 (6.8) 119.7 (0.1) 107.2 (6.3) 115.7 (4.9) 96.7 (9.4) 86.2 (6.7)

82.4 104.1 99.8 113.6 106.8 107.7 97.4 89.2

a b

Average of triplicate determinations. Values in parentheses are the coefficient of variation (CV, %).

content (5.21 mg/100 g) greater than the 5.0 mg/100 g allowable limit suggested by the US Food and Drug Administration (USFDA (US Food & Drug Administration), 2001, chap. 7). Therefore, based on the content of histamine in the test product, a 2.3% (1/43) unacceptable rate was obtained with these tuna sandwich samples. The occurrence of low levels of biogenic amines in tuna sandwich samples in this study could be resulted from the low levels of biogenic amines in commercial canned tuna products in Taiwan reported by Tsai et al. (2005). This result was similar to the report that 18 tuna sandwich samples were shown to contain histamine ranging from 0.6 mg/kg to 20 mg/kg for the 20 tuna sandwich samples in Hong Kong (Anonymous, 2005). Table 4 listed the identity of these five histamine-forming bacteria as determined by 16S rDNA sequences following comparison to reference strains using NCBI database analysis. The PCR amplicons from strains TS2-2 had a 99% homology with H. alvei, while that from strains TS25-1 aligned with Raoultella planticola at 100%. The PCR amplicons from strains TS3-2, TS9-1 and TS40-2 had a 100% homology with R. ornithinolyticus (Table 4). These five histamine-forming isolates as H. alvei (one strain), R. planticola

(one strain) and R. ornithinolyticus (three strains) by 16S rDNA sequencing produced substantial amounts of histamine (42.1– 595.4 ppm) in TSBH medium (Table 4). Some of them also produced different amounts of putrescine, cadaverine, 2-phenylethylamine and tyramine through the actions of their respective decarboxylase enzymes on various amino acids that also existed in the TSBH medium (Table 4). In this study, all of histamine-forming isolates belonged to Enterobacteriaceae. Enterobacteriaceae are generally thought to be the primary cause of histamine development in scombroid fish (Ten Brink, Damink, Joosten, & Huis in’t Veld, 1990). Omura, Proce, and Olcott (1978) also isolated histamine-producing M. morganii, Proteus spp., and Klebsiella spp., as well as H. alvei, from skipjack tuna and jack mackerel. Kanki, Yoda, Tsukamoto, and Shibata (2002) recently discovered that several histamine-producing cultures believed to be K. pneumoniae were incorrectly identified strains of Raoultella planticola. The R. planticola strain TS25-1 and R. ornithinolytica strains TS3-2, 9-1 and 40-2 that were isolated in this study were also potent histamine-formers and produced 560.5–595.4 ppm of histamine in TSBH (Table 4), accounting for 80% (4/5) of histamine-forming isolates. R. planticola and R. ornithinolytica isolated from tuna, bonito, and sardines produced 2610–5250 ppm of histamine in culture broth (Kanki et al., 2002). R. planticola isolated by Tsai et al. (2004) and Ababouch, Afila, Rhafiri, and Busta (1991) had histamine production rates between 1000 and 4000 ppm in 24 h at 37 °C. Recently, the R. ornithinolytica, isolated from dried milkfish implicated in a food-borne poisoning was a potent histamine-former capable of producing 1243 ppm of histamine in TSBH (Tsai, Kung, et al., 2007). Although high histamine concentration (>5 mg/100 g) was detected from one sample purchased at a diner chain-A store, histamine-forming bacteria were not isolated from the sample. In contrast, histamineforming bacteria were isolated from samples purchased at diner chain-B and other diners stores but high histamine concentration was not detected from the samples. Since original histidine was not analyzed, it was very difficult to predict existence of histamine

Table 3 Contents of biogenic amines in tested tuna sandwiches. Sample sources

No. of samples

Levels of biogenic amine (mg/100 g) Trya

Phe

Put

Cad

His

Tyr

Spd

Spm

Diner chainA Diner chainB Convenience store Other diners

10

NDb

8

ND

ND  0.22 (0.02 ± 0.07)cA ND

3

ND

ND ND  0.58 (0.03 ± 0.12)A

ND  5.21 (0.81 ± 1.6)A ND  1.15 (0.87 ± 1.6)A ND  0.7 (0.21 ± 0.37)A ND  2.49 (1.04 ± 0.82)A

ND  0.38 (0.22 ± 0.15)A ND  22.09 (2.99 ± 7.72)A ND

ND

ND  3.03 (0.61 ± 0.88)A ND  4.96 (1.02 ± 1.62)A ND  0.65 (0.22 ± 0.38)A ND  3.76 (0.79 ± 0.86)A

ND  0.48 (0.15 ± 0.18)A ND  9.91 (1.63 ± 3.39)A ND

22

ND  1.68 (0.51 ± 0.49)A ND  11.1 (2.42 ± 3.53)A 0.3–0.99 (0.55 ± 0.38)A ND  8.97 (0.74 ± 1.84)A

ND  7.28 (0.63 ± 1.51)A

ND  0.31 (0.20 ± 0.13)A

ND  0.53 (0.22 ± 0.21)A ND  13.01 (1.82 ± 4.52)A ND  0.99 (0.33 ± 0.57)A ND  5.29 (0.48 ± 1.09)A

a b c

Try, tryptamine; Phe, 2-phenylethylamine; Put, putrescine; Cad, cadaverine; His, histamine; Tyr, tyramine; Spd, spermidine; Spm, spermine. ND, not detected (amine levels is less than 0.05 mg/100 g). Means ± SD. Value in the same column with same letters are not statistically different (p > 0.05).

