Food Control 42 (2014) 165e171

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Food Control
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Histamine content and histamine-forming bacteria in mahi-mahi

(Coryphaena hippurus) fillets and dried products
Chung-Saint Lin a, **, Hsin-Chuan Tsai b, Chia-Min Lin b, Chun-Yung Huang b,
Hsien-Feng Kung c, Yung-Hsiang Tsai b, *
a
Department of Food Science, Yuanpei University, Hsin-Chu, Taiwan, ROC
b
Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung 811, Taiwan, ROC
c
Department of Biotechnology, Tajen University, Pingtung, Taiwan, ROC

a r t i c l e i n f o a b s t r a c t

Article history: Forty-two mahi-mahi fillets and 17 dried products sold in retail markets in Taiwan were tested for
Received 4 September 2013 histamine and histamine-forming bacteria. The levels of pH, salt content, water content, Aw, TVBN, APC,
Received in revised form TC and Escherichia coli in mahi-mahi fillet samples ranged from 5.6 to 6.5, 0.05 to 2.44%, 70.9 to 82.8%,
26 January 2014
0.95 to 0.99, 5.9 to 23.5 mg/100 g, 3.1 to 7.0 log CFU/g, <3 to 1650 MPN/g and <3 to 45 MPN/g,
Accepted 1 February 2014
Available online 11 February 2014
respectively. The levels of pH, salt content, water content, Aw, TVBN, APC, TC and E. coli in dried mahi-
mahi samples ranged from 5.7 to 6.4, 0.63 to 20.13%, 7.1 to 42.9%, 0.51 to 0.85, 21.4 to 133.9 mg/100 g, 3.6
to 8.7 log CFU/g, <3 to 5900 MPN/g and <3 to 2500 MPN/g, respectively. The average content of various
Keywords:
Histamine
biogenic amines in fillets samples was less than 0.3 mg/100 g. Four of the 17 dried samples (23.4%) had
Mahi-mahi histamine levels greater than the FDA guideline of 5 mg/100 g for scombroid fish and/or product with
Histamine-forming bacteria one of them containing 68.15 mg/100 g of histamine, which is greater than the 50 mg/100 g hazard
Hygienic quality action level. Eight histamine-producing bacterial isolates, capable of producing 12.6 ppme562 ppm of
histamine in trypticase soy broth supplemented with 1.0% L-histidine (TSBH), were identified as Raoul-
tella ornithinolytica (three isolates), Pantoea agglomerans (two isolates), Proteus vulgaris (two isolates) and
Enterobacter amnigenus (one isolate), by 16S rDNA sequencing with PCR amplification.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction in incidents of scombroid poisoning. In Taiwan, scombroid

Histamine is the causative agent of scombroid poisoning and a
food-borne chemical hazard. Although 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 amounts of histamine ingested and 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 can also be implicated
* Corresponding author. Department of Seafood Science, National Kaohsiung
Marine University, No. 142, Hai-Chuan Rd. Nan-Tzu, Kaohsiung City 811, Taiwan,
ROC. Tel.: þ886 7 3617141 3609; fax: þ886 7 3640634.
** Corresponding author. Tel.: þ886 3 5381183 8477; fax: þ886 3 7740213.
E-mail addresses: chungsl@mail.ypu.edu.tw (C.-S. Lin), yhtsai01@seed.net.tw
(Y.-H. Tsai).
poisoning occurs occasionally (Chen et al., 2008; Chen & Malison,
1987; Tsai, Kung, et al., 2005) and has commonly been associated
with tuna, mackerel, and black marlin. Recently, sailfish, swordfish
and marlin have also been implicated in several scombroid out-
breaks in Taiwan (Chang, Kung, Chen, Lin, & Tsai, 2008; Chen,
Huang, et al., 2010; Chen, Lee, Lin, Hwang, & Tsai, 2010; Tsai,
Hsieh, et al., 2007).
Biogenic amines are formed mainly through the decarboxyl-
ation of certain free amino acids by exogenous decarboxylases
produced by a number of bacteria associated with seafood. Many
species of bacteria can produce histidine decarboxylase which can
convert free histidine to histamine through decarboxylation (An &
Ben-Gigirey, 1998). In addition to Morganella morganii, Klebsiella
pneumoniae and Hafnia alvei which have been isolated from the fish
incriminated in scombroid poisoning (Stratton & Taylor, 1991),
several species of the enteric bacteria capable of producing hista-
mine have also been isolated from fish (Middlebrooks, Toom,
Douglas, Harrison, & McDowell, 1988; Taylor & Speckard, 1983).
These include Proteus vulgaris, Proteus mirabilis, Enterobacter

