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Radiation Physics and Chemistry 79 (2010) 731–734

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Radiation Physics and Chemistry

0969-80

doi:10.1

n Corr

E-m

journal homepage: www.elsevier.com/locate/radphyschem

Effects of gamma and electron beam irradiation on the survival of pathogensinoculated into sliced and pizza cheeses

Hyun-Joo Kim a, Jun-Sang Ham b, Ju-Woon Lee c, Keehyuk Kim d, Sang-Do Ha a, Cheorun Jo e,n

a Department of Food Science and Technology, Chung-Ang University, Ansung, Gyunggi-do 456-756, Republic of Koreab Animal Products Processing Division, National Livestock Research Institute, RDA, Suwon 441-706, Republic of Koreac Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185, Republic of Koread Department of Culinary Nutrition, Woosong University, Daejeon 300-718, Republic of Koreae Department of Animal Science and Biotechnology, Chungnam National University, Daejeon 305-764, Republic of Korea

a r t i c l e i n f o

Article history:

Received 14 October 2009

Accepted 20 December 2009

Keywords:

Gamma irradiation

Electron beam irradiation

Cheese

D10 value

Pathogen

Inoculation study

6X/$ - see front matter & 2009 Elsevier Ltd. A

016/j.radphyschem.2009.12.016

esponding author. Tel.: +82 42 821 5774; fax

ail address: cheorun@cnu.ac.kr (C. Jo).

a b s t r a c t

The objective of this study was to identify the efficacy of gamma and electron beam irradiation of the

food-borne pathogens (Listeria monocytogenes and Staphylococcus aureus) in sliced and pizza cheeses

commercially available in the Korean market. Total aerobic bacteria and yeast/mold in the cheeses

ranged from 102 to 103 Log CFU/g. Irradiation of 1 kGy for sliced cheese and 3 kGy for pizza cheese were

sufficient to lower the total aerobic bacteria to undetectable levels (101 CFU/g). Pathogen inoculation

test revealed that gamma irradiation was more effective than electron beam irradiation at the same

absorbed dose, and the ranges of the D10 values were from 0.84 to 0.93 kGy for L. monocytogenes and

from 0.60 to 0.63 kGy for S. aureus. Results suggest that a low dose irradiation can improve significantly

the microbial quality and reduce the risk of contamination of sliced and pizza cheeses by the food-borne

pathogens which can potentially occur during processing.

& 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Cheese is one of the major manufactured goods of the dairyindustry and has become a predominant consumer product forlarge segments of the population (IDFA, 2004). Cheese is a goodmedium for microbial growth due to its nutrient contents andlong storage duration. Pasteurization has been used to preventfood-borne diseases associated with dairy products (Lalaguna,2003). During the processing, however, a potential hazard mayoccur that can be caused by addition of contaminated ingredientsor improper handling of the final products, including an abuse ofthe storage temperature (Jo et al., 2007; Kaan Tekinsen andOzdemir, 2006).

There are some reports on the levels of Staphylococcus aureus

and Escherichia coli and the incidence of Salmonella, E. coli

O157:H7 and Listeria monocytogenes in various cheeses (KaanTekinsen and Ozdemir, 2006; Rudolf and Scherer, 2001). However,infants, children, and immunosuppresed patients, in particular,are very likely to acquire food-borne pathogens (Kabakna et al.,2004). Therefore, the sanitation treatment is needed for thisparticular food product.

ll rights reserved.

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Food irradiation technology can be used to improve themicrobiological safety and extend the shelf-life of a widerange of foods. Sources of ionizing radiation that have beenused include gamma rays, electron beams and X-rays(WHO, 1988). Gamma rays produced from radioisotope such as60Co and 137Cs and electron beams produced from machinesources operated at or below an energy level of 10 MeV areused in most commercial plants (Morehouse and Komolprasert,2004). The major differences between gamma and electronbeam irradiation are the penetration power and dose rate. Itoand Islam (1994) reported that electron beam and converted X-rays had slightly higher D10 values than gamma ray due to largedifference of dose rate. Halls (1994) indicated, however, the doserate has no effect on the radiation response of microbialpopulation.

On the other hand, a significant effect has been observed whengamma irradiation was applied to dairy products such as cheeseand ice cream for control the presence of pathogens (Kim et al.,2007, 2008; Jo et al., 2007). However, the information on theeffectiveness and elimination of food-borne pathogens andradiation sensitivity by electron beam of cheeses has not beenwell documented.

