Histamine formation by Morganella morganii isolated from Trachurus murphyii (Chilean mackerel)
Silvia Torres(1), Marlene Roeckel(1) and M. Cristina Martí(2*)
(1) Dto. de Ing. Química, Univ. de Concepción, Concepción, Chile. mroeckel@diq.udec.cl
(2) Dto. de Farmacología, Univ. de Concepción, Concepción, Chile. cmarti@udec.cl
* 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.
I. INTRODUCTION
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.
Batch assays
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:
![]() |
(1) |
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):
![]() |
(2) |
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:
![]() |
(3) |
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.
ACKNOWLEDGEMENTS
This work was possible through grants from Dirección de Investigación, Universidad de Concepción (Proyecto N° 95.32.01-1.2) and FONDEF 2-75.
REFERENCES
1. Ababouch, L., L. Souibri, K. Rhaliby, O. Ouahdi, M.
Battal, and F. Busta. "Quality changes in sardines
(Sardina pilchardus) stored in ice and at ambient
temperature." Food Microbiol., 13, 123-132 (1996).
2. Anson, M.L. "The extinction of pepsin, trypsin, papain
and cathepsin with hemoglobin." J. Gen. Physiol.,
22, 79-89 (1938)
3. Bailey, J. and D. Ollis. Biochemical Engineering
Fundamentals. 2nd Edition. McGraw-Hill, Inc. New
York, USA (1986).
4. Behling, A. and S. Taylor. "Bacterial histamine
production as a function of temperature and time of
incubation." J. Food Sci., 47, 1311-1315 (1982).
5. Belitz, H.D. and W. Gosch. Lehrbuch der
Lebensmittelchemie, 2nd Edition, Springer Verlag,
Berlin, Germany, pp.480 (1985).
6. Bergey's Manual of Systematic Bacteriology, Vol. II.
The Williams & Wilkins Co., Baltimore, USA (1984).
7. Carlucci, F.V. and E. Karmas. "Liquid chromatographic
determination of some amines and their aminoacids
in protein food." J.Am.Oil Chemist's, 71, 564-568
(1988).
8. Collins, C.H. Métodos Micro-Biológicos, Editorial
Acribia, Zaragoza, España, pp. 155 (1969).
9.
Eitenmiller, R., J. Wallis, J. Orr, and R. Philips. "Production of histidine decarboxylase and
histamine by Proteus morganii." J. Food Protection,
44, 815-820 (1981).
10. Fernández-Salguero, J. and Y. Mackie. "Histidine
metabolism in mackerel (Scomber scombrus). Studies
on histidine decarboxylase activity and histamine
formation during storage of fresh and liver under
sterile and nonsteril conditions." J. Food Technol.,
14, 131-139 (1979).
11. Gale, E.F. "The bacterial amino acid decarboxylases." Adv. in Enzymology, 6, 1-32 (1946).
12. Harry, E. and J. Tucker. "The effect of ora lly
administered histamine on the weight gain and
development of gizzard lesions in chicks." Vet. Rec.,
99, 206-207 (1976).
13. Ibe, A., T. Nishima, and N. Kasai. "Bacteriological
properties of and amine-production conditions for tyramine and histamine-producing bacterial strains
isolated from soybean paste miso starting materials ."
Jap. J. Toxico & Environm. Health, 38, 403-409
(1992).
14. Instituto de Fomento Pesquero. "Catálogo tecnológico
de las principales materias primas pesqueras-
Chile." Corporación de Fomento de la Producción,
Santiago, Chile (1983).
15. Klausen, N. and H. Huss. "Growth and histamine
production by Morganella morganii under various
temperatures conditions." Int. J. Food Microbiol., 5,
147-156 (1987).
16. López-Sabater, E., J. Rodríguez-Jerez, M. Hernández-
Herrero, A. Roig-Sagués and M. Mora-Ventura."Sensory quality and histamine formation during
controlled decomposition of tuna (Thunnus
thynnus)." J. Food Protection, 59, 167-174 (1996).
17. Lowry, H., N. Rosebrough, A. Farr and R. Randall."Protein measurement with the Folin phenol
reagent." J. Biol. Chem., 193, 265-276 (1951).
18.
Maijala, R. "Histamine and tyramine production by a
Lactobacillus strain subjected to external pH
decrease." J. Food Protection, 57(3), 259-262 (1994).
19. Moser, A. "Formalkinetik von Bioprozessen." In: Bioprozesstechnik. pp. 129-168. Springer Verlag,
Wien, Austria (1981).
20. Middlebrooks, B., P Toom, W. Douglas, R. Harrison and
S. McDowell. "Effects of storage time and
temperature on the microflora and amine
development in Spanish Mackerel (Scombromorus
maculatus)." J. Food Sci., 4, 1024-1029 (1988).
21. Niven, C., M. Jeffrey and D. Corlett. "Differencial plating
medium for quantitative detection of histamineproducing
bacteria." Applied Environm. Microbiol.,
41, 321-322 (1981).
22. Omura, Y., R. Price and H. Olcott. "Histamine-forming
bacteria isolated from spoiled skipjack tuna and jack
mackerel." J. Food Sci ., 43, 1779-1781 (1978).
23. Pirt, J. Principles of Microbe and Cell Cultivation.
Halsted Press. Blackwell Scientific Publications. New
Jersey (1975).
24. Rodríguez-Jerez, J., E. López-Sabater, M. Hernández-
Herrero and M. Mora-Ventura. "Histamine,
putrescine, and cadaverine formation in Spanish
semipreserved anchovies as affected by
time/temperature." J. Food Sci., 59, 993-997 (1994a).
25. Rodríguez-Jerez, J., M. Mora-Ventura, E. López-Sabater
and M. Hernández-Herrero. "Histidine, lysine and
ornithine decarboxylase bacteria in Spanish salted
semipreserved anchovies." J.Food Protection, 57,
784-787 (1994b).
26. Sato, T., M. Okuzumi, T. Masuda and T. Fujii."Distribution and genus/species composition if
histamine-decomposition bacteria during storage of
common mackerel." Fisheries Sci. (Tokyo), 61, 83-85
(1995).
27. Smith, A.M., M.A. Hayden, S.G. McCay, F.A. Zapatka
and M.K. Hamdy. "Detection and confirmation of
histamine producing bacteria." Bull. Environm.
Contamination and Toxicol., 29, 618-623 (1982).
28. Warne, D. Aplicación del concepto de punto crítico de
análisis de riesgos. Sun Pan, B. and James, D. (eds.).
In: La histamina en los productos marinos:
producción por bacterias, medición y predicción de
su formación. FAO Fisheries Reports 252. Hobart,
Australia (1985).
Received: July 17, 2000.
Accepted for publication: December 4, 2000.
Recommended by Subject Editor A. Cukierman.