- Email: [email protected]
Inactivation of Enterobacter sakazakii, Bacillus cereus, and Salmonella typhimurium in powdered weaning food by electron-beam irradiation
ARTICLE IN PRESS Radiation Physics and Chemistry 77 (2008) 1097– 1100
Contents lists available at ScienceDirect
Radiation Physics and Chemistry journal homepage: www.elsevier.com/locate/radphyschem
Inactivation of Enterobacter sakazakii, Bacillus cereus, and Salmonella typhimurium in powdered weaning food by electron-beam irradiation Yun-Hee Hong a, Ji-Yong Park b, Jong-Hyun Park c, Myong-Soo Chung d, Ki-Sung Kwon e, Kyungsook Chung f, Misun Won f, Kyung-Bin Song a, a
Department of Food Science and Technology, College of Agriculture and Life Science, Chungnam National University, Yuseong-Gu, Daejeon 305-764, Republic of Korea Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea c Department of Food Science and Biotechnology, Kyungwon University, Sungnam 461-701, Republic of Korea d Department of Food Science, Ehwa Women’s University, Seoul 120-750, Republic of Korea e Center for Food safety Evaluation, Korea Food and Drug Administration, Seoul 122-704, Republic of Korea f Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Republic of Korea b
a r t i c l e in fo
abstract
Article history: Received 11 February 2008 Accepted 12 May 2008
Inactivation of Enterobacter sakazakii, Bacillus cereus, and Salmonella typhimurium were evaluated in powdered weaning food using electron-beam irradiation. E. sakazakii, B. cereus, and S. typhimurium were eliminated by irradiation at 16, 8, and 8 kGy, respectively. The D10-vlaues of E. sakazakii, B. cereus, and S. typhimurium inoculated on powdered weaning food were 4.83, 1.22, and 0.98 kGy, respectively. The results suggest that electron-beam irradiation should inhibit the growth of pathogenic bacteria on baby food without impairing qualities. & 2008 Elsevier Ltd. All rights reserved.
Keywords: Powdered weaning food Electron-beam irradiation Microbial growth
1. Introduction Enterobacter sakazakii, Bacillus cereus, and Salmonella typhimurium are the major pathogenic bacteria that have been associated with food poisoning in powdered weaning foods. In particular, E. sakazakii in infant foods has been implicated in infections among high-risk infants (Iverson and Forsythe, 2003). Necrotizing enterocolitis and meningitis caused by E. sakazakii have high mortality rates (Lucas and Cole, 1990). In addition, dried weaning foods are known to be easily contaminated with B. cereus spores (Kramer and Gilbert, 1989), causing diarrheal and emetic type of disease (Agata et al., 2002). Salmonella spp. has been also associated with weaning foods, and the presence of Salmonella spp. was responsible for disease among infants (Leuschner Renata et al., 2004). As a food preservation method, ionizing irradiation is considered as an effective method in providing hygienic quality by reducing microbial spoilage. There are two most common types of ionizing radiation: gamma ray and electron beam. Electron-beam irradiation is used to inactivate food borne microorganisms during storage, and to guarantee the hygienic quality of foods (Sarrias et al., 2003). Electron-beam irradiation has been shown to destroy 99.9% of the major food pathogenic bacteria (Rodriguez et al., 2006), and it requires relatively short processing time and low
Corresponding author. Tel.: +82 42 821 6723; fax: +82 42 825 2664.
E-mail address: [email protected] (K.-B. Song). 0969-806X/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2008.05.004
equipment cost. Thus, it can be applicable as a preservation method for powdered weaning food to achieve microbial decontamination. In particular, application of electron-beam irradiation to food products can avoid the negative image toward gamma irradiation on foods since it does not use a radioisotope source like 60Co (Sarrias et al., 2003). Therefore, the objectives of this study were to examine the effect of electron-beam irradiation on the microbial safety and qualities of powdered weaning food during storage, and to provide appropriate processing condition for its safety.
2. Experimental 2.1. Materials Powdered weaning food (Namyang Co., Gongju, Korea) was purchased from a local market in Daejeon, Korea. Ingredients of powdered weaning food are uncleaned rice, defatted dried milk, a-rice flour, concentrated milk protein, sunflower powder, fructose, sunflower oil, peas powder, milk protein isolate, safflower oil, beef, concentrated rice protein, concentrated whey protein, whole milk powder, chicken, millet, oatmeal, dextrin, powdered uncleaned rice syrup, malt extract, pineapple powder, elderberry powder, banana powder, apple powder, mandarin powder, strawberry powder, celery, broccoli, tomato, carrot powder, pumpkin powder, spinach powder, mushroom, safflower seed extract, docosahexaenoic acid (DHA), chlorella extract (C, G, F),
ARTICLE IN PRESS 1098
Y.-H. Hong et al. / Radiation Physics and Chemistry 77 (2008) 1097–1100
egg yolk, casein phosphopeptides, vitamin E, calcium phosphate tribasic, potassium chloride, eggshell calcium, taurine, dried yeast, L-methionine, L-carnitine, magnesium hydroxide, lactoferrin Concentrates, vitamin premix (vitamin C, commercial enrichments, dextrin, betacarotine), lactic acid bacteria, copper sulfate, sulfate of zinc, ferrous lactate, yoghurt, cod fish, tuna, anchovy, kelp extract powder, embryo bud, jujube extract, and chestnut powder.
