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Effectiveness of gamma irradiation in the inactivation of histamine-producing bacteria
Food Control 28 (2012) 143e146
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Short communication
Effectiveness of gamma irradiation in the inactivation of histamine-producing bacteria Daisuke Nei a, *, Susumu Kawasaki a, Yasuhiro Inatsu a, Kazutaka Yamamoto a, Masataka Satomi b a b
National Food Research Institute, Kannondai 2-1-12, Tsukuba 305-8642, Japan National Research Institute of Fisheries Science, Fisheries Research Agency, 2-12-4 Fukuura, Kanazawa-ku, Yokohama 2368648, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 November 2011 Received in revised form 25 April 2012 Accepted 1 May 2012
Histamine is a cause of scombroid foodborne poisoning. The control of histamine-producing bacteria is a method that can be used to avoid the accumulation of histamine. This study examined the effectiveness of gamma irradiation in inactivating histamine-producing bacteria. The histamine-producing bacteria Morganella morganii (JCM 1672), Enterobacter aerogenes (ATCC 43175) and Raoultella planticola (ATCC 43176) were suspended in tryptic soy broth and were gamma-irradiated at 0.5e4.0 kGy at room temperature. The bacterial populations declined with higher absorbance doses, and the radiation D10 (the dose of radiation required to cause a 90% reduction in the number of survivors) values ranged from 0.32 to 0.42 kGy. Histamine-producing bacteria were inoculated on tuna and were gamma-irradiated; the D10 values were between 0.31 and 0.34 kGy, depending on the type of bacteria. Complete inactivation of the histamine-producing bacteria that were inoculated on tuna was achieved at 4.0 kGy. Although gamma irradiation was effective in controlling histamine-producing bacteria in order to reduce the risk of histamine poisoning, excessive doses were associated with color changes in the tuna. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Gamma ray Histamine Morganella morganii Enterobacter aerogenes Raoultella planticola Tuna meat
1. Introduction Foodborne illnesses caused by histamine occur after the consumption of foods containing histamine. The symptoms of histamine poisoning include difficulty in breathing, itching, rash, vomiting, fever and hypertension (Naila, Flint, Fletcher, Bremer, & Meerdink, 2010). One of the difficult problems in histamine poisoning is the difficulty in judging histamine accumulation based on the appearance of fish meats (Lehane & Olley, 2000). In total, 187 histamine poisoning incidents and 752 incidents were reported from 2000 to 2006 in the USA (Toda, Yamamoto, Uneyama, & Morikawa, 2009). In Japan, 89 outbreaks and 1577 cases were reported between 1998 and 2008 (Toda et al., 2009). The actual number of cases could be higher because there may be unreported incidents due to the mildness of the disease (Lehane & Olley, 2000). Thus, histamine poisoning is a large problem for public health and food safety. Accordingly, methods controlling the production and accumulation of histamine in foods play an important role in reducing the risk of histamine poisoning. Bacterial decarboxylation of amino acids leads to the formation of biogenic amines such as histamine (Santos, 1996), and the
* Corresponding author. Tel.: þ81 29 838 8021; fax: þ81 29 838 7996. E-mail address: [email protected] (D. Nei). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.05.006
inhibition of bacterial growth is an effective way to reduce the risk of histamine poisoning. Low temperature storage is a basic strategy for reducing histamine accumulation because histamine formation is temperature dependent (Bakar, Yassoralipour, Bakar, & Rahman, 2010; Emborg & Dalgaard, 2008; Visciano, Campana, Annunziata, Vergara, & Ianieri, 2007). However, some bacteria, such as Photobacterium spp., are able to grow and produce histidine decarboxylase at low temperatures (Emborg, Laursen, & Dalgaard, 2005), and temperature control alone is not always sufficient to inhibit the accumulation of histamine. In addition, low temperature control throughout the food chain is difficult to achieve and temperature abuse is usually observed during distribution (Nei, Uchino, Sakai, & Tanaka, 2005). The combination of decontamination treatments and subsequent temperature control would be more effective to avoid histamine poisoning. Irradiation is an attractive technology that reduces the risk of biogenic amine poisoning (Naila et al., 2010). Although the degree of public acceptance of irradiated foods is not always high in some consumer communities (Gunes & Tekin, 2006), irradiation of several types of foods, such as meat products, fish and seafood, and fresh vegetables, is widely recognized and is approved in more than 50 countries. The mechanism of microbial inactivation by ionizing radiation is primarily damage to nucleic acids. Direct or indirect damage is caused by oxidative radicals originating from the radiolysis of water. The gamma irradiation is a safe, efficient, environmentally
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D. Nei et al. / Food Control 28 (2012) 143e146
clean and energy-efficient process and is particularly valuable as a decontamination procedure (Farkas,1998). Irradiation is reported to control biogenic amines through radiolysis of the amines. Kim et al. (2004) indicated that 5e100% of biogenic amines were destroyed by irradiation in the range of 2.5e25 kGy in a model system. Kim et al. (2003) irradiated materials of fermented soybean paste at 5e15 kGy and reported significant decreases in microbial populations of major bacteria in the paste such as Bacillus spp. and lactic-acid bacteria. In addition, they indicated that the accumulation of biogenic amines was reduced during the fermentation of soybean pastes. Thus, some research on the effectiveness of irradiation in controlling biogenic amines and the bacteria that produce them has been conducted. However, data on the resistance to gamma ray irradiation by other biogenic amine-producing bacteria, such as Morganella morganii, are not available. The objectives of this study are (1) to evaluate the resistance of histamine-producing bacteria (M. morganii, Enterobacter aerogenes and Raoultella planticola) to gamma irradiation in pure cultures and on tuna fillets and (2) to evaluate the visible quality changes in tuna after irradiation. 2. Materials and methods 2.1. Test strain The histamine-producing bacteria M. morganii (JCM 1672), E. aerogenes (ATCC 43175) and R. planticola (ATCC 43176) were used in this study. To minimize the growth of microorganisms naturally present on tuna, the bacteria were adapted to growth in tryptic soy broth (TSB, Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 50 mg/ml of rifampicin (TSBR). Plating on media containing rifampicin greatly minimized interference by naturally occurring microorganisms and facilitated the detection of test bacteria on the recovery media (Inatsu, Bari, Kawasaki, Isshiki, & Kawamoto, 2005). 2.2. Preparation of inocula Each bacterium was cultured at 37 C in 20 ml of TSB medium (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 50 mg/ml rifampicin. The cultures were transferred to the TSBR by loop at three successive 24 h intervals immediately before they were used as inocula. The cells of each strain were collected by centrifugation (3000 g, 5 min, 20 C) and resuspended in 5 ml of sterile phosphate-buffered saline (PBS, pH 7.2). The final suspension, containing approximately 9 log CFU/ml, was maintained at 22 2 C, and the suspension was applied to tuna within 30 min after preparation.