Table 4 Identification of histamine-forming bacteria isolated from tuna sandwiches by 16S rDNA, basing on the output results from NCBI database analysis, and their production of histamine and other biogenic amines (ppm) in culture broth. Strain

Organism identified

Percentage identity (%)

Source

Gene bank accession number

Hisa

Phe

Put

Cad

Tyr

TS TS TS TS TS

Hafnia alvei Raoultella planticola R. ornithinolytica R. ornithinolytica R. ornithinolytica

99 100 100 100 100

Diner chain-B Diner chain-B Diner chain-B Other diners Other diners

AY253922.1 AB099407.1 AF129441.1 EF057396.1 AM184250.1

42.1 560.5 593.5 593.2 595.4

ND 0.5 2.5 4.8 3.6

8.5 NDb ND ND ND

280.0 ND 242.0 259.8 224.3

1.1 0.9 ND 1.5 1.4

a b

2-2 25-1 3-2 9-1 40-2

His, histamine; Phe, 2-phenylethylamine; Put, putrescine; Cad, cadaverine; Tyr, tyramine. ND, not detected (amine level less than 0.05 ppm).

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based on the presence of histamine-forming bacteria. However, presence of histamine-forming bacteria is a valuable indicator for prevention of histamine poisoning. 4. Conclusion This study, to determine the safety of 43 tuna sandwiches sold in Taiwan, showed that the total coliform and E. coli content in 17 samples (39.5%) and 4 samples (9.3%), respectively, exceeded the Taiwanese regulatory limit. The average content for each of the eight tested biogenic amines in these samples was less than 3 mg/100 g, although one of them had a histamine content at 5.21 mg/100 g greater than the 5 mg/100 g USFDA guideline value. While the bacterial isolate H. alvei (one strain) was identified to be a weak histamine-former, the R. planticola (one strain) and R. ornithinolytica (three strains) isolates were proven to be prolific histamine-formers with ability to produce >500 ppm histamine in TSBH medium. Since tuna was obtained from commercial cans, the most likely source of histamine-forming bacteria could be from preparation of sandwiches. Acknowledgements The study was supported by the National Science Council, ROC (Contract No. NSC 96-2313-B-127-002). 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 Microbiology, 8, 127–136. An, H., & Ben-Gigirey, B. (1998). Scombrotoxin poisoning. In I. Millar, D. Gray, & N. Strachan (Eds.), Microbiology of seafoods (pp. 68–89). London: Chapman & Hall Ltd.. Anonymous (2005). Joint study shows histamine content in canned fish and tuna sandwiches is low – CHOICE #347. The Consumer Council of Hong Kong. . AOAC (1995). Official methods of analysis of AOAC international (16th ed.). Arlington, VA: AOAC International. Chang, S. C., Kung, H. F., Chen, H. C., Lin, C. S., & Tsai, Y. H. (2008). Determination of histamine and bacterial isolation in swordfish fillets (Xiphias gladius) implicated in a food borne poisoning. Food Control, 19, 16–21. Chen, H. C., Kung, H. F., Chen, W. C., Lin, W. F., Hwang, D. F., Lee, Y. C., et al. (2008). Determination of histamine and histamine-forming bacteria in tuna dumpling implicated in a food-borne poisoning. Food Chemistry, 106, 612–618. Chen, K. T., & Malison, M. D. (1987). Outbreak of scombroid fish poisoning, Taiwan. American Journal of Public Health, 77, 1335–1336. 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. Eerola, S., Hinkkanen, R., Lindfors, E., & Hirvi, T. (1993). Liquid chromatographic determination of biogenic amines in dry sausages. Journal of AOAC International, 76, 575–577. FDA (1992). Bacteriological analytical manual. Arlington, VA: AOAC International. Hwang, D. F., Chang, S. H., Shiau, C. Y., & Chai, T. (1997). High-performance liquid chromatographic determination of biogenic amines in fish implicated in food poisoning. Journal of Chromatography B, 693, 23–30. Hwang, D. F., Chang, S. H., Shiau, C. Y., & Cheng, C. C. (1995). Biogenic amines in the flesh of sailfish (Istiophorus platypterus) responsible for scombroid poisoning. Journal of Food Science, 60, 926–928.

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