2.6. Color measurement Color of the samples was analyzed using a colorimeter (CR-300 Minolta Chroma Meter, Minolta Camera Co., Osaka, Japan). Samples were placed on a white standard plate and Hunter’s color values (L, a, b) were measured and total color difference values were expressed as E value. Hunter’s L, a, and b values for the standard plate were 98.34, 0.03, and 1.62, respectively. Five measurements were taken at different locations of each sample.
2.2. Culture conditions 2.7. Sensory evaluation E. sakazakii (ATCC 51329), B. cereus (KCCM 40935), and S. typhimurium (ATCC 14028) were used in this study. E. sakazakii, B. cereus, and S. typhimurium in 20% (w/v) glycerol solution were kept individually at 70 1C in 5 ml vials. E. sakazakii, B. cereus, and S. typhimurium cultures were grown at 37 1C for 24 h in 50 ml tubes containing 25 ml of Enterobacteriaceae enrichment broth (Oxoid, Basingstoke, U.K), brain heart infusion (BHI) (Oxoid), and Salmosyst (Merck, Darmseadt, Germany), respectively.
Samples were analyzed for their color, odor, and overall acceptability by 8 trained panelists (2 men and 6 women; age range 24–27). Sensory qualities of samples were evaluated using five point scoring method. Sensory scores were 5, very good; 4, good; 3, fair; 2, poor; and 1, very poor. 2.8. Statistical analysis
2.3. Inoculation on weaning food
Analysis of variance and Duncan’s multiple range tests with significance at po0.05 were performed using a SAS program (SAS Inst. Version 8.2, 2001).
Powdered weaning food (10 g) was placed into a sterile bag and inoculated with 200 ml of E. sakazakii, B. cereus, and S. typhimurium to obtain an initial level of 108–109 CFU/g, respectively. After inoculation, the samples were shaken for 1 min to evenly spread the bacteria on the food.
3. Results and discussion
2.4. Irradiation treatments Electron-beam irradiation was performed using an electronbeam accelerator (Model ELV-4, 1 MeV, Eb-Tech, Yuseong, Daejeon, Korea). Samples were individually packaged in 100 mm 170 mm low-density polyethylene bags (thickness, below 5 mm) and they were exposed to 3 dose levels of 2, 8, and 16 kGy at 1 MeV (beam current 2–8 mA, beam dimension 600 mm 600 mm, conveyer velocity 5–10 m/min). Absorption dose was determined using a cellulose triacetate (CTA) dosimeter. After irradiation, samples were stored at 20 1C for 12 days.
3.1. Microbiological analysis Electron-beam treatment decreased the microbial populations with increasing radiation dose (Fig. 1). In particular, E. sakazakii, B. cereus, and S. typhimurium were eliminated at 16, 8, and 8 kGy, respectively. Louise et al. (2006) reported that inactivation of microorganism by electron-beam irradiation comes from the inhibition of DNA repair mechanism by increased energy demand of homeostasis on the cell. Among the bacteria used in this study, E. sakazakii was the most resistant to irradiation. Populations of E. sakazakii treated with electron beam at 2 and 8 kGy were reduced to 5.49 and 4.47 log CFU/g, respectively, compared to 6.29 log CFU/g for the non-irradiated sample (Fig. 1). E. sakazakii has been known to be more heat-resistant than any other Enterobacteriaceae associated with infant milk formula (Lin and Beuchat, 2007). Our results are
2.5. Microbiological analysis and calculation of D10-value
7 6 5 Log CFU/g
After irradiation, samples were placed in 90 ml peptone water (0.1% sterile peptone, w/v) in a sterile stomacher bag. Samples were then homogenized using a Stomacher (MIX 2, AES Laboratoire, France) for 3 min, and diluted with peptone water (0.1% sterile peptone, w/v) for microbial count. Serial dilutions were performed in triplicate on each selective agar plate. E. sakazakii counts were determined by plating appropriately diluted samples onto E. sakazakii agar (Oxoid). Samples were evenly spread on the surface of the plates with a sterile glass rod. S. typhimurium were plated onto Xylose lysine deoxychlorate (XLD) Agar (Difco Laboratoires, Detroit, MI, USA). B. cereus counts were plated onto Mannitol egg yolk polymyxin (MYP) Agar (Oxoid). All plates were incubated at 37 1C for 24 h. Each microbial count was the mean of three determinations. Microbial counts were expressed as log CFU/g. The D10-values (the radiation dose necessary to reduce 1 log cycle of the bacterial populations) were determined from the linear regression of the survival curve for the inactivation of E. sakazakii, B. cereus, and S. typhimurium inoculated on powdered weaning food by electron-beam treatment.
8
4 3 2 1 0 0
2
8 Dose (kGy)
16
Fig. 1. Effect of electron beam irradiation on the survival of pathogenic bacteria on powdered weaning food. Bars represent standard error.K: Enterobacter sakazakii, W: Bacillus cereus, ’: Salmonella typhimurium.