http://dx.doi.org/10.1016/j.foodcont.2014.02.004
0956-7135/Ó 2014 Elsevier Ltd. All rights reserved.
166 C.-S. Lin et al. / Food Control 42 (2014) 165e171
aerogenes, Enterobacter cloacae, Serratia fonticola, Serratia liquefa-
ciens and Citrobacter freundii (Kim et al., 2003; Tsai, Lin, et al., 2005).
Other than the enteric bacteria, Clostridium spp., Vibrio alginolyti-
cus, Acinetobacter lowffi, Plesiomonas shigelloides, Pseudomonas
putida, Pseudomonas fluorescens, Aeromonas spp. and Photo-
bacterium spp. have also been reported as histamine producers
(Middlebrooks et al., 1988; Okuzumi, Hiraishi, Kobayashi, & Fujii,
1994). Recently, we also isolated several prolific histamine-
forming bacteria, including Enterobacter, Klebsiella, Raoultella and
Citrobacter spp. from sailfish fillets, dried milkfish, tuna dumpling
and tuna sandwich in Taiwan (Hsu et al., 2009; Kung et al., 2010;
Kung et al., 2009; Tsai, Kung, Lee, Lin, & Hwang, 2004).
In January 2009, an incident of food-borne poisoning causing
illness in 53 victims due to ingestion of mahi-mahi fillets occurred
in southern Taiwan (Chen, Lee, Hwang, Chiou, & Tsai, 2011). This is
the first report that mahi-mahi was associated with histamine
intoxication in Taiwan. However, there exists no report on the
occurrence of biogenic amines, including histamine, histamine-
forming bacteria and related bacteria in mahi-mahi fillets and
dried mahi-mahi products. Therefore, this research was undertaken
by testing 42 mahi-mahi fillets and 17 dried mahi-mahi products
sold in retail markets in Taiwan so that a better understanding of
the safety quality of the products could be accomplished to better
protect the consumers.
2. Materials and methods
2.1. Samples
Forty-two mahi-mahi fillets and 17 dried mahi-mahi products
were purchased from seven retail markets every two weeks in
Taiwan. Troll-caught mahi-mahi (Coryphaena hippurus) (5e12 kg
each) were commercially harvested off the Taiwan coast and
delivered to fish processing factories from March to May 2011. The
mahi-mahi fish were all degutted and filleted on processing fac-
tories, stored in ice and brought to retail markets for sale (250e
350 g each fillet). If mahi-mahi fish or fillets were harvested or left
too much, the retailers processed the mahi-mahi fillets to dried
products with salting and sun-drying for several days (150e300 g
each dried sample). Therefore, the number of dried samples
collected was less than that of fillet samples in this study. In gen-
eral, the fillet samples were kept in frozen or refrigeration tem-
perature, while the dried products were no packed and kept in
room temperature at retail stores before purchase. All samples
were wrapped in aseptic bags, placed in ice, and immediately
transported to the laboratory for use within 8 h. The experiment
was triplicate for each analysis.
2.2. pH value, salt content, water content and water activity
determination
Mahi-mahi samples (10 g) were 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 salt content in each sample
was determined according to the AOAC procedures (1995) by ho-
mogenizing 2 g of mahi-mahi 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 analyzed
with the standard gravimetric method by drying 1e3 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 mahi-mahi sample was homogenized at
high speed for 2 min in a sterile blender with 225 ml sterile po-
tassium phosphate buffer (0.05 M, pH 7.0). The blender was ster-
ilized 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 poured 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.
Triplicates were taken for bacterial analysis. Bacterial numbers in
the mahi-mahi samples were expressed as log10 colony forming
units (CFU)/g.
To isolate histamine-forming bacteria, a 0.1 ml aliquot of the
sample dilute was spread on histamine-forming bacterium isola-
tion 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. Each pure culture was grown 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 was used to analyze production of