The objective of this study was to evaluate and compare theefficacy of gamma and electron beam irradiation in order to evaluatethe microbial contamination in sliced and pizza cheese commerciallyavailable in the Korean market and determine the radiation sensitivity

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H.-J. Kim et al. / Radiation Physics and Chemistry 79 (2010) 731–734732

of the food-borne pathogens including L. monocytogenes and S. aureus

inoculated into cheeses.

2. Materials and methods

2.1. Sample preparation

Sliced and pizza cheeses produced from the same companywere purchased from a local market in Daejeon, Korea. Thesamples (approximately 10 g) were transferred into a sterilizedoxygen-impermeable nylon bags (2 mL O2/m2/24 h at 0 1C,0.09 mm thickness; Sunkyung Co. Ltd., Seoul, Korea) with asterilized spoon in a clean bench. The packs were sealed andtransferred to a refrigerator (4 1C) and stored.

2.2. Gamma and electron beam irradiation

Packed samples were irradiated in a cobalt-60 gammairradiator (point source, ACEL, IR-79, MDS Nordion, Ontrario,Canada) at the Korea Atomic Energy Research Institute, Jeongeup,Korea. The applied doses in this study were 0, 1, 3 and 5 kGy. Thesource strength was approximately 11.1 PBq with a dose rate of10 kGy/h. Cheeses were irradiated to 0.5 cm of thickness.Dosimetry was performed using 5 mm diameter alanine dosi-meters (Bruker Instruments, Rheinstetten, Germany), and the freeradical signal was measured using a Brucker EMS 104 EPRAnalyzer. The dosimeters were calibrated against an internationalstandard set by the International Atomic Energy Agency (Vienna,Austria).

At the same day, the prepared samples were also irradiatedin a linear electron beam RF accelerator (energy 2.5 MeV,beam power 40 kW, EB Tech, Daejeon, Korea) at 1070.5 1C.Irradiation was performed in the presence of air, 2.5 MeVof energy and a conveyor velocity of 10 m/min. The doserates were 0.55, 1.5 and 2.5 kGy/s, respectively. Cheeses wereirradiated with the same thickness (0.5 cm) with the gammairradiated sample. To confirm the target dose, alanine dosi-meters attached to the top and bottom surfaces of the samplepack were read using a 104 Electron Paramagnetic Resornanceunit (EMS-104, Bruker Instruments Inc., Bullerica, MA). Foreffective process, the samples were placed evenly to haveminimum thickness.

After irradiation, the samples were stored in a refrigerator setat 4 1C until required for further analysis.

2.3. Microbial analysis

The prepared sample (10 g) was homogenized for 2 min in asterile stomacher bag containing 90 mL of sterile saline solutionusing a stomacher (bag mixers 400, Interscience Co, France).Media for the enumeration of the total aerobic bacteria and yeastand mold were prepared by a total plate count agar (DifcoLaboratories, Detroit, MI, USA), and a potato dextrose agar (Difco),respectively. The plates were incubated at 37 1C for 48 hr (totalaerobic bacteria) or at 30 1C for 5 days (yeast and mold). Thecolony forming units (CFU) per gram were counted at a dilution of30–300 CFU per plate.

2.4. Inoculation test

2.4.1. Sterilization and irradiation

For the inoculation test of pathogens, the sliced and pizzacheeses were sterilized by gamma irradiation (40 kGy)with the same irradiation conditions explained in Section 2.2.

Radiation-sterilized cheeses were found to be devoid of any viablemicroorganisms as determined by the analysis.

2.4.2. Test pathogens and culture condition

Two pathogens, L. monocytogenes (KCTC 3569) and S. aureus

(KCTC 1916) were used in this study and obtained fromthe Korean Collection for Type Cultures (KCTC, Daejeon, Korea).L. monocytogenes and S. aureus were grown in a tryptic soybroth (Difco, Laboratories, Sparks, MD, USA). Incubation for L.

monocytogenes was performed at 30 1C and that of S. aureus at37 1C for 48 hr. The activated cell cultures were centrifuged(2795g for 10 min at 4 1C) in a refrigerated centrifuge (Vs-5500,Vision Scientific, Co., Seoul, Korea) and the cultures were washedtwice with sterile saline solution. The pellet was finally suspendedin sterile saline solution to a cell density of approximately 107–108 CFU/mL levels.

2.4.3. Inoculation of the test microorganism and irradiation

Sterile cheeses samples were inoculated with each cellsuspension of the two pathogens, respectively. The test culturesuspension (200 mL) was spread aseptically on to the sliced andpizza cheeses. Then, it was kept in a sterile workstation for 1 minto allow it to be absorbed. The inoculated samples in thestomacher bag were irradiated with the same conditionspreviously described. The applied doses were 0, 1, 3 and 5 kGytreated by gamma and electron beam irradiation. After irradia-tion, the samples were stored in a refrigerator set at 4 1C forfurther analysis.