ARTICLE IN PRESS
7
7
6
6
5
5 Log CFU/g
Log CFU/g
Y.-H. Hong et al. / Radiation Physics and Chemistry 77 (2008) 1097–1100
4 3 2
4 3 2
1
1
0
0 0
3 6 9 Storage time (days)
1099
12
0
3 6 9 Storage time (days)
12
7 6 Log CFU/g
5 4 3 2 1 0 0
3 6 9 Storage time (days)
12
Fig. 2. Effect of electron beam irradiation on the microbial growth on powdered weaning food during storage. Bars represent standard error: (a) Enterobacter sakazakii, (b) Bacillus cereus and (c) Salmonella typhimurium K: control, W: 2 kGy, ’: 8 kGy J: 16 kGy.
in good agreement with the fact that E. sakazakii is the most troublesome pathogen in powdered weaning food. For B. cereus, radiation at 2 kGy decreased the populations from 5.82 to 4.18 log CFU/g, resulting in a reduction by 1.64 log CFU/g, and the bacteria were eliminated at 8 kGy (Fig. 1). In addition, in the case of S. typhimurium, radiation at 2 kGy decreased the populations from 6.40 to 4.36 log CFU/g, resulting in a reduction by 2.04 log CFU/g, and radiation at 8 kGy eliminated the bacteria (Fig. 1). Effect of irradiation treatment on powdered weaning food was sustained during storage (Fig. 2). Populations of E. sakazakii for the non-irradiated sample increased to 6.21 log CFU/g after 12 days of storage, while the irradiated sample at 2 and 8 kGy reached 5.30 and 3.50 log CFU/g, respectively (Fig. 2a). For B. cereus, there was not much change in the populations of the bacteria among treatments after 12 days of storage (Fig. 2b), yet it should be noted that radiation above 8 kGy eliminated the bacteria. In the case of S. typhimurium, irradiation at 2 kGy had 2.00 log CFU/g after 12 days of storage, compared to 3.78 log CFU/g for the control sample (Fig. 2c). Only a few studies have been reported on the effect of ionizing irradiation on the inactivation of pathogenic bacteria in infant foods. Osaili et al. (2007) have reported that E. sakazakii in dehydrated infant formula needs gamma irradiation of 5.13 kGy to obtain 3 log reduction. Sarrias et al. (2003) also have reported that B. cereus in raw rice was eliminated by radiation at 7.5 kGy. Lee et al. (1998) have investigated that electron-beam irradiation treatment can be useful in enhancing the shelf life of ginseng powder by reducing microorganisms. Those reports are in good agreement with our results.
3.2. D10-values The D10-values of E. sakazakii, B. cereus, and S. typhimurium inoculated on powdered weaning food were 4.83, 1.22, and
Table 1 The D10-values of the major pathogenic bacteria in powdered weaning food Pathogenic bacteria
D10-valuea (kGy)
R2
Enterobacter sakazakii Bacillus cereus Salmonella typhimurium
4.83 1.22 0.98
0.9586 0.9839 0.9966
a
Decimal reduction dose.
0.98 kGy, respectively (Table 1). Ko et al. (2005) have reported that D10-values of total aerobic bacteria in whole black pepper powder and commercial Sunsik (powdered cereals) were 5.32 and 1.56 kGy, respectively. Lee et al. (1998) also have reported that D10-values of total aerobic bacteria in white and red ginseng powder were 3.75 and 2.85 kGy, respectively. These results indicate that D10-value of the microorganisms depend on sample thickness, initial microbial load, physicochemical properties of the sample, and irradiation and storage conditions (Lee et al., 1998, 2000). In particular, the D10-value for E. sakazakii in our study is higher than those of other studies (Lee et al., 2007; Osaili et al., 2007). Lee et al. (2007) have reported that the D10-value for E. sakazakii is 0.76 kGy for dehydrated infant formula using gamma irradiation. Osaili et al. (2007) also have reported that the D10-value is 1.71 kGy for dehydrated infant milk formula. However, it should be noted that the sample in our study was different, because powdered weaning food made from a particular formula containing rice and rice flour was used as a sample. The difference in D10-value for E. sakazakii in our study may be attributed to different formulation. Thus, radiation sensitivity of the microorganisms is also affected by the factor like the composition of the sample (Molins, 2001).
ARTICLE IN PRESS 1100
Y.-H. Hong et al. / Radiation Physics and Chemistry 77 (2008) 1097–1100
Table 2 Change in Hunter color values of electron beam irradiated weaning food during storage Color Storage Irradiation dose (kGy) parameter time (day) 0 2 L
96.9670.66a 83.9971.40a 84.8470.36a 84.8870.53a 83.3570.94a
0 3 6 9 12
a
0 3 6 9 12
3.2170.06b 2.1870.09c 1.7470.12b 1.8170.18a 1.6170.24ba
b
0 3 6 9 12
19.1570.31cb 20.2970.36b 21.2470.42b 20.8870.42c 22.4570.72ba
8
16
95.5470.04ba 83.8570.77a 83.9170.22ba 84.4070.32ba 83.5571.05a
94.7771.20b 83.1870.33a 81.9470.79b 83.1270.39b 83.7270.59a
94.7371.09b 82.1670.21a 81.8570.83b 81.4571.90c 82.7370.80a
2.2770.04a 1.9970.16c 1.3070.30a 1.8070.78a 1.6770.26ba
2.6370.29a 1.7270.03b 1.4770.19a 1.2770.27a 1.8170.09b
2.4870.26a 1.4670.10a 1.3170.11a 1.4570.21a 1.4970.06a
20.1771.16b 21.4470.14a 23.0270.23a 23.2270.52b 22.4770.39ba
21.5870.12a 22.4070.19a 23.3670.64a 24.0770.80a 23.0070.46a
18.7770.10c 21.6271.07a 21.5870.26b 21.5770.53c 22.0770.43b
Any means in the same row followed by different letters are significantly (po0.05) different by Duncan’s multiple range test.
are also observed to be acceptable among treatments except at 16 kGy, where there was a change in quality (Table 3). These results are similar with those of our previous report (Ko et al., 2005), where whole black pepper and commercial Sunsik were irradiated by electron beam.