biogenic amines by each isolate.
Analyses of total coliform and Escherichia coli in these mahi-
mahi samples were conducted using the three-tube most prob-
able number (MPN) methods (FDA, 1998). Lauryl sulfate tryptose
broth (LST broth) and brilliant green lactose bile (2%) broth (BGLB
broth) incubated at 35  C for 48 h were used for presumptive and
confirmation tests for total coliform, respectively. E. coli was
determined by using the LST broth and EC broth incubated at 35  C
and 44.5  C for 48 h, respectively. Cultures that showed positive
production of gas in BGLB or 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 examined on
the basis of morphology, Gram stain, endospore stain, catalase and
oxidase reaction. The identity of histamine-forming isolates was
confirmed by amplifying and sequencing approximately 1400 bp of
the 16S ribosomal DNA (rDNA) of bacteria (Kuhnert, Capaul, Nicolet,
& Frey, 1996; Kuhnert, Heyberger-Meyer, Nicolet, & Frey, 2000).
Amplification of the 16S rDNA of histamine-forming bacteria was
performed using the universal primers UNI-L (50 -AGAGTTTGAT-
CATGGCTCAG-30 ) and UNI-R (50 -GTGTGACGGGCGGTGTGTAC-30 )
(Kuhnert et al., 1996, 2000). Cells of presumptive histamine-
forming isolates were cultured overnight in 2 ml of TSB at 35  C
and then centrifuged at 5000 g for 10 min. The cell pellet was
washed and resuspended in 0.5 ml of TE-buffer (10 mM TriseHCl,
1 mM EDTA; pH 8.0), and then lysed by 20% sodium dodecyl sulfate
(SDS). The solution was boiled for 20 min and the supernatant was
collected following centrifugation at 13,000g for 3 min at 4  C, the
total DNA in the supernatant was precipitated with 70% ethanol
and used as template DNA for PCR.
PCR amplification was performed in 20 ml reaction mixture
containing 10 mM TriseHCl (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 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,
 C.-S. Lin et al. / Food Control 42 (2014) 165e171
2000). Amplicons were detected by electrophoresis on a 1.5%
agarose gel followed by staining with ethidium bromide. Ampli-
cons were purified using a QIAquick PCR Purification Kit (Qiagen,
Valencia, CA, USA) eluted in TriseHCl (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 se-
quences were analyzed with the BLAST (NCBI) for identification of
histamine-forming bacteria.
2.5. Determination of total volatile base nitrogen (TVBN)
The TVBN contents of each mahi-mahi sample were measured
by the method of Conway’s dish (Cobb, Aoaniz, & Thompson, 1973).
The TVBN extract of each sample in 6% trichloroacetic acid (TCA,
Sigma, St. Louis, Mo, USA) was absorbed by boric acid and then
titrated with 0.02 N HCl. Results of TVBN contents were expressed
in mg/100 g fish.
2.6. Biogenic amine analysis
Each mahi-mahi 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
(10,000g, 10 min, 4  C) and filtered through Whatman No. 2 filter
paper (Whatman, Maidstone, England). The sample filtrate was
collected in volumetric flasks and mixed with TCA to a final volume
of 50 ml. Samples of standard biogenic amine solutions and 1 ml
aliquots of the mahi-mahi extracts were derivatized with dansyl
chloride according to the previously described method (Chen,
Huang, et al., 2010). One milliliter of each culture TSBH broth of
presumptive histamine-forming isolate was also dansylated using
the same procedures for mahi-mahi extracts. The dansyl de-
rivatives were dissolved in 5 ml of acetonitrile, and an aliquot of
20 ml were used for HPLC injection.
The contents of biogenic amines in the mahi-mahi samples 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 UVeVis detector
(set at 254 nm), and a Model D-2500 Chromato-integrator. A
LiChrospher 100 RP-18 reversed-phase column (5 mm,
125  4.6 mm, E. Merck, Damstadt, Germany) was used for sepa-
ration. 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) in the next
10 min. The acetonitrile:water mix was then decreased to 50:50