Cell-220, Nordion International, Inc., Kanata, Ontario, Canada). The inoculated tuna were also irradiated after the samples were placed into a polyethylene bag. Dosimetry was performed using a 5-mmdiameter alanine dosimeter (Bruker Instruments, Rheinstetten, Germany), and the free radical signal was measured using an ESR analyzer (EMX-Plus, Bruker Instruments, Rheinstetten, Germany) to evaluate the actual doses. The actual doses were within 3% of the targeted doses. 2.5. Microbial analysis After irradiation, serial decimal dilutions were prepared with PBS to enumerate the microbial population in the TSBR. When the microbial population on the tuna was counted, 25 g of tuna was placed in a stomacher bag; 225 ml of PBS (pH 7.2) was added and the mixture was pummeled for 60 s. Serial decimal dilutions were prepared with PBS, and the appropriately diluted samples were pour-plated in quadruplicate on tryptic soy agar plates (TSA; Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 50 mg/ml rifampicin (TSAR). All of the ingredients except rifampicin were combined and sterilized by heating at 121 C for 15 min. The rifampicin solution was added to the molten agar before pouring the medium into petri dishes. Inoculated enumeration media were incubated at 37 C for 24 h before the presumptive colonies of pathogen were counted. 2.6. Detection of surviving histamine-producing bacteria The survival of histamine-producing bacteria was confirmed when the population was below detection limits (<1.0 log CFU/g for tuna meats and <1.0 log CFU/mL for TSBR experiments). In total, 20 ml of the irradiated suspension culture was transferred to 180 ml of TSBR and incubated at 37 C for 24 h. For the irradiation experiments in tuna, the homogenized mixture containing 25 g tuna meat with 225 ml of TSB in a stomacher bag was kept in an incubator at 37 C for 24 h for enrichment. Subsequently, 0.1 ml of suspensions or homogenates was plated onto TSAR. All of the plates were incubated at 37 C for 24 h, and the surviving pathogen colonies were counted. 2.7. Color measurements The color of the tuna was evaluated before and after gamma irradiation. A portable color meter (model SP-62, X-Rite, Grandville, Mich.) was calibrated with a white standard tile before use. The samples were placed on the tiles, which were then placed on a white sheet. The Commission Internationale de l’Eclairage (CIE) parameters (L*, a* and b* values) were determined in six blocks of the tuna for each experimental condition.
2.3. Procedure for inoculation 2.8. Statistical analysis The cell suspension (200 ml) in PBS was applied with a micropipette to the surface of 25 g of tuna (approximately 1.3 cm 1.3 cm 1.3 cm), and the inoculated tuna were dried at room temperature (22 2 C) for 30 min. After drying, the tuna were immediately treated with irradiation. 2.4. Treatments of histamine-producing bacteria in pure culture and on tuna The centrifuge tubes (diameter of 30 mm) containing 20 ml of TSBR suspensions of M. morganii, E. aerogenes and R. planticola were prepared as described above and were irradiated at room temperature. The targeted doses were 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 or 4.0 kGy, at 5.6 kGy/h, using a cobalt-60 gamma source (Gamma
The experiments were repeated three times, and the data are shown as the means and standard deviations. Significant differences in average values were established by the TukeyeKramer multiple-comparison method at the 5% level of significance using SPSS (SPSS Inc., Chicago). 3. Results and discussion 3.1. Effect of gamma irradiation on the population of histamineproducing bacteria in pure culture The populations of histamine-producing bacteria in TSBR after gamma irradiation are shown in Table 1. Approximately
D. Nei et al. / Food Control 28 (2012) 143e146 Table 1 The populations of histamine-producing bacteria in TSBR after gamma irradiation at various absorbance doses. Absorbance dose (kGy)
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
R. planticola
E. aerogenes
8.4 0.6A 8.0 0.2B 6.1 0.2C 4.2 0.2D 2.6 0.3E ND (3/3)b ND (3/3) ND (0/3) ND (0/3)
8.6 0.3A 7.6 0.4B 6.3 0.0C 5.7 0.4C 4.2 0.1D 2.8 0.1E ND (3/3) ND (0/3) ND (0/3)
8.6 0.1A 8.1 0.1B 7.1 0.1C 5.9 0.2D 4.0 0.1E 3.6 0.1F 1.2 0.2G ND (3/3) ND (1/3)
a The population data are represented by the mean of three independent trials and standard deviations (n ¼ 3). Within individual columns, the values followed by different letters are significantly different (p < 0.05). b ND indicates a level below the detection limit (<1.0 log CFU/mL). The number of positive samples for histamine-forming bacteria following enrichment is provided.