4. Conclusions Electron-beam irradiation inactivated the major pathogenic bacteria such as E. sakazakii, B. cereus, and S. typhimurium inoculated on powdered weaning food. In particular, irradiation below 16 kGy eliminated all the pathogenic bacteria. Therefore, electron-beam treatment of powdered weaning food appears to achieve microbial decontamination, without affecting the quality change such as color and flavor.
Acknowledgments This study was supported by the Korea Food and Drug Administration. References
Table 3 Sensory evaluation of electron beam irradiated weaning food during storage Organoleptic parameter
Storage time (day)
Irradiation dose (kGy) 0
2 a
8 a
16 a
Color
0 3 6 9 12
5.0070.00 4.6370.74a 4.0070.76ba 3.8870.84a 3.3870.92a
5.0070.00 4.3870.52a 4.1370.64a 3.7570.71a 3.6370.52a
4.6370.52b 4.1270.64a 4.0070.54ba 3.50701.54a 3.0070.54a
4.2570.46b 4.0070.76a 3.3870.74b 3.1370.84a 2.8870.84a
Odor
0 3 6 9 12
5.0070.00a 4.6370.52a 4.0070.76a 3.8870.64a 3.6370.52a
4.7570.46a 4.5070.54a 3.7570.71ba 3.6370.52ba 3.1370.35ba
4.8870.35a 4.0070.54ba 3.1370.64bc 3.1370.64bc 2.5070.54bc
4.2570.46b 3.5070.76b 2.8870.64c 2.6370.52c 2.2570.46c
Overall
0 3 6 9 12
5.0070.00a 4.7570.46a 3.8870.84a 3.7570.71a 3.5070.76a
5.0070.00a 4.5070.54ba 3.8870.64a 3.6370.52a 3.6370.52a
4.7570.46a 4.0070.54bc 3.7570.46a 3.5070.76a 3.0070.54ba
4.3870.52b 3.6370.74c 3.2570.71a 3.1370.64a 2.7570.71b
Any means in the same row followed by different letters are significantly (po0.05) different by Duncan’s multiple range test.
3.3. Color measurement and sensory evaluation Color of powdered weaning food was determined using a colorimeter during storage. Hunter L, a, and b values of the food treated with electron beam were not significantly altered among treatments during storage (Table 2). Our results clearly indicate that electron-beam treatment does not alter the color change of powdered weaning food significantly. Sensory qualities of the food
Agata, N., Ohta, M., Yokoyama, K., 2002. Production of Bacillus cereus emetic toxin in various foods. Int. J. Food Microbiol. 73, 23–27. Iverson, C., Forsythe, S., 2003. Risk profile of Enterobacter sakazakii an emergent pathogen associated with infant milk formula. Trends Food Sci. Technol. 14, 443–454. Ko, J.K., Ma, Y.H., Song, K.B., 2005. Effect of electron beam irradiation on microbial qualities of whole black pepper powder and commercial Sunsik. Korean J. Food Sci. Technol. 37, 308–312. Kramer, J.M., Gilbert, J.L., 1989. Bacillus cereus and other species. In: Doyle, M.P. (Ed.), Foodborne Bacterial Pathogens. Marcel Dekker, New York, pp. 21–70. Lee, M.H., Lee, M.K., Kwon, J.H., 1998. Sterilizing effect of electron beam on ginseng powders. Korean J. Food Sci. Technol. 30, 1362–2366. Lee, J.E., Kwon, O.J., Kwon, J.H., 2000. Effects of electron beam irradiation on microbiological and organoleptic qualities of powdered red pepper and ginger. Korean J. Food Sci. Technol. 32, 380–386. Lee, J.W., Oh, S.H., Byun, E.B., Kim, J.H., Woon, J.H., Byun, M.W., 2007. Inactivation of Enterobacter sakazakii of dehydrated infant formula by gamma-irradiation. Rad. Phys. Chem. 76, 1858–1861. Leuschner Renata, G.K., Bew, J., Boughtflower, M.P., 2004. A collaborative study to evaluate qualitatively powdered weaning food validation sample artificially contaminated with Salmonella anatum. Int. J. Food Microbiol. 97, 43–51. Lin, L.C., Beuchat, L.R., 2007. Survival of Enterobacter sakazakii in infant cereal as affected by composition, water activity, and temperature. Food Microbiol. 24, 767–777. Louise, M.F., Paul, E.C., Alistair, S.G., 2006. The effect of electron beam irradiation, combined with acetic acid, on the survival and recovery of Escherichia coli and Lactobacillus curvatus. Int. J. Food Microbiol. 35, 259–265. Lucas, A., Cole, T.J., 1990. Breast milk and neonatal necrotizing enterocolitis. Lancet 336, 1519–1523. Molins, R.A., 2001. Irradiation of meat and poultry. In: Molins, R.A. (Ed.), Food Irradiation—Principles and Applications. Wiley Interscience, New York, pp. 131–191. Osaili, T.M., Shaker, R.R., Abu, A.S., Ayyash, M.M., Martin, E.M., 2007. Inactivation of Enterobacter sakazakii in infant milk formula by gamma irradiation: determination of D10-value. J. Food Sci. 72, M85–M88. Rodriguez, O., Castell-Perez, M.E., Ekpanyaskun, N., Moreira, R.G., Castillo, A., 2006. Surrogates of validation of electron beam irradiation of foods. Int. J. Food Microbiol. 110, 117–122. Sarrias, J.A., Valero, M., Salmeron, M.C., 2003. Elimination of Bacillus cereus contamination in raw rice by electron beam irradiation. Food Microbiol. 20, 327–332. SAS Institute, 2001. SAS system for Windows. Version 8.2. SAS Institute Inc., Cary, NC.