(1.0 ml/min) during the next 10 min. All samples were analyzed in
duplicate.
2.7. Free histidine analysis of fresh mahi-mahi fillet
Free histidine in fresh mahi-mahi fillet was determined ac-
cording to the method described by Konosu, Watanabe, and
Table 1
Values of pH, salt content, water content, water activity (Aw), total volatile basic nitrogen (TVBN), aerobic plate count (APC), total coliform (TC) and Escherichia coli (E. coli) in
the commercial mahi-mahi fillets and dried mahi-mahi products.
Samples No. of pH Salt content (%)
samples
Mahi-mahi fillets 42 5.6e6.5 0.05e2.44
(6.0  0.2)a,A (0.22  0.36)B
Dried mahi-mahi 17 5.7e6.4 0.63e20.13
Products (6.1  0.2)A (2.92   2.70)A
a
Mean  SD. Values in the same column with different superscript letters are statistically different (P < 0.05).
167
Shimizu (1974) in triplicate. Ten grams of sample were homoge-
nized for 2 min in 20 ml of 7% trichloroacetic acid (TCA) and
analyzed by postcolumn derivatization with ninhydrin using an L-
8500 high-speed amino acid analyzer attached with Hitachi
2622SC packed column (4.6  60 mm; Hitachi, Tokyo, Japan). The
buffers used were the standard lithium citrate buffers. Postcolumn
derivatization with ninhydrin yielded histidine derivatives which
were measured by the absorbance at 570 and 440 nm. Analytical
conditions and procedures were performed according to the
manual provided by the manufacturer (Hitachi, Ltd., Tokyo, Japan).
The histidine content in a sample was estimated on the basis of
peak area of known concentrations of standard (Wako, Osaka,
Japan) using Hitachi D-2850 Chromato-integrator.
2.8. Effect of NaCl content on histamine-forming bacteria
The effect of NaCl content on histamine production by
histamine-forming bacteria was determined in 50 ml of TSBH
medium in flasks containing 0.5%, 5%, 10%, 15% or 20% of NaCl. One
hundred microliters of the 20-h-old bacterial cultures in 5 ml of
TSBH medium (0.5% NaCl) at 35  C were inoculated into fresh TSBH
to obtain an initial concentration of about 6.0 log CFU/ml. Bacterial
growth and histamine production in TSBH were determined after
incubation at 35  C for 12, 24, 36, 48 and 72 h.
2.9. Statistical analysis
Pearson correlation was carried out to determine relationships
between pH, salt content, water content, Aw, TVBN, APC, total
coliform, E. coli and histamine contents in the 59 samples. All sta-
tistical 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, salt content, water content, water activity
(Aw), total volatile basic nitrogen (TVBN), aerobic plate count (APC),
total coliform (TC) and E. coli in the 42 mahi-mahi fillets and 17
dried mahi-mahi products from the retail markets were presented
in Table 1. The average levels of salt content (2.92%), TVBN (39.6 mg/
100 g), TC (1098.5 MPN/g) and E. coli (162.9 MPN/g) in dried sam-
ples were significantly (P < 0.05) higher than those of fillet samples,
whereas the average levels of Aw (0.69) and water content (24.9%)
in dried samples were lower than those of fillet samples (P < 0.05)
(Table 1).
The rates of unacceptable dried mahi-mahi samples were 76.4%
(13/17) for TVBN, based on the decomposition limit level of 25 mg/
100 g for fish quality determination using Taiwanese regulatory
standard, and 29.0% (5/17) and 19.0% (3/17), using the Taiwanese
regulatory standard of 6.47 log CFU/g for APC and 10 MPN/g for
E. coli, respectively. However, the rates of unacceptable fillet sam-
ples were 2.4% (1/42) and 7.1% (3/42) for APC and E. coli, respec-
tively. The dried samples were made by sun-drying for several days,
Water content Aw TVBN APC TC (MPN/g) E. coli (MPN/g)
(%) (mg/100 g) (log CFU/g)
70.9e82.8 0.95e0.99 5.9e23.5 3.1e7.0 <3e1650 <3e45
(77.6  2.1)A (0.98  0.01)A (13.7  4.1)B (4.7  0.9)A (144.9  296.4)B (1.8  7.2)B
7.1e42.9 0.51e0.85 21.4e133.9 3.6e8.7 <3e5900 <3e2500
(24.9  10.3)B (0.69  0.09)B (39.6  16.3)A (5.9  0.9)A (1098.5  1224.7)A (162.9  274.9)A
168 C.-S. Lin et al. / Food Control 42 (2014) 165e171

