versión impresa ISSN 0327-0793
Lat. Am. appl. res. v.32 n.2 Bahía Blanca abr./jun. 2002
Histamine formation by Morganella morganii isolated from Trachurus murphyii (Chilean mackerel)
(1) Dto. de Ing. Química, Univ. de Concepción, Concepción, Chile. firstname.lastname@example.org
(2) Dto. de Farmacología, Univ. de Concepción, Concepción, Chile. email@example.com
* To whom all correspondence should be addressed
Abstract Morganella morganii, the most histaminogenic bacteria in chilean mackerel, was isolated and used to study the effect of temperature on h istamine production in this fish. Growth and histamine production rates at different initial pH (4.0, 5.5, 6.0, 7.0) and temperatures (10, 15, 20, 30 °C) were studied in batch cultures of M. morganii in a synthetic medium (TSBH- 2% histidine). An Arrhenius- type relationship between growth rate and temperature was found; its activation energy was 88.49 kJ mol-1. Bacterial growth and histamine formation were negligible below pH 5.0. The Leudeking-Piret expression for product formation rate was used; the α parameter was not significantly different at 30, 20 and 15 °C and its average value was 0.1 [mg histamine (mg cell)-1]. Assuming an initial protein content of 188 g (kg fish)-1, a level of 500 mg histamine (kg fish)-1 would be produced after 43 h at 15 °C or after 24 h at 20 °C. The prediction of histamine level at different temperatures can be used as a strategy of catch management.
Keywords Trachurus murphyii; Morganella morganii; Histamine production.
Chile is the world's second fishmeal producer and this product comes mostly from the reduction of jack mackerel (Trachurus murphyii).
Fishmeal is a valuable protein source due to its protein quality and omega-3 fatty acid content. However, its fraction of inclusion in feeds is limited by its histamine content as high levels of this amine can induce deleterious effects and even scombroid poisoning and death of animals fed with it (Harry and Tucker, 1976; Omura et al., 1978). Thus, the quality of fishmeal and its price relates, between others, to its histamine content. Prime quality fishmeal, with less than 500 mg histamine kg-1 of fishmeal, will cost an average of 25 % more per ton than the common product (Pesquera Itata, Chile, personal communication).
Gale (1946) showed that specific amino acid bacterial decarboxylases produce amines and that these enzymes are formed within bacterial cells in response to certain well-defined conditions of growth (temperature and acid medium). Scombroid fish, such as mackerel, have high levels of free histidine within their muscle tissue that could amount to 2 % of the total free amino acids (Belitz and Gosch, 1985).
Amino acids decarboxylases are expressed in the presence of the specific substrate, provided that the enzyme is present in the potential enzymic constitution of the organism (Gale, 1946). Several bacteria isolated from the Scomberiscida and Scombridae families ( e.g. , mackerel , tuna and sardine), have been identified as histaminogenic (Fernández-Salguero and Mackie, 1979; Niven et al., 1981; Eitenmiller et al., 1981; Klausen and Huss., 1987; Middlebrooks et al., 1988; Rodríguez-Jerez et al., 1994a, 1994b). These bacteria belong mainly to the proteolytic intestinal flora (Fernández-Salguero and Mackie, 1979).
Histaminergic bacteria do not survive the temperatures applied during fishmeal production. However, histamine is thermoestable (Warne, 1985) and its level remains relatively constant throughout the process (unpublished results); thus, the control of histamine levels in fishmeal must focused on a reduction of the bacterial degradation of histidine before the fish processing. A temperature reduction decreases histamine formation, however the literature does not report a direct expression to relate either histamine formation as a function of temperature and pH or between histamine-production and the histaminogenic bacteria growth rate (Behling and Taylor, 1982; Ibe et al., 1992).
Therefore, the goal of this work was to analyze the effect of temperature and initial pH of the medium on the growth rate of histaminergic bacteria and its relationship with histamine formation as a way to control the histamine level in fishmeal produced from chilean mackerel. The study was carried out using as a model the most histaminogenic bacterium able to grow at the catch handling temperatures.