2.4.4. Microbiological analysis for inoculated samples

A 10 g sample was aseptically homogenized for 2 min in asterile stomacher bag containing 90 mL of sterile salinesolution using a stomacher (bag mixers 400, Interscience Co,France). After serial dilutions, a 100 mL aliquot from an appro-priate dilution was plated onto the media. Media for themicrobial count of the L. monocytogenes and S. aureus wereprepared by a tryptic soy agar (Difco). Plates were incubated atthe optimal temperature of the bacteria for 48 h and the CFU pergram were counted at 30–300 CFU per plate. D10 values (the doserequired to inactivate 90% of a population) for each of theorganisms tested were determined by the linear fit of thelogarithmic survivors versus irradiation dose points (Kim et al.,2007).

2.5. Statistical analysis

All the experiments were carried out in triplicate with 3observation numbers per trial. One way analysis of variance wasperformed using SPSS (1997) software system and the Duncan’smultiple range test was used to compare the differences amongthe mean values. Mean values and the standard deviations (SD)were reported.

3. Results and discussions

3.1. Microbial contamination of commercial cheeses

The total aerobic bacterial population of the commercial slicedand pizza cheeses were 2.7870.05 and 3.3770.09 log CFU/g,respectively. The sliced cheese did not show any yeast and moldpopulation but pizza cheese showed 2.8770.05 log CFU/g(Table 1). Gamma irradiation was more effective than electronbeam irradiation on elimination of microorganisms in the pizzacheese. Hayaloglu and Kirbag (2007) discussed that total

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Table 1Microbial population of irradiated sliced and pizza cheeses commercially available in Korea.

Sample Irradiation dose (kGy) Viable cell counts (log CFU/g)

Gamma ray Electron beam

Total aerobic bacteria Sliced cheese 0 2.7870.05a 2.7870.05

1 NGb NG

3 NG NG

5 NG NG

Pizza cheese 0 3.3770.09 3.3770.09

1 2.13d70.07 2.75c70.15

3 NG NG

5 NG NG

Yeast and mold Sliced cheese 0 NG NG

1 NG NG

3 NG NG

5 NG NG

Pizza cheese 0 2.8770.05 2.8770.05

1 1.83d70.12 2.15c70.09

3 NG NG

5 NG NG

a Means7standard deviation (n=3).b Viables with no growth at a detection limit o101 CFU/g.c,d Values with different letters within the same row differ significantly (Po0.05).

Table 2Effects of gamma and electron beam irradiation on Listeria monocytogenes and Staphylococcus aureus inoculated into sliced and pizza cheeses.

Microorganisms Sample Irradiation. dose (kGy) Viable cell counts (log CFU/g)

Gamma ray Electron beam

Listeria monocytogenes Sliced cheese 0 5.6970.04a 5.6970.04

1 3.97d70.04 4.85c 70.08

2 3.32d70.01 3.98c70.04

3 2.47d70.09 2.81c70.06

5 NGb NG

Pizza cheese 0 5.8470.09 5.8470.09

1 4.8370.04 3.8970.04

2 3.96d70.04 3.03c70.02

3 2.53d70.04 2.75c70.03

5 NG NG

Staphylococcus aureus Sliced cheese 0 4.9970.01a) 4.9970.01

1 3.67d70.06 4.06c70.04

2 2.43d70.07 3.13c70.04

3 NGb) NG

5 NG NG

Pizza cheese 0 5.1470.04 5.1470.04

1 3.99d70.02 4.10c70.01

2 2.80d70.09 3.06c70.05

3 NG NG

5 NG NG

a Means7standard deviation (n=3).b Viables with no growth at a detection limit o101 CFU/g.c,d Values with different letters within the same row differ significantly (Po0.05).

H.-J. Kim et al. / Radiation Physics and Chemistry 79 (2010) 731–734 733

mesophilic bacterial counts were high (ranged from 8.26 to10.69 log CFU/g) in cheeses, probably due to the use of raw milkwith high microbial load in their manufacture and uncontrolledripening conditions. The number of total aerobic bacteria wasmuch lower in the sample presently analyzed.

No viable cells were observed in the irradiated slicedcheese. However, aerobic bacteria and yeast and mold weredetected when irradiated at 1 kGy but not at 3 kGy in thepizza cheese. Irradiation of 1 and 2 kGy reduced the respectivemicrobial load by approximately a 1 or 2 decimal reduction fromthe original microbial load (Kamat et al., 2000).