and stored at room temperature, allowing the products being Table 3

contaminated under poor sanitary conditions with low quality. Distribution of histamine in the 42 mahi-mahi fillets and 17 dried mahi-mahi
products.
Concerning the hygienic quality, Hsu et al. (2009) also reported that
dried milkfish product had the unacceptable rates of 68.8% for Content of histamine Mahi-mahi fillets Dried mahi-mahi
TVBN, 46.9% for APC and 12.5% for E. coli. (mg/100 g) products

Table 2 summarized the contents of biogenic amines in the
tested mahi-mahi fillets and dried products. Except for dried
samples, the average content of various biogenic amines in tested
fillet samples was less than 0.3 mg/100 g. The average contents of
cadaverine and histamine in tested dried samples were 3.68 mg/
100 g and 7.38 mg/100 g, respectively, whereas the average content
of each of the remaining six biogenic amines was less than 0.66 mg/
100 g (Table 2). Table 3 showed the distribution of histamine con-
tents in tested mahi-mahi fillets and dried products, with 23.5% (4/
17) dried 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 product. One dried sample (68.15 mg/
100 g of histamine) with greater than 50 mg/100 g of histamine
could be hazardous to the health of consumers based on the data
collected from numerous outbreak reports (USFDA, 2001). It is also
observed that histamine at 20 mg/100 g may be sufficient to cause
the symptoms of scombroid poisoning (CDC, 2000). In a previous
study, a high content of histamine at 61.6 mg/100 g in the suspected
dried milkfish product was considered the etiological factor for a
histamine poisoning in Taiwan (Tsai, Kung, et al., 2007). 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). Based on the finding that higher levels of histamine,
TVBN, TC and E. coli occurred in dried mahi-mahi samples, we
postulate that these samples had been seriously contaminated
during food preparation and processing. The average tyramine
content in 42 mahi-mahi fillets was 0.2 mg/100 g, whereas none of
the 17 dried mahi-mahi products contained tyramine (Table 2). The
formation of tyramine in fish depends on the quantities of free
tyrosine and the presence of microorganisms with tyrosine decar-
boxylase activities (tyramine-producing bacteria) (Silla-Santos,
1996). Therefore, no tyramine content in dried mahi-mahi prod-
ucts observed in this work may be related to these samples con-
tained less tyramine-producing bacteria.
The free histidine content in fresh mahi-mahi fillet was 705 mg/
100 g in this study (data not shown). This result corresponds with
that of free histidine content ranged from 490 to 940 mg/100 g in
white muscle of mahi-mahi (Baranowski, Brust, & Frank, 1985).
However, Antoine et al. (2002) reported free histidine level ranged
from 290 to 334 mg/100 g in mahi-mahi. Such variations are a
result of several factors which include feeding, season, sex and
stage of maturity (Shiau, Pong, Chiou, & Chai, 1997). The minimum
histidine concentration required for bacterial histidine decarbox-
ylase activity was estimated to be 100e200 mg/100 g (Chen, Wei,
Koburger, & Marshall, 1989). Therefore, mahi-mahi fillet sample
containing free histidine at a level of 705 mg/100 g may become a
<5 42 (100%) 13 (76.5%)
5e50 0 (0%) 3 (17.6%)
>50 0 (0%) 1 (5.9%)
Total 42 17
vehicle for scombroid poisoning if it is contaminated with
