Arch Med Vet 40, 95-98 (2008)

COMMUNICATION

Histamine formation and microbiological changes in endemic

Chalcalburnus tarichi Pallas 1811 (Inci Kefali) stored at 4 °C

Formación de histamina y cambios microbiológicos en

Chalcalburnus tarichi Pallas 1811 (Inci Kefali) endémico almacenado a 4 °C

K Ekici*, M Alisarli
University of Yüzüncü Yil Veterinary College, Department of Food Hygiene and Technology, Van, Turkey

resumen

El objetivo de este estudio fue medir la formación de histamina y los cambios microbiológicos en Chalcalburnus tarichi fresco procedente del lago
Van y almacenado a 4 oC por un período de hasta 15 días. Muestras del músculo de los pescados fueron tomadas en intervalos de tiempo (días 1, 5, 7, 9, 11,
13 y 15) durante el almacenaje. El contenido de la histamina fue determinado usando un método espectrofluorométrico y el conteo total y características
de las Enterobacteriaceae y Pseudomonas spp. presentes en las muestras fueron establecidas por procedimientos microbiológicos estándares.
La concentración inicial de histamina era de 27,5 mg/kg, aumentando gradualmente hasta 134,38 mg/kg en el día 15. El conteo bacteriano viable
total varió de 8,0x102 a 9,0x109 ufc/g. Las Enterobacteriaceae estaban en el rango de 2,0 x 102 a 6,5 x 109 ufc/g, mientras las Pseudomonas spp. estaban
entre 3,0 x 102 a 7,3 x 109 ufc/g.

Palabras clave: histamina, calidad microbiológica, Chalcalburnus tarichi

Key words: histamine, microbiological quality, Chalcalburnus tarichi

INTRODUCTION al 1985, Stratton et al 1991, Santos et al 1998). Histamine

may be involved in the onset of migraine attacks in sus-
The ingestion of foods containing substantial amounts ceptible subjects and may produce hypertensive crises in
of histamine is the cause of food poisoning episodes, a type patients treated with monoamine oxidase inhibitor-type
of foodborne illness commonly associated to consump- drugs (Khalid and Marth 1990).
tion of scombroid fish such as tuna and mackerel and In addition to its toxicological properties, histamine is
some nonscombroid fish such as bluefish or mahi-mahi of interest as an indicator of food quality (Bover-Cid et al
that have begun to spoil by the growth of particular types 2001, Ekici et al 2004) and of food spoilage (Valsamaki
of bacteria (Merson et al 1974, Beling and Taylor 1982, et al 2000). The minimal concentration of histamine in
Hughes et al 1991). foodstuffs that would elicit a toxic response has been
Biologically active amines have been found in many estimated to be 100 mg/100g (Taylor et al 1978).
foods such as fish and fish products (Merson et al 1974, Fish are of great importance for human nutrition
Shalaby 1994, Becker et al 2001). Biogenic amines are worldwide. Non-pathogenic and pathogenic bacteria
organic bases with an aliphatic, aromatic, or heterocyclic have been found on the skin, gills and intestines of fish
structure that has been found in many foods that include fish (Feldhusen 2000).
and fish products, cheese, wine, beer and other fermented Lake Van is the largest soda lake and fourth largest
foods (Stratton et al 1991, Hernández-Jover et al 1997). closed lake on earth. It is situated at 1,648 m above sea
Histamine is formed by bacterial enzymatic decarboxyla- level in Eastern Anatolia, Turkey. Many springs and
tion of histidine during the final steps of protein breakdown freshwater rivers flow into this lake. Its high carbonate
(Koehler and Eitenmiller 1978). Bacterial strains known to content makes it extremely alkaline, with a pH = 9,8 that
be capable of histamine production include Escherichia, is unsuitable for many fresh water species with exception
Enterobacter, Pseudomonads, Salmonella, Shigella, of a fish locally known as “Inci Kefali” or Pearl Mullet,
Clostridium perfringens, Streptococcus, Lactobacillus Chalcalburnus tarichi, a member of the cyprinidae family
and Leuconostoc (Edwards and Sandine 1981, Chang et (Arabaci and Sari 2004).
To date, there is no detailed information available
on histamine formation and microbiological quality of
Inci Kefali. The purpose of this study was to measure the
Accepted: 03.04.2007. amount of histamine and microbiological changes in pearl
* Yüzüncü Yil University, Veterinary College, Department mullet for 15 days while stored at 4 °C to determine if
of Food Hygiene and Technology, TR-65080 Van, Turkey;
[email protected]
this is a safe practice for handling this product, in order