8.4e8.6 log CFU/mL of bacteria was recovered before irradiation. The population of M. morganii was decreased by gamma irradiation (p < 0.05), and the reduction in the population of the bacteria was increased by higher doses of irradiation. A reduction of 5.8 log CFU/ mL was obtained at 2.0 kGy. The radiation D10 (the radiation dose required to reduce by 90% the number of survivors) value for M. morganii calculated from the negative reciprocal of the slope of the survivor curve (the slope was obtained by linear regression) was 0.32 kGy (Table 3). Gamma irradiation at 2.5e3.0 kGy reduced the M. morganii population to less than the detection limit of the plate counting method (1.0 log CFU/mL); however, M. morganii was detected after enrichment. The complete elimination of the bacteria was achieved at irradiation levels above 3.5 kGy, and no survivors were detected after enrichment. Similarly, gamma irradiation reduced the population of R. planticola and E. aerogenes. The populations were reduced by 4.4e4.6 log CFU/mL after gamma irradiation at 2.0 kGy. the radiation D10 values of R. planticola and E. aerogenes were 0.42 and 0.41 kGy, respectively. The complete elimination of R. planticola was achieved at doses greater than 3.5 kGy. However, it was difficult to eliminate E. aerogenes even after irradiation at 4.0 kGy, and higher doses would be required to achieve complete elimination. The accumulation of histamine has been reported in liquid foods such as fish sauces, milk for cheese production and fermented beverages (Halász, Baráth, Simon-Sarkadi, & Holzapfel, 1994; Magwamba, Matsheka, Mpuchane, & Gashe, 2010). The results obtained in this study indicate that irradiation is an effective method Table 2 The populations of histamine-producing bacteria inoculated on tuna meats after gamma irradiation at room temperature. Absorbance dose (kGy)
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
Table 3 The radiation D10 values of histamine-producing bacteria. D10 valuesa
Population recovered (log CFU/mL)a M. morganii
Population recovered (log CFU/g)a M. morganii
R. planticola
E. aerogenes
7.6 0.0A 6.7 0.6A 5.3 0.0B 2.5 0.5C 1.6 0.2C ND (2/3)b ND (2/3) ND (0/3) ND (0/3)
7.5 0.2A 5.5 0.6B 3.5 0.3C 2.6 0.3CD 1.6 0.6D ND (3/3) ND (3/3) ND (0/3) ND (0/3)
7.5 0.1A 6.0 0.1B 4.8 0.5C 3.6 0.3D 1.4 0.2E ND (3/3) ND (2/3) ND (1/3) ND (0/3)
a The population data are represented by the mean of three independent trials and standard deviations (n ¼ 3). Within individual columns, the values followed by different letters are significantly different (p < 0.05). b ND indicates a level below the detection limit (<1.0 log CFU/g). The number of positive samples for histamine-forming bacteria following enrichment is provided.
145
Tryptic soy broth Tuna meats
M. morganii
R. planticola
E. aerogenes
0.32 0.01 0.30 0.01
0.42 0.03 0.34 0.02
0.41 0.02 0.34 0.02
a The radiation D10 values are represented by the mean of three independent trials and standard deviations (n ¼ 3).
to reduce the population of histamine-producing bacteria, and more than 7.0 log CFU/mL reduction could be obtained. Since initial microbial population is important factor affecting accumulation of biogenic amines (Bover-Cid, Izquierdo-Pulido, & Vidal-Carou, 2000), gamma irradiation should be considered as an intervention technology to reduce the risk of histamine poisoning in liquid foods. 3.2. Gamma irradiation of histamine-producing bacteria inoculated on tuna Changes in the populations of histamine-producing bacteria after gamma irradiation are shown in Table 2. The uninoculated tuna was analyzed before the irradiation treatment, and no bacteria were recovered on the TSAR plates. In control samples, 7.5e7.6 log CFU/g of histamine-producing bacteria were recovered. The population of M. morganii was significantly decreased after gamma irradiation (p < 0.05), and an approximate 6.0 log reduction was obtained at 2.0 kGy irradiation. The D10 value obtained by linear regression analysis was 0.30 kGy, as shown in Table 3. Significant reductions in the populations of the other bacteria tested (R. planticola and E. aerogenes) were observed (p < 0.05); 5.9e6.1 log CFU/g reductions were obtained at 2.0 kGy. The radiation D10 values of R. planticola and E. aerogenes were 0.34 kGy and 0.34 kGy, respectively (Table 3). The populations of M. morganii and R. planticola were below the detection limits (<1.0 log CFU/g) when inoculated tuna was irradiated at 2.5e3.0 kGy. However, inoculated bacteria could be recovered from tuna after enrichment, and complete elimination could not be achieved. On the other hand, gamma irradiation at 3.5 kGy succeeded in eliminating the histamine-producing bacteria inoculated on tuna; no survivors were detected after enrichment. An absorbance dose of 4.0 kGy was required to achieve the complete elimination of E. aerogenes inoculated on tuna. The radiation sensitivities of the histamine-producing bacteria on tuna meats were higher than those in TSBR. The cause of the difference could not be clearly explained, but the differences in water and oxygen conditions between TSBR and tuna meats were suggested as the causes, because these factors significantly affect the radiation sensitivities of bacteria (Gomes, Moreira, & Castell-Perez, 2011; Thayer, Boyd, Fox, & Lakritz, 1995). Radiation resistance has been studied primarily for pathogenic bacteria such as Salmonella spp., Escherichia coli O157:H7, Listeria monocytogenes and Vibrio spp. (Jakobi et al., 2003; Kamat & Thomas, 1999; Molins, Motarjemi, & Käferstein, 2001). The reported radiation sensitivities were remarkably varied, depending on the strain, temperatures, pH and the water content of foods (Buchanan, Edelson, & Boyd, 1999; Mahmoud, 2009; Mayer-Miebach et al., 2005; Osaili, Shaker, Abu AlHasan, Ayyash, & Martin, 2007). However, there are no other reports on the radiation sensitivities of the histamine-producing bacteria, and future studies of histamine-producing bacteria will be important for clarifying radiation sensitivities under various conditions. 3.3. Changes in the skin color of tuna after irradiation The effect of gamma irradiation on the color of tuna is shown in Table 4. The remarkable changes in the L* values of the irradiated tuna
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D. Nei et al. / Food Control 28 (2012) 143e146
Table 4 The effect of gamma irradiation on the color changes of tuna meats.a Absorbance dose (kGy)
L*
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
31.9 33.7 31.7 31.7 32.5 33.5 33.3 31.7 30.3
a*
1.3 1.7 1.0 1.5 1.1 1.4 1.1 1.7 1.1
16.3 15.7 15.2 13.6 13.7 13.5 13.7 13.3 10.4
b*
1.5 1.3 2.0 1.7 1.8 2.2 2.0 2.4 1.4
6.3 5.1 5.1 4.4 4.4 3.9 4.2 4.1 2.4
0.8 0.9 1.3 0.6 0.9 0.8 0.9 1.2 0.8
a The L*, a* and b* values are represented by the mean of six blocks of tuna meats and standard deviations (n ¼ 6).