3.2. Inoculation test

The two pathogens inoculated into the commercial sliced andpizza cheeses are shown in Table 2. L. monocytogenes and S. aureus

were initially loaded at 107 and 106 CFU/g when inoculated,respectively (data not shown). Irradiation of 3 kGy caused about 3decimal reductions in the number of L. monocytogenes. Irradiationof 5 kGy was not detected (less than 101 CFU/g). Gammairradiation was more effective than electron beam irradiation inelimination of L. monocytogenes. It has been reported that L.

monocytogenes is capable of growing in a refrigerated or frozen

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Table 3D10 values (kGy) of Listeria monocytogenes and Staphylococcus aureus inoculated

into sliced and pizza cheeses.

Sample Pathogens Gamma ray Electron beam

Sliced cheese Listeria monocytogenes 0.86c70.02a 0.88b70.07

Staphylococcus aureus 0.6270.05 0.6370.08

Pizza cheese Listeria monocytogenes 0.84c70.02 0.93b70.05

Staphylococcus aureus 0.6070.05 0.6170.03

a Means7standard deviation (n=3).b,c Values with different letters within the same row differ significantly

(Po0.05).

H.-J. Kim et al. / Radiation Physics and Chemistry 79 (2010) 731–734734

conditions, and in a high salt environment (Warke et al., 2000;Han et al., 2008). Listeriosis is a relatively rare food-borne illnessbut it can be life threatening with high fatality rates (Okutaniet al., 2004). Konteles et al. (2009) reported that L. monocytogenes

was not detected when exposed to a 5 kGy in Feta cheese.The inhibition effects of irradiation on S. aureus inoculated into

the samples are presented in Table 2. Irradiation at 2 kGy showedabout 1 or 2 decimal reductions of S. aureus. No viable cell wasobserved in the 3 and 5 kGy irradiated samples. Similarly to L.

monocytogenes, gamma irradiation was more effective thanelectron beam irradiation in elimination of S. aureus. S. aureus isa common pathogen that causes food-borne illness. Sime~ao doCarmo et al. (2002) reported that food poisoning was occurreddue to enterotoxigenic strains of Staphylococcus present in Minascheese and raw milk in Brazil. Low-dose gamma irradiation iseffective in reducing pathogens in a variety of foods and may beeffective in reducing S. aureus in ready-to-eat foods includingcheese.

Table 3 shows the calculated D10 values of the irradiationtreatment of the samples. The range of the D10 value varies from0.84 to 0.93 for L. monocytogenes and from 0.60 to 0.63 for S. aureus.Kim et al. (2007) reported that the D10 value of L. monocytogenes andS. aureus were 0.64 and 0.54 kGy in commercial cheddar cheese,respectively. D10 values of bacteria in food are affected by the wateractivity, food composition, irradiation or storage temperature,presence of oxygen, and others (Mendoca, 2002). The indirect effectexerted by free radicals formed during an irradiation of cheese and icecreams at frozen temperature are expected to be less due to therestricted mobilization of the free radicals in the frozen product(Kamat et al., 2000). Tsiotsias et al. (2002) reported that D10 value of L.

monocytogenes was 1.38 kGy in Anthotyros cheese. Anthotyros cheesecontains approximately 17% fat and 10% protein, and thecomparatively high D10 value may be explained by a doubleprotective effect of complex protein and fat matrix of the productin combination.

Gamma irradiation was more effective than electron beamirradiation in terms of the D10 value for the L. monocytogenes.However, the D10 value of the S. aureus did not show anydifferences (Table 3). Previous studies demonstrated that higherD10 values were obtained in foods treated with electron beamcompared with gamma ray (Miller, 2005; Song et al., 2009; Wajeet al., 2009) and the authors explained as penetration powerbetween the two radiation sources. The one-side electron beamirradiation also might be the reason. On the other hand, Ito andIslam (1994) explained the difference of radiation sensitivitiesbetween electron beam or converted X-rays and gamma-rays bydose rate. The dose rate of electron beam irradiation is usually1000–10,000 times higher than gamma ray in commercial scale(Ito and Islam, 1994). Ito and Islam (1994) indicated that higherD10 values in high dose rate (electron beam) caused bysuppression of the oxidation damage of microorganisms at highpenetration speed of oxygen inside of cell walls. However, sincethis high dose rate irradiation can also suppress the oxidativedeterioration in food itself, the dose rate effect of electron beam

irradiation may not large and cause serious problems incommercial application.

4. