Archivos de medicina veterinaria
versión impresa ISSN 0301-732X
Arch. med. vet. v.40 n.1 Valdivia 2008
Arch Med Vet 40, 95-98 (2008)
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
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 °C 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 Enterohacteriaceae 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 Enterohacteriaceae 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
Histamine accumulates in food via microbial decarboxylation of histidine. Small amounts of histamine naturally occurring in food under normal circumstances do not pose a public health hazard. Certain microbial species such as Enterobacteriaceae and Pseudomonas spp. have considerable capacity for histamine formation and can proliferate during handling or processing of foodstuffs, possibly elevating the histamine content to a harmful level. The objective of this study was to measure the formation of histamine and microbiological changes in fresh Chalcalburnus tarichi from Van lake and stored at 4 °C for up to 15 days. Fish muscle samples were taken on day 1,3,5,7,9,11,13 and 15 of experiment, during storage. Histamine content was determined using a spectrofluorometric method and the total count and features of Enterobacteriaceae and Pseudomonas spp. present in the samples were established by standard microbiological procedures. The initial concentration of histamine was 27.5 mg/kg, increasing gradually up to 134.38 mg/kg on day 15. Total viable bacterial count varied from 8.0 x 102 to 9.0x109 cfu/g. Enterobacteriaceae was in the 2.0 x 102 to 6.5 x 109 cfu/g range, while Pseudomonas spp. was in the 3.0 x 102 to 7.3 x 109 cfu/g range.
Key words: histamine, microbiological quality, Chalcalburnus tarichi
The ingestión of foods containing substantial amounts of histamine is the cause of food poisoning episodes, a type of foodbome illness commonly associated to consumption of scombroid fish such as tuna and mackerel and some nonscombroid fish such as bluefish or mahi-mahi that have begun to spoil by the growth of particular types of bacteria (Merson et al 191 A, Beling and Taylor 1982, Hughes etal 1991).
Biologically active amines have been found in many foods such as fish and fish products (Merson et al 1974, Shalaby 1994, Becker et al 2001). Biogenic amines are organic bases with an aliphatic, aromatic, or heterocyclic structure that has been found in many foods that include fish and fish products, cheese, wine, beer and other fermented foods (Stratton et al 1991, Hernández-Jover et al 1997).
Histamine is formed by bacterial enzymatic decarboxylation of histidine during the final steps of protein breakdown (Koehler and Eitenmiller 1978). Bacterial strains known to be capable of histamine production include Escherichia, Enterobacter, Pseudomonads, Salmonella, Shigella, Clostridium perfringens, Streptococcus, Lactobacillus and Leuconostoc (Edwards and Sandine 1981, Chang et al 1985, Stratton et al 1991, Santos et al 1998). Histamine may be involved in the onset of migraine attacks in susceptible subjects and may produce hypertensive crises in patients treated with monoamine oxidase inhibitor-type drugs (Khalid and Marth 1990).
In addition to its toxicological properties, histamine is of interest as an indicator of food quality (Bover-Cid et al 2001, Ekici et al 2004) and of food spoilage (Valsamaki et al 2000). The minimal concentration of histamine in foodstuffs that would elicit a toxic response has been estimated to be 100 mg/100g (Taylor et al 1978).
Fish are of great importance for human nutrition worldwide. Non-pathogenic and pathogenic bacteria have been found on the skin, gills and intestines of fish (Feldhusen 2000).
Lake Van is the largest soda lake and fourth largest closed lake on earth. It is situated at 1,648 m above sea level in Eastern Anatolia, Turkey. Many springs and freshwater rivers flow into this lake. Its high carbonate content makes it extremely alkaline, with a pH = 9,8 that is unsuitable for many fresh water species with exception of a fish locally known as "Inci Kefali" or Pearl Mullet, Chalcalburnus tarichi, a member of the cyprinidae family (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 amount of histamine and microbiological changes in pearl mullet for 15 days while stored at 4 °C to determine if this is a safe practice for handling this product, in order to evaluate whether the level of histamine and microbial count would pose a health hazard.
MATERIAL AND METHODS
Inci Kefali were caught from Van lake. Whole and uneviscerated fresh fish were about 25 kg weight, placed immediately in an ice box and delivered to the laboratory 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 temperature of 4 °C. At predetermined times, samples of five to six Inci Kefali were randomly drawn from each lot for analyses. Samples were periodically taken every other day (1st, 3rd, 5th, 7th, 9th, 11th, 13th and 15th) throughout the experimental period and used for the determination of histamine and bacteriological analysis. All assays were done in duplicate and the results are given as average of these two values.
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 LS 50 B Wellesley, USA), using an excitation wavelength of 350 nm and an emission wavelength of 450 nm. Standard curves were automatically obtained by the spectrofluorometer from known solutions. The results were expressed as mg/kg wet weight of fresh fish muscle sample.