were not observed up to 4.0 kGy. On the other hand, decreases in the a* and b* values were detected at higher absorbance doses, and the lowest values were recorded at 4.0 kGy. Color changes caused by gamma irradiation have been reported in several kinds of foods, such as poultry, pork and beef meats (Brewer, 2004; Park et al., 2010). Yagiz et al. (2010) reported decreases in a* values of salmon muscle after gamma irradiation, and the same results were obtained in tuna in this study. These decreases in a* values were thought to be due to the oxidation of lipids (Yagiz et al., 2010). Although gamma irradiation is effective in the control of histamine-producing bacteria, excessive absorbances cause the loss of quality. The optimum balance between the safety and the quality of products should be considered. The population of histamine-producing bacteria in commercially available fish is maximally 3.0e4.0 log CFU/g (Yoguchi, Okuzumi, & Fujii, 1990). Low-dose irradiation at 1.0 kGy (2.3e4.0 log CFU/g reduction could be expected) would be practical and effective in controlling histamine-producing bacteria without changes in appearance. 4. Conclusion In this study, the effect of gamma irradiation on populations of histamine-producing bacteria was evaluated. Histamine-producing bacteria inoculated on tuna were remarkably reduced by gamma irradiation, and complete elimination could be achieved at 4.0 kGy. Histamine is accumulated by the actions of these bacteria, and the proper control of the bacteria is an effective way to avoid histamine illness. Gamma irradiation is an attractive method for supplying high-safety tuna without histamine accumulation. However, it should be taken into consideration that excessive doses were associated with the loss of quality of the tuna. Acknowledgment The authors thank the ‘‘Research and development projects for application in promoting new policy of Agriculture, Forestry and Fisheries’’ No. 22036 (funded by MAFF) for financial support. References Bakar, J., Yassoralipour, A., Bakar, F. A., & Rahman, R. A. (2010). Biogenic amine changes in barramundi (Lates calcarifer) slices stored at 0 C and 4 C. Food Chemistry, 119, 467e470. Bover-Cid, S., Izquierdo-Pulido, M., & Vidal-Carou, M. C. (2000). Influence of hygienic quality of raw materials on biogenic amine production during ripening and storage of dry fermented sausages. Journal of Food Protection, 63, 1544e1550. Brewer, S. (2004). Irradiation effects on meat color e a review. Meat Science, 68, 1e17.
Buchanan, R. L., Edelson, S. G., & Boyd, G. (1999). Effects of pH and acid resistance on the radiation resistance of enterohemorrhagic Escherichia coli. Journal of Food Protection, 62, 219e228. Emborg, J., & Dalgaard, P. (2008). Growth, inactivation and histamine formation of Morganella psychrotolerans and Morganella morganii e development and evaluation of predictive models. International Journal of Food Microbiology, 128, 234e243. Emborg, J., Laursen, B. G., & Dalgaard, P. (2005). Significant histamine formation in tuna (Thunnus albacares) at 2 C e effect of vacuum- and modified atmospherepackaging on psychrotolerant bacteria. International Journal of Food Microbiology, 101, 263e279. Farkas, J. (1998). Irradiation as a method for decontaminating food e a review. International Journal of Food Microbiology, 44, 189e204. Gomes, C., Moreira, R. G., & Castell-Perez, E. (2011). Radiosensitization of Salmonella spp. and Listeria spp. in ready-to-eat baby spinach leaves. Journal of Food Science, 76, E141eE148. Gunes, G., & Tekin, M. D. (2006). Consumer awareness and acceptance of irradiated foods: results of a survey conducted on Turkish consumers. LWTeFood Science and Technology, 39, 443e447. Halász, A., Baráth, Á., Simon-Sarkadi, L., & Holzapfel, W. (1994). Biogenic amines and their production by microorganisms in food. Trends in Food Science and Technology, 5, 42e49. Inatsu, Y., Bari, M. L., Kawasaki, S., Isshiki, K., & Kawamoto, S. (2005). Efficacy of acidified sodium chlorite treatments in reducing Escherichia coli O157:H7 on Chinese cabbage. Journal of Food Protection, 68, 251e255. Jakobi, M., Gelli, D. S., Torre, J. C. M. D., Rodas, M. A. B., Franco, B. D. G. M., Destro, M. T., et al. (2003). Inactivation by ionizing radiation of Salmonella enteritidis, Salmonella infantis, and Vibrio parahaemolyticus in oysters (Crassostrea brasiliana). Journal of Food Protection, 66, 1025e1029. Kamat, A., & Thomas, P. (1999). Recovery and growth of some food-borne pathogens in irradiated low and high fat fish during storage at 2e4 C. Journal of Food Safety, 19, 35e44. Kim, J. H., Ahn, H. J., Jo, C., Park, H. J., Chung, Y. J., & Byun, M. W. (2004). Radiolysis of biogenic amines in model system by gamma irradiation. Food Control, 15, 405e408. Kim, J. H., Ahn, H. J., Kim, D. H., Jo, C., Yook, H. S., Park, H. J., et al. (2003). Irradiation effects on biogenic amines in Korean fermented soybean paste during fermentation. Journal of Food Science, 68, 80e84. Lehane, L., & Olley, J. (2000). Histamine fish poisoning revisited. International Journal of Food Microbiology, 58, 1e37. Magwamba, C., Matsheka, M. I., Mpuchane, S., & Gashe, B. A. (2010). Detection and quantification of biogenic amines in fermented food products sold in Botswana. Journal of Food Protection, 73, 1703e1708. Mahmoud, B. S. M. (2009). Inactivation effect of X-ray treatments on Cronobacter species (Enterobacter sakazakii) in tryptic soy broth, skim milk, low-fat milk and whole-fat milk. Letters in Applied Microbiology, 49, 562e567. Mayer-Miebach, E., Stahl, M. R., Eschrig, U., Deniaud, L., Ehlermann, D. A. E., & Schuchmann, H. P. (2005). Inactivation of a non-pathogenic strain of E. coli by ionizing radiation. Food Control, 16, 701e705. Molins, R. A., Motarjemi, Y., & Käferstein, F. K. (2001). Inactivation: a critical control point in ensuring the microbiological safety of raw foods. Food Control, 12, 347e356. Naila, A., Flint, S., Fletcher, G., Bremer, P., & Meerdink, G. (2010). Control of biogenic amines in food-existing and emerging approaches. Journal of Food Science, 75, R139eR150. Nei, D., Uchino, T., Sakai, N., & Tanaka, S. (2005). The effect of temperature on the quality of tomato and eggplant fruits during distribution. Journal of the Faculty of Agriculture Kyushu University, 50, 213e221. Osaili, T. M., Shaker, R. R., Abu Al-Hasan, A. S., Ayyash, M. M., & Martin, E. M. (2007). Inactivation of Enterobacter sakazakii in infant milk formula by gamma irradiation: determination of D10-value. Journal of Food Science, 72, M85eM88. Park, J. G., Yoon, Y., Park, J. N., Han, I. J., Song, B. S., Kim, J. H., et al. (2010). Effects of gamma irradiation and electron beam irradiation on quality, sensory, and bacterial populations in beef sausage patties. Meat Science, 85, 368e372. Santos, M. H. S. (1996). Biogenic amines: their importance in foods. International Journal of Food Microbiology, 29, 213e231. Thayer, D. W., Boyd, G., Fox, J. B., & Lakritz, L. (1995). Effects of NaCl, sucrose, and water content on the survival of Salmonella typhimurium on irradiated pork and chicken. Journal of Food Protection, 58, 490e496. Toda, M., Yamamoto, M., Uneyama, C., & Morikawa, K. (2009). Histamine food poisonings in Japan and other countries. Bulletin of National Institute of Health Sciences, 127, 31e38. 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Contents lists available at SciVerse ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Short communication
Effectiveness of gamma irradiation in the inactivation of histamine-producing bacteria Daisuke Nei a, *, Susumu Kawasaki a, Yasuhiro Inatsu a, Kazutaka Yamamoto a, Masataka Satomi b a b
National Food Research Institute, Kannondai 2-1-12, Tsukuba 305-8642, Japan National Research Institute of Fisheries Science, Fisheries Research Agency, 2-12-4 Fukuura, Kanazawa-ku, Yokohama 2368648, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 November 2011 Received in revised form 25 April 2012 Accepted 1 May 2012
Histamine is a cause of scombroid foodborne poisoning. The control of histamine-producing bacteria is a method that can be used to avoid the accumulation of histamine. This study examined the effectiveness of gamma irradiation in inactivating histamine-producing bacteria. The histamine-producing bacteria Morganella morganii (JCM 1672), Enterobacter aerogenes (ATCC 43175) and Raoultella planticola (ATCC 43176) were suspended in tryptic soy broth and were gamma-irradiated at 0.5e4.0 kGy at room temperature. The bacterial populations declined with higher absorbance doses, and the radiation D10 (the dose of radiation required to cause a 90% reduction in the number of survivors) values ranged from 0.32 to 0.42 kGy. Histamine-producing bacteria were inoculated on tuna and were gamma-irradiated; the D10 values were between 0.31 and 0.34 kGy, depending on the type of bacteria. Complete inactivation of the histamine-producing bacteria that were inoculated on tuna was achieved at 4.0 kGy. Although gamma irradiation was effective in controlling histamine-producing bacteria in order to reduce the risk of histamine poisoning, excessive doses were associated with color changes in the tuna. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Gamma ray Histamine Morganella morganii Enterobacter aerogenes Raoultella planticola Tuna meat
1. Introduction Foodborne illnesses caused by histamine occur after the consumption of foods containing histamine. The symptoms of histamine poisoning include difficulty in breathing, itching, rash, vomiting, fever and hypertension (Naila, Flint, Fletcher, Bremer, & Meerdink, 2010). One of the difficult problems in histamine poisoning is the difficulty in judging histamine accumulation based on the appearance of fish meats (Lehane & Olley, 2000). In total, 187 histamine poisoning incidents and 752 incidents were reported from 2000 to 2006 in the USA (Toda, Yamamoto, Uneyama, & Morikawa, 2009). In Japan, 89 outbreaks and 1577 cases were reported between 1998 and 2008 (Toda et al., 2009). The actual number of cases could be higher because there may be unreported incidents due to the mildness of the disease (Lehane & Olley, 2000). Thus, histamine poisoning is a large problem for public health and food safety. Accordingly, methods controlling the production and accumulation of histamine in foods play an important role in reducing the risk of histamine poisoning. Bacterial decarboxylation of amino acids leads to the formation of biogenic amines such as histamine (Santos, 1996), and the
* Corresponding author. Tel.: þ81 29 838 8021; fax: þ81 29 838 7996. E-mail address: [email protected] (D. Nei). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.05.006
inhibition of bacterial growth is an effective way to reduce the risk of histamine poisoning. Low temperature storage is a basic strategy for reducing histamine accumulation because histamine formation is temperature dependent (Bakar, Yassoralipour, Bakar, & Rahman, 2010; Emborg & Dalgaard, 2008; Visciano, Campana, Annunziata, Vergara, & Ianieri, 2007). However, some bacteria, such as Photobacterium spp., are able to grow and produce histidine decarboxylase at low temperatures (Emborg, Laursen, & Dalgaard, 2005), and temperature control alone is not always sufficient to inhibit the accumulation of histamine. In addition, low temperature control throughout the food chain is difficult to achieve and temperature abuse is usually observed during distribution (Nei, Uchino, Sakai, & Tanaka, 2005). The combination of decontamination treatments and subsequent temperature control would be more effective to avoid histamine poisoning. Irradiation is an attractive technology that reduces the risk of biogenic amine poisoning (Naila et al., 2010). Although the degree of public acceptance of irradiated foods is not always high in some consumer communities (Gunes & Tekin, 2006), irradiation of several types of foods, such as meat products, fish and seafood, and fresh vegetables, is widely recognized and is approved in more than 50 countries. The mechanism of microbial inactivation by ionizing radiation is primarily damage to nucleic acids. Direct or indirect damage is caused by oxidative radicals originating from the radiolysis of water. The gamma irradiation is a safe, efficient, environmentally