Contents lists available at ScienceDirect
Radiation Physics and Chemistry journal homepage: www.elsevier.com/locate/radphyschem
Inactivation of Enterobacter sakazakii, Bacillus cereus, and Salmonella typhimurium in powdered weaning food by electron-beam irradiation Yun-Hee Hong a, Ji-Yong Park b, Jong-Hyun Park c, Myong-Soo Chung d, Ki-Sung Kwon e, Kyungsook Chung f, Misun Won f, Kyung-Bin Song a, a
Department of Food Science and Technology, College of Agriculture and Life Science, Chungnam National University, Yuseong-Gu, Daejeon 305-764, Republic of Korea Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea c Department of Food Science and Biotechnology, Kyungwon University, Sungnam 461-701, Republic of Korea d Department of Food Science, Ehwa Women’s University, Seoul 120-750, Republic of Korea e Center for Food safety Evaluation, Korea Food and Drug Administration, Seoul 122-704, Republic of Korea f Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Republic of Korea b
a r t i c l e in fo
abstract
Article history: Received 11 February 2008 Accepted 12 May 2008
Inactivation of Enterobacter sakazakii, Bacillus cereus, and Salmonella typhimurium were evaluated in powdered weaning food using electron-beam irradiation. E. sakazakii, B. cereus, and S. typhimurium were eliminated by irradiation at 16, 8, and 8 kGy, respectively. The D10-vlaues of E. sakazakii, B. cereus, and S. typhimurium inoculated on powdered weaning food were 4.83, 1.22, and 0.98 kGy, respectively. The results suggest that electron-beam irradiation should inhibit the growth of pathogenic bacteria on baby food without impairing qualities. & 2008 Elsevier Ltd. All rights reserved.
Keywords: Powdered weaning food Electron-beam irradiation Microbial growth
1. Introduction Enterobacter sakazakii, Bacillus cereus, and Salmonella typhimurium are the major pathogenic bacteria that have been associated with food poisoning in powdered weaning foods. In particular, E. sakazakii in infant foods has been implicated in infections among high-risk infants (Iverson and Forsythe, 2003). Necrotizing enterocolitis and meningitis caused by E. sakazakii have high mortality rates (Lucas and Cole, 1990). In addition, dried weaning foods are known to be easily contaminated with B. cereus spores (Kramer and Gilbert, 1989), causing diarrheal and emetic type of disease (Agata et al., 2002). Salmonella spp. has been also associated with weaning foods, and the presence of Salmonella spp. was responsible for disease among infants (Leuschner Renata et al., 2004). As a food preservation method, ionizing irradiation is considered as an effective method in providing hygienic quality by reducing microbial spoilage. There are two most common types of ionizing radiation: gamma ray and electron beam. Electron-beam irradiation is used to inactivate food borne microorganisms during storage, and to guarantee the hygienic quality of foods (Sarrias et al., 2003). Electron-beam irradiation has been shown to destroy 99.9% of the major food pathogenic bacteria (Rodriguez et al., 2006), and it requires relatively short processing time and low
Corresponding author. Tel.: +82 42 821 6723; fax: +82 42 825 2664.
E-mail address: [email protected] (K.-B. Song). 0969-806X/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2008.05.004
equipment cost. Thus, it can be applicable as a preservation method for powdered weaning food to achieve microbial decontamination. In particular, application of electron-beam irradiation to food products can avoid the negative image toward gamma irradiation on foods since it does not use a radioisotope source like 60Co (Sarrias et al., 2003). Therefore, the objectives of this study were to examine the effect of electron-beam irradiation on the microbial safety and qualities of powdered weaning food during storage, and to provide appropriate processing condition for its safety.
2. Experimental 2.1. Materials Powdered weaning food (Namyang Co., Gongju, Korea) was purchased from a local market in Daejeon, Korea. Ingredients of powdered weaning food are uncleaned rice, defatted dried milk, a-rice flour, concentrated milk protein, sunflower powder, fructose, sunflower oil, peas powder, milk protein isolate, safflower oil, beef, concentrated rice protein, concentrated whey protein, whole milk powder, chicken, millet, oatmeal, dextrin, powdered uncleaned rice syrup, malt extract, pineapple powder, elderberry powder, banana powder, apple powder, mandarin powder, strawberry powder, celery, broccoli, tomato, carrot powder, pumpkin powder, spinach powder, mushroom, safflower seed extract, docosahexaenoic acid (DHA), chlorella extract (C, G, F),
ARTICLE IN PRESS 1098
Y.-H. Hong et al. / Radiation Physics and Chemistry 77 (2008) 1097–1100
egg yolk, casein phosphopeptides, vitamin E, calcium phosphate tribasic, potassium chloride, eggshell calcium, taurine, dried yeast, L-methionine, L-carnitine, magnesium hydroxide, lactoferrin Concentrates, vitamin premix (vitamin C, commercial enrichments, dextrin, betacarotine), lactic acid bacteria, copper sulfate, sulfate of zinc, ferrous lactate, yoghurt, cod fish, tuna, anchovy, kelp extract powder, embryo bud, jujube extract, and chestnut powder.
2.6. Color measurement Color of the samples was analyzed using a colorimeter (CR-300 Minolta Chroma Meter, Minolta Camera Co., Osaka, Japan). Samples were placed on a white standard plate and Hunter’s color values (L, a, b) were measured and total color difference values were expressed as E value. Hunter’s L, a, and b values for the standard plate were 98.34, 0.03, and 1.62, respectively. Five measurements were taken at different locations of each sample.