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k eKici, m alisarli

to evaluate whether the level of histamine and microbial Total viable counts Enterobacteriaceae Pseudomonas
count would pose a health hazard. 10

MATERIAL AND METHODS 8

Inci Kefali were caught from Van lake. Whole and

6

log cfu/g
uneviscerated fresh fish were about 25 kg weight, placed
immediately in an ice box and delivered to the laboratory
4
approximately 3-5 h later, where they were immediately
divided into eight lots weighing about 3 kg in sterile
bags each. The bags were stored for 15 days at a constant 2
temperature of 4 °C. At predetermined times, samples of
five to six Inci Kefali were randomly drawn from each lot 0
for analyses. Samples were periodically taken every other 1 3 5 7 9 11 13 15
day (1st, 3rd, 5th, 7th, 9th, 11th, 13th and 15th) throughout Storage Time (days)
the experimental period and used for the determination of Figure 1. Microbiological analyses of Chalcalburnus tarichi
histamine and bacteriological analysis. All assays were stored 1-15 days at 4°C.
done in duplicate and the results are given as average of Análisis microbiológicos de Chalcalburnus tarichi alma-
these two values. cenado 1-15 días a 4 °C.

Histamine analysis. Histamine was analyzed spectrofluo-

rimetrically following the procedure of Lerke and Bell
(1976). The fluorescence of the compound was measured by
means of a Luminescence spectrofluorometer (Perkin Elmer 150
LS 50 B Wellesley, USA), using an excitation wavelength Histamine content
120
of 350 nm and an emission wavelength of 450 nm. Standard
curves were automatically obtained by the spectrofluoro- 90
mg/kg

meter from known solutions. The results were expressed

as mg/kg wet weight of fresh fish muscle sample. 60

Microbiological analysis. For microbiological analysis 10 30

g fresh muscle fish samples were weighed in stomacher 0
bags to which 90 ml sterile saline solution with peptone 1 3 5 7 9 11 13 15
(0.85% NaCl + 0.1% peptone) were added. The samples Storage Time (day)
were then homogenized in the bags for 2 min. After ho- Figure 2. Histamine content of Chalcalburnus tarichi stored
mogenization, serial decimal dilutions were prepared up 1-15 days at 4°C
to 109 that were used for inoculating the growth media Contenido de histamina en Chalcalburnus tarichi almace-
in Petri dishes. For the total viable count, the drop plate nado 1-15 días a 4 °C.
technic on the plate count agar (PCA) (Oxoid CM325)
at 35 °C for 72 hours, Enterobacteriaceae violet red
bile agar (VRBA) (Oxoid CM485) pour plate technic at other bacteria have as great a capacity to form histamine
37 °C for 48 hours, Pseudomonas spp. the drop plate tech- (Eitenmiller et al 1981). Amine-degrading activities were
nic on pseudomonas agar base (PAB) (Oxoid CM559), at found in Brevibacterium linens and coryneform bacteria
25 °C for 48 hours was used. After inoculation, the colonies (Leuschnert and Hammes 1998). The presence of some
formed were counted (Pichhardt 1993). decarboxilase-positive spoiling microorganisms such as
Pseudomonas, Micrococci and Enterococci that can result
RESULTS AND DISCUSSION in histamine formation have been cited as possible cause
of this increase (Santos et al 1998).
The results are shown in figures 1 and 2. As can be seen There are several studies linking bacterial contamina-
from figures the concentration of histamine and microor- tion to histamine formation in aquatic species. Tekinsen et
ganisms increased steadily during the two weeks storage. al (1993) investigated histamine formation in three aquatic
Formation of biogenic amines depends on the concentra- species. After 3 days of storage at 4 °C, the histamine con-
tion of amino acids or peptides which act as precursors in tent in mackerel, bonito and anchovy was 6.28, 3.70 and
food (Voight et al 1974, Leuschnert and Hammes 1998). 3.25 mg/kg, respectively, well below toxic or dangerous
Recent studies have shown that Morganella morganii can levels. Edmunds et al (1975) also reported that histamine
be a contaminant of fish tissue during spoilage and that few formation in Spanish mackerel, white shrimp, common