histamine-forming bacteria.
Although higher content of histamine was detected in dried mahi-
mahi products tested in this study, only very few cases of food-borne
histamine intoxication were reported due to consumption of dried
mahi-mahi products. Symptoms of histamine poisoning are not
particularly definitive. Therefore, histamine intoxication is frequently
confused diagnostically with an allergic reaction. Also, histamine
poisoning is not a reportable illness even in those countries that keep
surveillance records (Taylor,1986). High levels of cadaverine were also
found in dried mahi-mahi products in this study (Table 2). Putrescine
and cadaverine have been shown to potentiate histamine toxicity
when present in spoiled fish by inhibiting the intestinal histamine
metabolizing enzyme (Antoine et al., 2002).
Pearson correlation was conducted to determine if there existed
any relationship among the pH, salt content, water content, Aw,
TVBN, APC, TC, E. coli, and histamine contents of the tested 59
samples. In general, the positive correlations existed between Aw
and APC (r ¼ 0.685, P < 0.05), Aw and histamine (r ¼ 0.602,
P < 0.05), water content and histamine (r ¼ 0.522, P < 0.05), pH and
TVBN (r ¼ 0.726, P < 0.05), histamine and TVBN (r ¼ 0.622,
P < 0.05), histamine and APC (r ¼ 0.551, P < 0.05), histamine and TC
(r ¼ 0.691, P < 0.05), and histamine and E. coli (r ¼ 0.589, P < 0.05).
However, the negative correlations were noted between salt con-
tent and APC (r ¼ 0.873, P < 0.01), and Aw and salt content
(r ¼ 0.590, P < 0.05). This result showed that the samples con-
taining higher Aw, water content, TVBN, APC, TC or E. coli levels had
more histamine content in these fish samples.
Table 4 listed the identity of eight histamine-forming bacteria
isolated from the mahi-mahi fillets and dried samples as deter-
mined by 16S rDNA sequences using NCBI database analysis. Eight
histamine-producing bacterial isolates, capable of producing
12.6 ppme561.7 ppm of histamine in trypticase soy broth supple-
mented with 1.0% L-histidine (TSBH) after incubation at 35  C for
24 h were identified as Raoultella ornithinolytica (three strains),
Pantoea agglomerans (two strains), P. vulgaris (two strains) and
Enterobacter amnigenus (one strain) (Table 4). Some of them also
produced different amounts of putrescine and cadaverine through
the action of their respective decarboxylase enzymes on various
amino acids that also existed in culture medium.
In this study, all histamine-forming isolates belonged to Enter-
obacteriaceae. Enterobacteriaceae are generally thought to be the

Table 2
Contents of biogenic in the 42 mahi-mahi fillets and 17 dried mahi-mahi products.

Samples No. of Levels of biogenic amines (mg/100 g)

sample
Trya Phe Put Cad His Tyr Spd Spm

Mahi-mahi fillets 42 NDb NDe1.04 NDe0.39 ND NDe0.77 NDe5.81 NDe1.44 NDe0.99

(0.11  0.26)c (0.05  0.11) (0.13  0.23) (0.20  0.98) (0.04  0.08) (0.30  0.24)
Dried mahi-mahi 17 0e0.40 NDe0.32 NDe0.31 NDe21.98 NDe68.15 ND NDe0.41 NDe1.41
products (0.02  0.04) (0.04  0.1) (0.44  0.79) (3.68  7.1) (7.38  17.46) (0.06  0.09) (0.66  0.34)
a
Try: tryptamine; Phe: 2-phenylethylamine; Put: putrescine; Cad: cadaverine; His: histamine; Tyr: tyramine; Spd: spermidine; Spm: spermine.
b
ND: Not detected (amine level less than 0.05 mg/100 g).
c
Mean  S.D.
 C.-S. Lin et al. / Food Control 42 (2014) 165e171 169