2.2. Culture conditions 2.7. Sensory evaluation E. sakazakii (ATCC 51329), B. cereus (KCCM 40935), and S. typhimurium (ATCC 14028) were used in this study. E. sakazakii, B. cereus, and S. typhimurium in 20% (w/v) glycerol solution were kept individually at 70 1C in 5 ml vials. E. sakazakii, B. cereus, and S. typhimurium cultures were grown at 37 1C for 24 h in 50 ml tubes containing 25 ml of Enterobacteriaceae enrichment broth (Oxoid, Basingstoke, U.K), brain heart infusion (BHI) (Oxoid), and Salmosyst (Merck, Darmseadt, Germany), respectively.
Samples were analyzed for their color, odor, and overall acceptability by 8 trained panelists (2 men and 6 women; age range 24–27). Sensory qualities of samples were evaluated using five point scoring method. Sensory scores were 5, very good; 4, good; 3, fair; 2, poor; and 1, very poor. 2.8. Statistical analysis
2.3. Inoculation on weaning food
Analysis of variance and Duncan’s multiple range tests with significance at po0.05 were performed using a SAS program (SAS Inst. Version 8.2, 2001).
Powdered weaning food (10 g) was placed into a sterile bag and inoculated with 200 ml of E. sakazakii, B. cereus, and S. typhimurium to obtain an initial level of 108–109 CFU/g, respectively. After inoculation, the samples were shaken for 1 min to evenly spread the bacteria on the food.
3. Results and discussion
2.4. Irradiation treatments Electron-beam irradiation was performed using an electronbeam accelerator (Model ELV-4, 1 MeV, Eb-Tech, Yuseong, Daejeon, Korea). Samples were individually packaged in 100 mm 170 mm low-density polyethylene bags (thickness, below 5 mm) and they were exposed to 3 dose levels of 2, 8, and 16 kGy at 1 MeV (beam current 2–8 mA, beam dimension 600 mm 600 mm, conveyer velocity 5–10 m/min). Absorption dose was determined using a cellulose triacetate (CTA) dosimeter. After irradiation, samples were stored at 20 1C for 12 days.
3.1. Microbiological analysis Electron-beam treatment decreased the microbial populations with increasing radiation dose (Fig. 1). In particular, E. sakazakii, B. cereus, and S. typhimurium were eliminated at 16, 8, and 8 kGy, respectively. Louise et al. (2006) reported that inactivation of microorganism by electron-beam irradiation comes from the inhibition of DNA repair mechanism by increased energy demand of homeostasis on the cell. Among the bacteria used in this study, E. sakazakii was the most resistant to irradiation. Populations of E. sakazakii treated with electron beam at 2 and 8 kGy were reduced to 5.49 and 4.47 log CFU/g, respectively, compared to 6.29 log CFU/g for the non-irradiated sample (Fig. 1). E. sakazakii has been known to be more heat-resistant than any other Enterobacteriaceae associated with infant milk formula (Lin and Beuchat, 2007). Our results are
2.5. Microbiological analysis and calculation of D10-value
7 6 5 Log CFU/g
After irradiation, samples were placed in 90 ml peptone water (0.1% sterile peptone, w/v) in a sterile stomacher bag. Samples were then homogenized using a Stomacher (MIX 2, AES Laboratoire, France) for 3 min, and diluted with peptone water (0.1% sterile peptone, w/v) for microbial count. Serial dilutions were performed in triplicate on each selective agar plate. E. sakazakii counts were determined by plating appropriately diluted samples onto E. sakazakii agar (Oxoid). Samples were evenly spread on the surface of the plates with a sterile glass rod. S. typhimurium were plated onto Xylose lysine deoxychlorate (XLD) Agar (Difco Laboratoires, Detroit, MI, USA). B. cereus counts were plated onto Mannitol egg yolk polymyxin (MYP) Agar (Oxoid). All plates were incubated at 37 1C for 24 h. Each microbial count was the mean of three determinations. Microbial counts were expressed as log CFU/g. The D10-values (the radiation dose necessary to reduce 1 log cycle of the bacterial populations) were determined from the linear regression of the survival curve for the inactivation of E. sakazakii, B. cereus, and S. typhimurium inoculated on powdered weaning food by electron-beam treatment.
8
4 3 2 1 0 0
2
8 Dose (kGy)
16
Fig. 1. Effect of electron beam irradiation on the survival of pathogenic bacteria on powdered weaning food. Bars represent standard error.K: Enterobacter sakazakii, W: Bacillus cereus, ’: Salmonella typhimurium.
ARTICLE IN PRESS
7
7
6
6
5
5 Log CFU/g
Log CFU/g
Y.-H. Hong et al. / Radiation Physics and Chemistry 77 (2008) 1097–1100
4 3 2
4 3 2
1
1
0
0 0
3 6 9 Storage time (days)
1099
12
0
3 6 9 Storage time (days)
12
7 6 Log CFU/g
5 4 3 2 1 0 0
3 6 9 Storage time (days)
12
Fig. 2. Effect of electron beam irradiation on the microbial growth on powdered weaning food during storage. Bars represent standard error: (a) Enterobacter sakazakii, (b) Bacillus cereus and (c) Salmonella typhimurium K: control, W: 2 kGy, ’: 8 kGy J: 16 kGy.