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clean and energy-efficient process and is particularly valuable as a decontamination procedure (Farkas,1998). Irradiation is reported to control biogenic amines through radiolysis of the amines. Kim et al. (2004) indicated that 5e100% of biogenic amines were destroyed by irradiation in the range of 2.5e25 kGy in a model system. Kim et al. (2003) irradiated materials of fermented soybean paste at 5e15 kGy and reported significant decreases in microbial populations of major bacteria in the paste such as Bacillus spp. and lactic-acid bacteria. In addition, they indicated that the accumulation of biogenic amines was reduced during the fermentation of soybean pastes. Thus, some research on the effectiveness of irradiation in controlling biogenic amines and the bacteria that produce them has been conducted. However, data on the resistance to gamma ray irradiation by other biogenic amine-producing bacteria, such as Morganella morganii, are not available. The objectives of this study are (1) to evaluate the resistance of histamine-producing bacteria (M. morganii, Enterobacter aerogenes and Raoultella planticola) to gamma irradiation in pure cultures and on tuna fillets and (2) to evaluate the visible quality changes in tuna after irradiation. 2. Materials and methods 2.1. Test strain The histamine-producing bacteria M. morganii (JCM 1672), E. aerogenes (ATCC 43175) and R. planticola (ATCC 43176) were used in this study. To minimize the growth of microorganisms naturally present on tuna, the bacteria were adapted to growth in tryptic soy broth (TSB, Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 50 mg/ml of rifampicin (TSBR). Plating on media containing rifampicin greatly minimized interference by naturally occurring microorganisms and facilitated the detection of test bacteria on the recovery media (Inatsu, Bari, Kawasaki, Isshiki, & Kawamoto, 2005). 2.2. Preparation of inocula Each bacterium was cultured at 37 C in 20 ml of TSB medium (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 50 mg/ml rifampicin. The cultures were transferred to the TSBR by loop at three successive 24 h intervals immediately before they were used as inocula. The cells of each strain were collected by centrifugation (3000 g, 5 min, 20 C) and resuspended in 5 ml of sterile phosphate-buffered saline (PBS, pH 7.2). The final suspension, containing approximately 9 log CFU/ml, was maintained at 22 2 C, and the suspension was applied to tuna within 30 min after preparation.
Cell-220, Nordion International, Inc., Kanata, Ontario, Canada). The inoculated tuna were also irradiated after the samples were placed into a polyethylene bag. Dosimetry was performed using a 5-mmdiameter alanine dosimeter (Bruker Instruments, Rheinstetten, Germany), and the free radical signal was measured using an ESR analyzer (EMX-Plus, Bruker Instruments, Rheinstetten, Germany) to evaluate the actual doses. The actual doses were within 3% of the targeted doses. 2.5. Microbial analysis After irradiation, serial decimal dilutions were prepared with PBS to enumerate the microbial population in the TSBR. When the microbial population on the tuna was counted, 25 g of tuna was placed in a stomacher bag; 225 ml of PBS (pH 7.2) was added and the mixture was pummeled for 60 s. Serial decimal dilutions were prepared with PBS, and the appropriately diluted samples were pour-plated in quadruplicate on tryptic soy agar plates (TSA; Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 50 mg/ml rifampicin (TSAR). All of the ingredients except rifampicin were combined and sterilized by heating at 121 C for 15 min. The rifampicin solution was added to the molten agar before pouring the medium into petri dishes. Inoculated enumeration media were incubated at 37 C for 24 h before the presumptive colonies of pathogen were counted. 2.6. Detection of surviving histamine-producing bacteria The survival of histamine-producing bacteria was confirmed when the population was below detection limits (<1.0 log CFU/g for tuna meats and <1.0 log CFU/mL for TSBR experiments). In total, 20 ml of the irradiated suspension culture was transferred to 180 ml of TSBR and incubated at 37 C for 24 h. For the irradiation experiments in tuna, the homogenized mixture containing 25 g tuna meat with 225 ml of TSB in a stomacher bag was kept in an incubator at 37 C for 24 h for enrichment. Subsequently, 0.1 ml of suspensions or homogenates was plated onto TSAR. All of the plates were incubated at 37 C for 24 h, and the surviving pathogen colonies were counted. 2.7. Color measurements The color of the tuna was evaluated before and after gamma irradiation. A portable color meter (model SP-62, X-Rite, Grandville, Mich.) was calibrated with a white standard tile before use. The samples were placed on the tiles, which were then placed on a white sheet. The Commission Internationale de l’Eclairage (CIE) parameters (L*, a* and b* values) were determined in six blocks of the tuna for each experimental condition.