Microbiological analysis. For microbiological analysis 10 g fresh muscle fish samples were weighed in stomacher bags to which 90 ml sterile saline solution with peptone (0.85% NaCl + 0.1% peptone) were added. The samples were then homogenized in the bags for 2 min. After ho-mogenization, serial decimal dilutions were prepared up to 109 that were used for inoculating the growth media in Petri dishes. For the total viable count, the drop plate 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 37 °C for 48 hours, Pseudomonas spp. the drop plate technic on pseudomonas agar base (PAB) (Oxoid CM559), at 25 °C for 48 hours was used. After inoculation, the colonies formed were counted (Pichhardt 1993).
RESULTS AND DISCUSSION
The results are shown in figures 1 and 2. As can be seen from figures the concentration of histamine and microorganisms increased steadily during the two weeks storage. Formation of biogenic amines depends on the concentration of amino acids or peptides which act as precursors in food (Voight et al 1974, Leuschnert and Hammes 1998). Recent studies have shown that Morganella morganii can be a contaminant of fish tissue during spoilage and that few other bacteria have as great a capacity to form histamine (Eitenmiller et al 1981). Amine-degrading activities were found in Brevibacterium linens and coryneform bacteria (Leuschnert and Hammes 1998). The presence of some decarboxilase-positive spoiling microorganisms such as Pseudomonas, Micrococci and Enterococci that can result in histamine formation have been cited as possible cause of this increase (Santos et al 1998).
There are several studies linking bacterial contamination to histamine formation in aquatic species. Tekinsen et al (1993) investigated histamine formation in three aquatic species. After 3 days of storage at 4 °C, the histamine content in mackerel, bonito and anchovy was 6.28, 3.70 and 3.25 mg/kg, respectively, well below toxic or dangerous levels. Edmunds et al (1975) also reported that histamine formation in Spanish mackerel, white shrimp, common mullet, speckled trout and channel catfish did not increase in any of these species after 14 days storage even though extensive psychrophilic spoilage had occurred judged by appearance and odor evolution. Lopez-Sabater et al (1995) found that under some conditions the level of histamine 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. Enterobacteriaceae and Pseudomonas ssp. are not part of the normal microflora in fresh fish, but they grow rather quickly in aerobically stored fish (Lougovois et al 2003, Behling and Taylor 1982). Pseudomonas can be the dominant spoilage bacteria in fish from warmer waters and the maximum histidine decarboxylase activity was observed during the late logarithmic phase of growth (Falcao et al 2002).
Establishment of a precise toxicity threshold of histamine in individuals is extremely difficult because the toxic dose is strongly dependent on the efficiency of the detoxification mechanisms of each individual. Nevertheless, toxicological levels have been proposed, such as 10-100 mg of histamine per 100 g of food (Stratton et al 1991, Hernandez-Jover et al 1997). The FDA has set the hazardous level for histamine in tuna at 50 mg histamine/100g (Sumner et al 1990, Stratton et al 1991). The histamine content of Inci Kefali in our study was lower than the FDA's hazardous level for histamine in tuna at 50 mg histamine/100g. At present, there is no limitation of histamine for fish in theTurkish food codex.
The initial quality of the pearl mullet in our study was good, as indicated by a low initial bacterial count before storage. All counts increased during storage and spoilage was mainly due to psychrotrophic bacteria.
The number of total viable counts on the first day was 8.0 x 102, increasing to 9.0 x 109 cfu/g. Enterobacteriaceae increased from 2.0 x 102 on the 1st day to 6.5 x 109 cfu/g and that of Pseudomonas from 3.0 x 102 to 7.3 x 109 cfu/g by the 15th day. These results are comparable to those reported for tuna by Lopez-Sabater et al (1995).
Total viable count is an important tool to assess the quality of foodstufffs. It is considered an indicator for the lifecycle of fish, constituting a criterion in the determination of the general microbiological quality of the product (Altug and Bayrak 2003). It has been stated that a total viable count above 108 cfu/g may lead to food spoilge in smoked fish (Pichhardt 1993). The total viable count values here reported indicate that the pearl mullet samples were on the threshold of critical limits at the 11th day of storage.
It can be concluded that the histamine levels in Inci Kefali samples hygienically stored for up to 15 days at 4°C do not seem to pose a health hazard. As microorganisms readily propagate in fish, proper sanitary care should be taken during handling. There is, however, a potential risk depending from individual susceptibility to histamine, some gastrointestinal conditions, alcohol consumption or the use of certain medicines that may influence the biogenic amines detoxification routes.
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* Yüzüncü Yil University, Veterinary College, Department of Food Hygiene and Technology, TR-65080 Van, Turkey; firstname.lastname@example.org