in good agreement with the fact that E. sakazakii is the most troublesome pathogen in powdered weaning food. For B. cereus, radiation at 2 kGy decreased the populations from 5.82 to 4.18 log CFU/g, resulting in a reduction by 1.64 log CFU/g, and the bacteria were eliminated at 8 kGy (Fig. 1). In addition, in the case of S. typhimurium, radiation at 2 kGy decreased the populations from 6.40 to 4.36 log CFU/g, resulting in a reduction by 2.04 log CFU/g, and radiation at 8 kGy eliminated the bacteria (Fig. 1). Effect of irradiation treatment on powdered weaning food was sustained during storage (Fig. 2). Populations of E. sakazakii for the non-irradiated sample increased to 6.21 log CFU/g after 12 days of storage, while the irradiated sample at 2 and 8 kGy reached 5.30 and 3.50 log CFU/g, respectively (Fig. 2a). For B. cereus, there was not much change in the populations of the bacteria among treatments after 12 days of storage (Fig. 2b), yet it should be noted that radiation above 8 kGy eliminated the bacteria. In the case of S. typhimurium, irradiation at 2 kGy had 2.00 log CFU/g after 12 days of storage, compared to 3.78 log CFU/g for the control sample (Fig. 2c). Only a few studies have been reported on the effect of ionizing irradiation on the inactivation of pathogenic bacteria in infant foods. Osaili et al. (2007) have reported that E. sakazakii in dehydrated infant formula needs gamma irradiation of 5.13 kGy to obtain 3 log reduction. Sarrias et al. (2003) also have reported that B. cereus in raw rice was eliminated by radiation at 7.5 kGy. Lee et al. (1998) have investigated that electron-beam irradiation treatment can be useful in enhancing the shelf life of ginseng powder by reducing microorganisms. Those reports are in good agreement with our results.
3.2. D10-values The D10-values of E. sakazakii, B. cereus, and S. typhimurium inoculated on powdered weaning food were 4.83, 1.22, and
Table 1 The D10-values of the major pathogenic bacteria in powdered weaning food Pathogenic bacteria
D10-valuea (kGy)
R2
Enterobacter sakazakii Bacillus cereus Salmonella typhimurium
4.83 1.22 0.98
0.9586 0.9839 0.9966
a
Decimal reduction dose.
0.98 kGy, respectively (Table 1). Ko et al. (2005) have reported that D10-values of total aerobic bacteria in whole black pepper powder and commercial Sunsik (powdered cereals) were 5.32 and 1.56 kGy, respectively. Lee et al. (1998) also have reported that D10-values of total aerobic bacteria in white and red ginseng powder were 3.75 and 2.85 kGy, respectively. These results indicate that D10-value of the microorganisms depend on sample thickness, initial microbial load, physicochemical properties of the sample, and irradiation and storage conditions (Lee et al., 1998, 2000). In particular, the D10-value for E. sakazakii in our study is higher than those of other studies (Lee et al., 2007; Osaili et al., 2007). Lee et al. (2007) have reported that the D10-value for E. sakazakii is 0.76 kGy for dehydrated infant formula using gamma irradiation. Osaili et al. (2007) also have reported that the D10-value is 1.71 kGy for dehydrated infant milk formula. However, it should be noted that the sample in our study was different, because powdered weaning food made from a particular formula containing rice and rice flour was used as a sample. The difference in D10-value for E. sakazakii in our study may be attributed to different formulation. Thus, radiation sensitivity of the microorganisms is also affected by the factor like the composition of the sample (Molins, 2001).
ARTICLE IN PRESS 1100
Y.-H. Hong et al. / Radiation Physics and Chemistry 77 (2008) 1097–1100
Table 2 Change in Hunter color values of electron beam irradiated weaning food during storage Color Storage Irradiation dose (kGy) parameter time (day) 0 2 L
96.9670.66a 83.9971.40a 84.8470.36a 84.8870.53a 83.3570.94a
0 3 6 9 12
a
0 3 6 9 12
3.2170.06b 2.1870.09c 1.7470.12b 1.8170.18a 1.6170.24ba
b
0 3 6 9 12
19.1570.31cb 20.2970.36b 21.2470.42b 20.8870.42c 22.4570.72ba
8
16
95.5470.04ba 83.8570.77a 83.9170.22ba 84.4070.32ba 83.5571.05a
94.7771.20b 83.1870.33a 81.9470.79b 83.1270.39b 83.7270.59a
94.7371.09b 82.1670.21a 81.8570.83b 81.4571.90c 82.7370.80a
2.2770.04a 1.9970.16c 1.3070.30a 1.8070.78a 1.6770.26ba
2.6370.29a 1.7270.03b 1.4770.19a 1.2770.27a 1.8170.09b
2.4870.26a 1.4670.10a 1.3170.11a 1.4570.21a 1.4970.06a
20.1771.16b 21.4470.14a 23.0270.23a 23.2270.52b 22.4770.39ba
21.5870.12a 22.4070.19a 23.3670.64a 24.0770.80a 23.0070.46a
18.7770.10c 21.6271.07a 21.5870.26b 21.5770.53c 22.0770.43b
Any means in the same row followed by different letters are significantly (po0.05) different by Duncan’s multiple range test.
are also observed to be acceptable among treatments except at 16 kGy, where there was a change in quality (Table 3). These results are similar with those of our previous report (Ko et al., 2005), where whole black pepper and commercial Sunsik were irradiated by electron beam.
4. Conclusions Electron-beam irradiation inactivated the major pathogenic bacteria such as E. sakazakii, B. cereus, and S. typhimurium inoculated on powdered weaning food. In particular, irradiation below 16 kGy eliminated all the pathogenic bacteria. Therefore, electron-beam treatment of powdered weaning food appears to achieve microbial decontamination, without affecting the quality change such as color and flavor.