Table 4
Identification of histamine-forming bacteria isolated from the mahi-mahi fillets and dried mahi-mahi products collected from retail stores by 16S rDNA, based on the output
results from NCBI database analysis, and their production of histamine and other biogenic amines (ppm) in culture broth.

Sources Strain Organism identified Percentage GenBank accession Histamine content Hisa Put Cad
identity (%) number in original mahi-mahi
sample (mg/100 g)

Dried mahi-mahi product Lc22-2 Raoultella ornithinolytica 100% JF701186.1 68.15 561.7 NDb 205.9
Dried mahi-mahi product Lc22-1 Raoultella ornithinolytica 100% JF701186.1 68.15 526.7 ND 125.8
Dried mahi-mahi product Lc22-3 Raoultella ornithinolytica 100% JF701186.1 68.15 426.8 ND 285.9
Mahi-mahi fillet Lc1-8 Pantoea agglomerans 100% HM130693.1 <0.05 197.5 1.34 ND
Mahi-mahi fillet Lc5-4 Pantoea agglomerans 100% HM130693.1 <0.05 29.2 ND ND
Mahi-mahi fillet Ka3-3 Proteus vulgaris 100% DQ826507.1 0.13 156.0 9.3 ND
Mahi-mahi fillet Ka3-7 Proteus vulgaris 100% JN409462.1 0.13 72.5 ND ND
Mahi-mahi fillet Lc7-1 Enterobacter amnigenus 100% EU275356.1 <0.05 12.6 0.1 ND
a
Put: putrescine; His: histamine; Cad: cadaverine.
b
ND: Not detected (amine level less than 0.05 ppm).