II. MATERIALS AND METHODS
Bacterial Strains. Mackerels were obtained immediately after catching, aseptically handled, and refrigerated at 4 °C. They were homogenized (1 g fish: 9 ml water) by Ultra -Turrax for 5 min with sterile water; the resultant paste was diluted 10,000 times with water. Histaminergic bacteria were isolated using the histidine decarboxylase medium developed by Niven et al. (1981). 0.1 ml aliquots were inoculated in duplicate plates of Niven's medium and incubated under aerobic conditions for 48-72 h at 25 °C (Smith et al., 1982). Colonies that changed the bromocresol pH indicator in Niven's medium were isolated and identified by the API 20E diagnostic kit for Enterobacteriaceaes (Anlytab Products Inc.), as well as by descriptions in Bergey's Manual of Systematic Bacteriology (1984).
The histaminogenic ability was assessed by culturing each bacterium at 30 °C for 18-24 h in a 2% histidine-trypticase soy broth (2% TSBH), pH 5.3, which mimics the scombroid fish protein characteristics (Behling and Taylor, 1982). Aliquots of these cultures, with identical optical density at 620 nm, were transferred to fresh medium, incubated at 30 °C and 5 ml aliquots were withdrawn at 30 min intervals. Viable bacterial count was carried out in a Petroff-Hauser chamber (Collins, 1969) and histamine concentration measured in cell free aliquots of the growth medium. Histamine concentration was determined by HPLC (Merck Hitachi L-4250 UV-VIS Detector) after sterilization of the medium through a 0.22 nm membrane filter (Millipore) and extraction with 0.4 N perchloric acid. Histamine was derivatized with dansyl chloride (Carlucci and Karmas, 1988); chromatographic conditions were: Pico-Tag C-18 column (Waters); temperature of 50 °C; 1:1 methanolwater as mobile phase; flow of 0.017 ml s-1; detection at 254 nm.
Batch culture. M. morganii was cultured in 300 ml Erlenmeyer flasks containing 100 ml of growth medium at different temperatures and under aeration and agitation (G24 Environmental Incubator Shaker, New Brunswick Scientific, USA) at 144 rpm until the late exponential growth period was reached. The inoculum was similar to the original bacterium concentration in fish (0.0039 mg cells [kg fish]-1). The 2% histidine-TSB growth medium composition was (g l-1): Trypticase Soy Broth (Difco), 30; L-histidine HCl x H20 (Sigma), 20; pyridoxal monohydrate (Merck), 0.005. The pH was adjusted to 5.5 and sterilized at 120 °C for 15 min. The TSB (Difco) contains 47% of protein, dextrose, NaCl and K2HPO4.
Growth rate. The specific growth rate was measured so as to relate the bacterial growth with histamine formation. It was measured after the lag period as the slope of log cell concentration vs. time. Bacterial concentration (X) was determined by measurin g the absorbance of the bacterial solution at 620 nm (UV-VIS 1203, Shimadzu, Japan). A calibration curve relating the bacterium dry weight and the absorbance in the 2%- TSBH medium was performed. A relationship of 0.5 [(mg dry cells ml-1) OD-1] was found fo r the bacterium grown in 2%-TSBH.
Limiting substrate. To determine if protein was the growth limiting substrate, M. morganni was grown at different initial TSB concentrations plus 2 % histidine and the maximum growth measured (Bailey and Ollis, 1986).
Protein concentration. Protein was measured in cell free aliquots of the growth medium by the Lowry method (Lowry et al., 1951)
Enzymatic activity. Extracellular proteolytic activity was measured in cell free aliquots of the growth medium. Proteolytic activity was measured by the Anson method (Anson, 1938) using 2 ml aliquots of each fraction as enzymatic source; the reaction was carried out under agitation at 30 °C, stopped after 14 h by the addition of 5 % trichloroacetic acid..
Temperature effect on growth rate. The bacterium was grown at 30, 20, 15 and 10 °C. Temperature was kept constant by means of a cryogenic bath (Heto, CB 7, Denmark). Aliquots were withdrawn every 2 h; after bacterial concentration was measured, protein and histamine concentrations were measured in the cell free fraction.
Effect of the initial pH on growth rate. The bacterium was grown at an initial pH of 4.0, 6.0 or 7.0 by modifying the initial pH of the medium (5.5) by adding 1N HCl or 1N NaOH. Aliquots were withdrawn every 2 h; after bacterial concentration was measured, protein and histamine concentrations were measured in the cell free fraction.
Experimental data was obtained with at most a 10 % error and a confidence interval of 95 %.
III. RESULTS AND DISCUSSION
Histaminogenic strains. Table 1 shows the isolated and identified histaminogenic microorganisms, their bacterial count and the histamine concentration found after a 48-h incubation at 30 °C of each strain.
As shown, M. morganii was the largest producer of histamine; thus, this bacterium was chosen as a model of the mesophilic histaminogenic flora of chilean mackerel to carry out subsequent experiments.
Limiting substrate for growth. Fig. 1A shows the effect of the initial substrate concentration (TSB, S0) on M. morganii cellular concentration. Substrate consumption was determined by measuring soluble protein concentration in cell free aliquots as no extracellular proteolytic activity was found in the growth medium. As shown, up to 6 g I1 protein is the limiting substrate for growth, larger protein concentrations do not affect the final cell concentration; thus, further experiments were carried out at saturating substrate concentrations (30 g TSB l-1).
Fig. 1B shows the saturation-type relationship found between the initial substrate concentration (S0) and the specific growth rate (µ). M. morganii exhibited an approximate maximum growth rate of 0.77 h-1.
Effect of temperature on growth rate and histamine formation. Fig. 2 shows that, as expected for a mesophilic bacterium, the growth rate increased with temperature; thus, within the assayed temperatures, the maximum growth temperature for M. morganii was 30 °C. Several functions have been used to correlate the effect of temperature on the bacterial growth kinetics; the most used ones have been of the hyperbolic and Arrhenius type (Pirt, 1975).
According to Moser (1981), the constant rates are dependent on the temperature and the water activity; by assuming the same water activity for cells and reacting volumes, the following Arrhenius-type relationship can be applied:
where µ is the specific growth rate at the different assay temperatures, A, a constant, R, the gas constant, T, temperature, and Ea, energy of activation. Plotting lnµ vs. the reciprocal of the temperature, a straight line of the form y = -10636 x + 38.128 (R2 = 0.9543) was obtained. Values of 3.62 × 1016 (day-1) and 88.49 (kJ mol-1) were calculated for A and Ea, respectively.
An average value for the activation energy of a mesophilic aerobic bacterium is 58.6 kJ mol-1, thus M. morganii is more sensitive to temperature changes than most bacteria (Pirt, 1975). Figure 3 shows the effect of temperature on histamine production. At the stationary phase, the cell concentration (1241 - 1296 mg dried cells l- 1), histamine yield (0.0066-0.007 mg histamine (mg protein)-1) and final histamine concentration (0.213-0.197 g l-1) were quite similar at 30, 20 and 15 °C. Histamine production started 12 h earlier at 20 °C than at 15 °C; the latter reflects the effect of temperature on the duration of the lag period, but it may as well reflect the effect of temperature on the histidine decarboxylase production and its activity. The growth rate of M. morganii was greatly reduced at 10 °C and the small amount of histamine produced at this temperature fell below the sensitivity of the measurement technique.
Product formation can be described by the Luedeking and Piret model (Pirt, 1975; Bailey and Ollis, (1986):
As shown in Fig. 3, it was established that histamine is a primary catabolite, i.e., a decrease in growth rate produced a concomitant decrease in histamine production rate; therefore, the Luedeking and Piret equation is reduced to:
where P is histamine concentration, x, cell concentration,α ,a constant that relates to the cellular product (histamine) yield (YP/X) (Pirt, 1975)). Experimental α [mg histamine (mg cell)-1] values were α30 = 0.17, α25 = 0.1 and α15 = 0.09; the similarity between these values indicates a very low dependence on temperature.
Effect of the initial pH of the medium on growth rate and histamine formation. As shown in Fig.4, at the assayed pHs the optimum growth pH was around 6.0. The specific growth rate decreased 6.6 times by a reduction of the initial pH from 5.5 to 4.0 and, thus, histamine formation was also reduced. After 25 h of culture at an initial pH of 4.0, the histamine concentration was 15 mg l-1, whereas at an initial pH of5.5 the latter value was 200 mg l-1.
As histamine is a primary catabolite (β=0), by applying Eqn. (3) to the experimental data, the calculated αpH [mg histamine (mg cells)-1] values were α 4.0 ≈ 0, α5.5 = 0.17 and α7.0 = 0.165. The α4.0 approaches 0 as the bacterial growth at this pH was very small. The α values were similar at pH 5.5 and 7.0 as the media were not buffered and the initial pH increased as histamine formation increased; thus, both media had the same pH (@ 7.0) at the end of the experiment.
The normal muscle pH of fresh mackerel is 6.0 and is reduced to 5.5-5.7 after rigor mortis (Instituto de Fomento Pesquero, 1983). The reported optimal pH for histidine decarboxylase activity in a strain of M. morganii was 6.5 and at pH 5.0 its activity was minimal, thus greatly inhibiting histamine accumulation in the medium (Eitenmiller et al., 1981). Our findings indicate that even at a pH below 5.0 there is histamine formation. Although a pH reduction by addition of organic acids has been reported as effective in reducing histaminogenic bacterial growth (Maijala, 1994) and considerably cheaper than a temperature reduction, most histaminogenic bacteria are enteric microorganisms and the rate of diffusion of the acid to the gut is slow. Moreover, organic acids leave residues in fishmeal that affect weight gain in salmons fed with diets that include this fishmeal (I. Pyke, Director General of the International Fishmeal and Oil Manufacturers Association, personal communication). Thus, temperature and time of exposure of the catch to temperature seems to be the parameters to control for a reduction in histamine levels.
Temperature at the ship's holds varies between 5 °C and 15 ºC (refrigerated and non-refrigerated holds) and the catch remains 3 to 5 days at these holds. Once unloaded, the catch is stored at the plant's wells until its processing. The wells temperature varies between 15 and 25 º C, depending on the season of the year, and the catch remains an average of 20 h at the wells.
If it is assumed that histamine formation is the rate limiting process as compared to the transport rate of the bacteria to the muscle, the upper limit histamine concentration can be estimated in fish and extrapolated to the final concentration in fishmeal. Based on the histamine production by M. morganii at different assay temperatures (see Fig. 3) and assuming an initial protein content of 188 g (kg fish)-1, an inoculum size similar to the M. morganii concentration found in chilean mackerel and a 23 % fishmeal/fish process yield (Instituto de Fomento Pesquero, 1983), a histamine level of 500 mg of histamine (kg fishmeal)-1 would be produced after 43 h at 15 °C or after 24 h at 20 °C. Thus, if temperature is reduced in the wells from 20 to 15 °C, the catch can remain an extra 19 h without affecting the fishmeal quality. This prediction of histamine levels in fishmeal is in agreement with the levels actually found in a local industry, with mostly refrigerated ships' holds but without wells refrigeration (Pesquera Itata, personal communication, 1999). Thus, the assessment of histamine levels at the assayed temperature range using M. morganii as a model for histaminogenic bacteria could be used as a rough guide for a catch management strategy to minimize the histamine content and improve the quality of the product.
As the literature reports that psychrophilic bacteria may also play a role in histamine production (Sato et al., 1995; López-Sabater et al., 1996; Ababouch et al., 1996), the whole histaminogenic flora is presently being studied through the entire range of temperature at which the catch is actually handled.
This work was possible through grants from Dirección de Investigación, Universidad de Concepción (Proyecto N° 95.32.01-1.2) and FONDEF 2-75.
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Received: July 17, 2000.
Accepted for publication: December 4, 2000.
Recommended by Subject Editor A. Cukierman.