2.3. Procedure for inoculation 2.8. Statistical analysis The cell suspension (200 ml) in PBS was applied with a micropipette to the surface of 25 g of tuna (approximately 1.3 cm 1.3 cm 1.3 cm), and the inoculated tuna were dried at room temperature (22 2 C) for 30 min. After drying, the tuna were immediately treated with irradiation. 2.4. Treatments of histamine-producing bacteria in pure culture and on tuna The centrifuge tubes (diameter of 30 mm) containing 20 ml of TSBR suspensions of M. morganii, E. aerogenes and R. planticola were prepared as described above and were irradiated at room temperature. The targeted doses were 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 or 4.0 kGy, at 5.6 kGy/h, using a cobalt-60 gamma source (Gamma
The experiments were repeated three times, and the data are shown as the means and standard deviations. Significant differences in average values were established by the TukeyeKramer multiple-comparison method at the 5% level of significance using SPSS (SPSS Inc., Chicago). 3. Results and discussion 3.1. Effect of gamma irradiation on the population of histamineproducing bacteria in pure culture The populations of histamine-producing bacteria in TSBR after gamma irradiation are shown in Table 1. Approximately
D. Nei et al. / Food Control 28 (2012) 143e146 Table 1 The populations of histamine-producing bacteria in TSBR after gamma irradiation at various absorbance doses. Absorbance dose (kGy)
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
R. planticola
E. aerogenes
8.4 0.6A 8.0 0.2B 6.1 0.2C 4.2 0.2D 2.6 0.3E ND (3/3)b ND (3/3) ND (0/3) ND (0/3)
8.6 0.3A 7.6 0.4B 6.3 0.0C 5.7 0.4C 4.2 0.1D 2.8 0.1E ND (3/3) ND (0/3) ND (0/3)
8.6 0.1A 8.1 0.1B 7.1 0.1C 5.9 0.2D 4.0 0.1E 3.6 0.1F 1.2 0.2G ND (3/3) ND (1/3)
a The population data are represented by the mean of three independent trials and standard deviations (n ¼ 3). Within individual columns, the values followed by different letters are significantly different (p < 0.05). b ND indicates a level below the detection limit (<1.0 log CFU/mL). The number of positive samples for histamine-forming bacteria following enrichment is provided.
8.4e8.6 log CFU/mL of bacteria was recovered before irradiation. The population of M. morganii was decreased by gamma irradiation (p < 0.05), and the reduction in the population of the bacteria was increased by higher doses of irradiation. A reduction of 5.8 log CFU/ mL was obtained at 2.0 kGy. The radiation D10 (the radiation dose required to reduce by 90% the number of survivors) value for M. morganii calculated from the negative reciprocal of the slope of the survivor curve (the slope was obtained by linear regression) was 0.32 kGy (Table 3). Gamma irradiation at 2.5e3.0 kGy reduced the M. morganii population to less than the detection limit of the plate counting method (1.0 log CFU/mL); however, M. morganii was detected after enrichment. The complete elimination of the bacteria was achieved at irradiation levels above 3.5 kGy, and no survivors were detected after enrichment. Similarly, gamma irradiation reduced the population of R. planticola and E. aerogenes. The populations were reduced by 4.4e4.6 log CFU/mL after gamma irradiation at 2.0 kGy. the radiation D10 values of R. planticola and E. aerogenes were 0.42 and 0.41 kGy, respectively. The complete elimination of R. planticola was achieved at doses greater than 3.5 kGy. However, it was difficult to eliminate E. aerogenes even after irradiation at 4.0 kGy, and higher doses would be required to achieve complete elimination. The accumulation of histamine has been reported in liquid foods such as fish sauces, milk for cheese production and fermented beverages (Halász, Baráth, Simon-Sarkadi, & Holzapfel, 1994; Magwamba, Matsheka, Mpuchane, & Gashe, 2010). The results obtained in this study indicate that irradiation is an effective method Table 2 The populations of histamine-producing bacteria inoculated on tuna meats after gamma irradiation at room temperature. Absorbance dose (kGy)
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
Table 3 The radiation D10 values of histamine-producing bacteria. D10 valuesa
Population recovered (log CFU/mL)a M. morganii
Population recovered (log CFU/g)a M. morganii
R. planticola
E. aerogenes
7.6 0.0A 6.7 0.6A 5.3 0.0B 2.5 0.5C 1.6 0.2C ND (2/3)b ND (2/3) ND (0/3) ND (0/3)
7.5 0.2A 5.5 0.6B 3.5 0.3C 2.6 0.3CD 1.6 0.6D ND (3/3) ND (3/3) ND (0/3) ND (0/3)
7.5 0.1A 6.0 0.1B 4.8 0.5C 3.6 0.3D 1.4 0.2E ND (3/3) ND (2/3) ND (1/3) ND (0/3)
a The population data are represented by the mean of three independent trials and standard deviations (n ¼ 3). Within individual columns, the values followed by different letters are significantly different (p < 0.05). b ND indicates a level below the detection limit (<1.0 log CFU/g). The number of positive samples for histamine-forming bacteria following enrichment is provided.
145
Tryptic soy broth Tuna meats
M. morganii
R. planticola
E. aerogenes
0.32 0.01 0.30 0.01
0.42 0.03 0.34 0.02
0.41 0.02 0.34 0.02
a The radiation D10 values are represented by the mean of three independent trials and standard deviations (n ¼ 3).
to reduce the population of histamine-producing bacteria, and more than 7.0 log CFU/mL reduction could be obtained. Since initial microbial population is important factor affecting accumulation of biogenic amines (Bover-Cid, Izquierdo-Pulido, & Vidal-Carou, 2000), gamma irradiation should be considered as an intervention technology to reduce the risk of histamine poisoning in liquid foods. 3.2. Gamma irradiation of histamine-producing bacteria inoculated on tuna Changes in the populations of histamine-producing bacteria after gamma irradiation are shown in Table 2. The uninoculated tuna was analyzed before the irradiation treatment, and no bacteria were recovered on the TSAR plates. In control samples, 7.5e7.6 log CFU/g of histamine-producing bacteria were recovered. The population of M. morganii was significantly decreased after gamma irradiation (p < 0.05), and an approximate 6.0 log reduction was obtained at 2.0 kGy irradiation. The D10 value obtained by linear regression analysis was 0.30 kGy, as shown in Table 3. Significant reductions in the populations of the other bacteria tested (R. planticola and E. aerogenes) were observed (p < 0.05); 5.9e6.1 log CFU/g reductions were obtained at 2.0 kGy. The radiation D10 values of R. planticola and E. aerogenes were 0.34 kGy and 0.34 kGy, respectively (Table 3). The populations of M. morganii and R. planticola were below the detection limits (<1.0 log CFU/g) when inoculated tuna was irradiated at 2.5e3.0 kGy. However, inoculated bacteria could be recovered from tuna after enrichment, and complete elimination could not be achieved. On the other hand, gamma irradiation at 3.5 kGy succeeded in eliminating the histamine-producing bacteria inoculated on tuna; no survivors were detected after enrichment. An absorbance dose of 4.0 kGy was required to achieve the complete elimination of E. aerogenes inoculated on tuna. The radiation sensitivities of the histamine-producing bacteria on tuna meats were higher than those in TSBR. The cause of the difference could not be clearly explained, but the differences in water and oxygen conditions between TSBR and tuna meats were suggested as the causes, because these factors significantly affect the radiation sensitivities of bacteria (Gomes, Moreira, & Castell-Perez, 2011; Thayer, Boyd, Fox, & Lakritz, 1995). Radiation resistance has been studied primarily for pathogenic bacteria such as Salmonella spp., Escherichia coli O157:H7, Listeria monocytogenes and Vibrio spp. (Jakobi et al., 2003; Kamat & Thomas, 1999; Molins, Motarjemi, & Käferstein, 2001). The reported radiation sensitivities were remarkably varied, depending on the strain, temperatures, pH and the water content of foods (Buchanan, Edelson, & Boyd, 1999; Mahmoud, 2009; Mayer-Miebach et al., 2005; Osaili, Shaker, Abu AlHasan, Ayyash, & Martin, 2007). However, there are no other reports on the radiation sensitivities of the histamine-producing bacteria, and future studies of histamine-producing bacteria will be important for clarifying radiation sensitivities under various conditions. 3.3. Changes in the skin color of tuna after irradiation The effect of gamma irradiation on the color of tuna is shown in Table 4. The remarkable changes in the L* values of the irradiated tuna
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Table 4 The effect of gamma irradiation on the color changes of tuna meats.a Absorbance dose (kGy)
L*
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
31.9 33.7 31.7 31.7 32.5 33.5 33.3 31.7 30.3
a*
1.3 1.7 1.0 1.5 1.1 1.4 1.1 1.7 1.1
16.3 15.7 15.2 13.6 13.7 13.5 13.7 13.3 10.4
b*
1.5 1.3 2.0 1.7 1.8 2.2 2.0 2.4 1.4
6.3 5.1 5.1 4.4 4.4 3.9 4.2 4.1 2.4
0.8 0.9 1.3 0.6 0.9 0.8 0.9 1.2 0.8
a The L*, a* and b* values are represented by the mean of six blocks of tuna meats and standard deviations (n ¼ 6).
were not observed up to 4.0 kGy. On the other hand, decreases in the a* and b* values were detected at higher absorbance doses, and the lowest values were recorded at 4.0 kGy. Color changes caused by gamma irradiation have been reported in several kinds of foods, such as poultry, pork and beef meats (Brewer, 2004; Park et al., 2010). Yagiz et al. (2010) reported decreases in a* values of salmon muscle after gamma irradiation, and the same results were obtained in tuna in this study. These decreases in a* values were thought to be due to the oxidation of lipids (Yagiz et al., 2010). Although gamma irradiation is effective in the control of histamine-producing bacteria, excessive absorbances cause the loss of quality. The optimum balance between the safety and the quality of products should be considered. The population of histamine-producing bacteria in commercially available fish is maximally 3.0e4.0 log CFU/g (Yoguchi, Okuzumi, & Fujii, 1990). Low-dose irradiation at 1.0 kGy (2.3e4.0 log CFU/g reduction could be expected) would be practical and effective in controlling histamine-producing bacteria without changes in appearance. 4. Conclusion In this study, the effect of gamma irradiation on populations of histamine-producing bacteria was evaluated. Histamine-producing bacteria inoculated on tuna were remarkably reduced by gamma irradiation, and complete elimination could be achieved at 4.0 kGy. Histamine is accumulated by the actions of these bacteria, and the proper control of the bacteria is an effective way to avoid histamine illness. Gamma irradiation is an attractive method for supplying high-safety tuna without histamine accumulation. However, it should be taken into consideration that excessive doses were associated with the loss of quality of the tuna. Acknowledgment The authors thank the ‘‘Research and development projects for application in promoting new policy of Agriculture, Forestry and Fisheries’’ No. 22036 (funded by MAFF) for financial support. References Bakar, J., Yassoralipour, A., Bakar, F. A., & Rahman, R. A. (2010). Biogenic amine changes in barramundi (Lates calcarifer) slices stored at 0 C and 4 C. Food Chemistry, 119, 467e470. Bover-Cid, S., Izquierdo-Pulido, M., & Vidal-Carou, M. C. (2000). Influence of hygienic quality of raw materials on biogenic amine production during ripening and storage of dry fermented sausages. Journal of Food Protection, 63, 1544e1550. Brewer, S. (2004). Irradiation effects on meat color e a review. Meat Science, 68, 1e17.
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