Acknowledgments This study was supported by the Korea Food and Drug Administration. References
Table 3 Sensory evaluation of electron beam irradiated weaning food during storage Organoleptic parameter
Storage time (day)
Irradiation dose (kGy) 0
2 a
8 a
16 a
Color
0 3 6 9 12
5.0070.00 4.6370.74a 4.0070.76ba 3.8870.84a 3.3870.92a
5.0070.00 4.3870.52a 4.1370.64a 3.7570.71a 3.6370.52a
4.6370.52b 4.1270.64a 4.0070.54ba 3.50701.54a 3.0070.54a
4.2570.46b 4.0070.76a 3.3870.74b 3.1370.84a 2.8870.84a
Odor
0 3 6 9 12
5.0070.00a 4.6370.52a 4.0070.76a 3.8870.64a 3.6370.52a
4.7570.46a 4.5070.54a 3.7570.71ba 3.6370.52ba 3.1370.35ba
4.8870.35a 4.0070.54ba 3.1370.64bc 3.1370.64bc 2.5070.54bc
4.2570.46b 3.5070.76b 2.8870.64c 2.6370.52c 2.2570.46c
Overall
0 3 6 9 12
5.0070.00a 4.7570.46a 3.8870.84a 3.7570.71a 3.5070.76a
5.0070.00a 4.5070.54ba 3.8870.64a 3.6370.52a 3.6370.52a
4.7570.46a 4.0070.54bc 3.7570.46a 3.5070.76a 3.0070.54ba
4.3870.52b 3.6370.74c 3.2570.71a 3.1370.64a 2.7570.71b
Any means in the same row followed by different letters are significantly (po0.05) different by Duncan’s multiple range test.
3.3. Color measurement and sensory evaluation Color of powdered weaning food was determined using a colorimeter during storage. Hunter L, a, and b values of the food treated with electron beam were not significantly altered among treatments during storage (Table 2). Our results clearly indicate that electron-beam treatment does not alter the color change of powdered weaning food significantly. Sensory qualities of the food
Agata, N., Ohta, M., Yokoyama, K., 2002. Production of Bacillus cereus emetic toxin in various foods. Int. J. Food Microbiol. 73, 23–27. Iverson, C., Forsythe, S., 2003. Risk profile of Enterobacter sakazakii an emergent pathogen associated with infant milk formula. Trends Food Sci. Technol. 14, 443–454. Ko, J.K., Ma, Y.H., Song, K.B., 2005. Effect of electron beam irradiation on microbial qualities of whole black pepper powder and commercial Sunsik. Korean J. Food Sci. Technol. 37, 308–312. Kramer, J.M., Gilbert, J.L., 1989. Bacillus cereus and other species. In: Doyle, M.P. (Ed.), Foodborne Bacterial Pathogens. Marcel Dekker, New York, pp. 21–70. Lee, M.H., Lee, M.K., Kwon, J.H., 1998. Sterilizing effect of electron beam on ginseng powders. Korean J. Food Sci. Technol. 30, 1362–2366. Lee, J.E., Kwon, O.J., Kwon, J.H., 2000. Effects of electron beam irradiation on microbiological and organoleptic qualities of powdered red pepper and ginger. Korean J. Food Sci. Technol. 32, 380–386. Lee, J.W., Oh, S.H., Byun, E.B., Kim, J.H., Woon, J.H., Byun, M.W., 2007. Inactivation of Enterobacter sakazakii of dehydrated infant formula by gamma-irradiation. Rad. Phys. Chem. 76, 1858–1861. Leuschner Renata, G.K., Bew, J., Boughtflower, M.P., 2004. A collaborative study to evaluate qualitatively powdered weaning food validation sample artificially contaminated with Salmonella anatum. Int. J. Food Microbiol. 97, 43–51. Lin, L.C., Beuchat, L.R., 2007. Survival of Enterobacter sakazakii in infant cereal as affected by composition, water activity, and temperature. Food Microbiol. 24, 767–777. Louise, M.F., Paul, E.C., Alistair, S.G., 2006. The effect of electron beam irradiation, combined with acetic acid, on the survival and recovery of Escherichia coli and Lactobacillus curvatus. Int. J. Food Microbiol. 35, 259–265. Lucas, A., Cole, T.J., 1990. Breast milk and neonatal necrotizing enterocolitis. Lancet 336, 1519–1523. Molins, R.A., 2001. Irradiation of meat and poultry. In: Molins, R.A. (Ed.), Food Irradiation—Principles and Applications. Wiley Interscience, New York, pp. 131–191. Osaili, T.M., Shaker, R.R., Abu, A.S., Ayyash, M.M., Martin, E.M., 2007. Inactivation of Enterobacter sakazakii in infant milk formula by gamma irradiation: determination of D10-value. J. Food Sci. 72, M85–M88. Rodriguez, O., Castell-Perez, M.E., Ekpanyaskun, N., Moreira, R.G., Castillo, A., 2006. Surrogates of validation of electron beam irradiation of foods. Int. J. Food Microbiol. 110, 117–122. Sarrias, J.A., Valero, M., Salmeron, M.C., 2003. Elimination of Bacillus cereus contamination in raw rice by electron beam irradiation. Food Microbiol. 20, 327–332. SAS Institute, 2001. SAS system for Windows. Version 8.2. SAS Institute Inc., Cary, NC.