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 histamine, microbiolOgical quality, Chalcalburnus tarichi

Table 1. Results of microbiological analyses of Chalcalburnus tarichi tion of the general microbiological quality of the product
stored 1-15 days at 4°C.
Resultados de los análisis microbiológicos de Chalcalburnus
(Altug and Bayrak 2003). It has been stated that a total
tarichi almacenado 1-15 días a 4 °C. viable count above 108 cfu/g may lead to food spoilge
in smoked fish (Pichhardt 1993). The total viable count
Storage time Total viable counts Enterobacteiaceae Pseudomonads spp. values here reported indicate that the pearl mullet samples
Days cfu/g cfu/g cfu/g were on the threshold of critical limits at the 11th day of
1 8.0 x 102 2.0 x 102 3.0 x 102 storage.
3 2.2 x 103 3.0 x 102 3.0 x 102
5 3.4 x 104 2.6 x 104 3.3 x 104
CONCLUSIONS
7 5.2 x 105 2.8 x 104 2.0 x 105
It can be concluded that the histamine levels in Inci
9 4.2 x 106 4.5 x 106 3.0 x 106
Kefali samples hygienically stored for up to 15 days at 4°C
11 2.8 x 108 2.4 x 108 2.0 x 108
do not seem to pose a health hazard. As microorganisms
13 9.0 x 109 8.1 x 108 8.2 x 108 readily propagate in fish, proper sanitary care should be
15 9.0 x 109 6.5 x 109 7.3 x 109 taken during handling. There is, however, a potential risk
depending from individual susceptibility to histamine,
mullet, speckled trout and channel catfish did not increase some gastrointestinal conditions, alcohol consumption
in any of these species after 14 days storage even though or the use of certain medicines that may influence the
extensive psychrophilic spoilage had occurred judged by biogenic amines detoxification routes.
appearance and odor evolution. Lopez-Sabater et al (1995)
found that under some conditions the level of histamine summary
was higher than the 50 mg/100 g level established by the
FDA for tuna fish after 21 days storage at 0, 8 and 20 °C. Histamine accumulates in food via microbial decarboxylation
Enterobacteriaceae and Pseudomonas ssp. are not part of the of histidine. Small amounts of histamine naturally occurring in food
under normal circumstances do not pose a public health hazard. Certain
normal microflora in fresh fish, but they grow rather quickly
microbial species such as Enterobacteriaceae and Pseudomonas spp.
in aerobically stored fish (Lougovois et al 2003, Behling have considerable capacity for histamine formation and can proliferate
and Taylor 1982). Pseudomonas can be the dominant spoil- during handling or processing of foodstuffs, possibly elevating the
age bacteria in fish from warmer waters and the maximum histamine content to a harmful level. The objective of this study was
histidine decarboxylase activity was observed during the to measure the formation of histamine and microbiological changes in
fresh Chalcalburnus tarichi from Van lake and stored at 4 °C for up to
late logarithmic phase of growth (Falcao et al 2002).
15 days. Fish muscle samples were taken on day 1, 3, 5, 7, 9, 11, 13 and
Establishment of a precise toxicity threshold of histamine 15 of experiment, during storage. Histamine content was determined
in individuals is extremely difficult because the toxic dose using a spectrofluorometric method and the total count and features
is strongly dependent on the efficiency of the detoxification of Enterobacteriaceae and Pseudomonas spp. present in the samples
mechanisms of each individual. Nevertheless, toxicological were established by standard microbiological procedures. The initial
concentration of histamine was 27.5 mg/kg, increasing gradually up
levels have been proposed, such as 10-100 mg of histamine
to 134.38 mg/kg on day 15. Total viable bacterial count varied from
per 100 g of food (Stratton et al 1991, Hernandez-Jover 8.0 x 102 to 9.0x109 cfu/g. Enterobacteriaceae was in the 2.0 x 102 to
et al 1997). The FDA has set the hazardous level for 6.5 x 109 cfu/g range, while Pseudomonas spp. was in the 3.0 x 102 to
histamine in tuna at 50 mg histamine/100g (Sumner et al 7.3 x 109 cfu/g range.
1990, Stratton et al 1991). The histamine content of Inci
Kefali in our study was lower than the FDA’s hazardous
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