primary cause of histamine development in scombroid fish. the three days of incubation. No bacterial growth or histamine
Meanwhile, three out of eight histamine-producing bacterial iso- production occurred when the NaCl content in TSBH was increased
lates capable of producing >426 ppm of histamine in TSBH isolated to 20%. Taylor and Speckard (1983) reported that NaCl at 0.5e2.0%
from the dried mahi-mahi sample (68.15 mg/100 g of histamine) did not inhibit the growth of M. morganii and K. pneumoniae, and
were identified as R. ornithinolytica (three strains). Therefore, we their histamine production. The E. cloacae from salted anchovies
conclude that R. ornithinolytica were the bacteria to produce haz- (Hernandez-Herrero, Roig-Sagues, Rodriguez-Jerez, & Mora-
ardous levels of histamine in this dried mahi-mahi sample. Ventura, 1999) and salted mackerel (Tsai, Lin, et al., 2005) pro-
R. ornithinolytica has been frequently reported as a prolific hista- duced histamine in culture broths that had a NaCl content of 0.5 or
mine-former in tuna (Lopez-Sabater, Rodriguez-Jerez, Hernandez-
Herrero, Roig-Sagues, & Mora-Ventura, 1996), albacore (Kim et al.,
2001) and sailfish (Tsai et al., 2004). R. ornithinolytica isolated
from dried milkfish implicated in a food-borne poisoning was re-
ported to be potent histamine-former capable of producing
1243 ppm of histamine in TSBH at 35  C for 24 h (Tsai, Kung, et al.,
2007). Recently, R. ornithinolytica isolated from tuna dumpling,
tuna sandwich and tuna sausage products were also identified as
prolific histamine-formers (Kung et al., 2010; Kung, Tsai, Chang,
Hong, 2012; Kung et al., 2009). 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
tank with salting process for a long time.
Although P. agglomerans were rarely reported as histamine-
formers, they were the important histamine-producing bacteria
found in this study, accounting for 25% (2/8) of the total histamine-
forming isolates. The P. agglomerans that Kim et al. (2001) isolated
from temperature-abused albacore was a weak histamine-former.
The recently isolated P. agglomerans from salted mackerel prod-
ucts in Taiwan were identified as weak histamine-forming bacteria
(Tsai, Lin, et al., 2005). Similarly, two isolates of P. agglomerans (25%
of isolates) isolated from mahi-mahi fillet sample in this study were
identified as weak histamine-forming formers, capable of produc-
ing 197.5 and 29.2 ppm of histamine in TSBH, respectively (Table 4).
The growth and histamine production of R. ornithinolytica strain
Lc22-2 in TSBH medium containing 0.5%, 5%, 10%, 15%, or 20% of
NaCl are shown in Fig. 1. At 0.5% NaCl, histamine production was
accelerated along with bacterial growth, with histamine levels
exceeding 850 ppm being detected after 36 h of incubation. At 5%
NaCl, bacterial count increased gradually until they reached about
9.1 log CFU/ml after 24 h. The levels of histamine were below
100 ppm at 12 h of incubation but increased to above 500 ppm
thereafter at 36 h. Higher levels of histamine were detected in TSBH
containing 0.5% NaCl than 5% NaCl at each incubation time. How-
ever, as the medium NaCl content was increased to 10% or 15%, Fig. 1. The growth (A) and histamine production (B) of Raoultella ornithinolytica strain
bacterial growth was inhibited for three days. Low levels of hista- Lc22-2 at 35  C in TSBH medium containing 0.5%, 5%, 10%, 15% or 20% of NaCl. Each
mine at below 10 ppm were produced by this bacterial strain during value represents the mean  standard deviation in triplicate.
170 C.-S. Lin et al. / Food Control 42 (2014) 165e171
3%, but these histamine-forming bacterial isolates failed to produce
histamine in a broth containing 10 or 20% of NaCl. In previous study,
we reported that NaCl concentrations in 1.5 and 3.5% had a stim-
ulatory effect on histamine formation of R. ornithinolyticus, whereas
levels of NaCl in excess of 7.5% inhibited their growth and histamine
formation (Tsai, Kung, et al., 2007). Apparently, the decarboxylase
activity of enterobacteria is highly sensitive to elevated NaCl con-
tent (Rodriguez-Jerez, Lopez-Sabater, Roig-Sagues, & Mora-
Ventura, 1994; Rodriguez-Jerez, Mora-Ventura, Lopez-Sabater, &
Hernandez-Herrero, 1994). All these reports supported our finding
that higher content of NaCl at about 10% reduced both the growth
and histidine decarboxylase activity of enterobacteria. It is inter-
esting to note that the dried mahi-mahi samples tested in this study
had salt contents from 0.63% to 20.13% and average salt contents of
2.92% (Table 1), while the optimal NaCl concentration for histamine
formation in TSBH for R. ornithinolytica strain Lc22-2 was 0.5e5%. It
is very possible that once the processed dried fish containing 0.5e
5% of salt contents is contaminated with R. ornithinolytica during
manufacturing, the bacteria will grow under these conditions with
optimal salt content and produce hazardous levels of histamine.
4. Conclusion
This study analyzed 42 mahi-mahi fillets and 17 dried mahi-
mahi samples sold in Taiwan and showed that the TVBN content
in 13 dried fish samples exceeded the 25 mg/100 g decomposition
limit. Four of the 17 dried fish samples contained histamine at
levels of greater than the USFDA guideline of 5 mg/100 g for human
consumption. R. ornithinolytica isolated from one dried fish sample
containing the highest histamine content of 68.15 mg/100 g was
identified as a prolific histamine-former capable of producing
>500 ppm of histamine in TSBH medium containing 5% NaCl.
Acknowledgment
The study was supported by the National Science Council, ROC
(Contract No. NSC 101-2313-B-022-002-MY3).
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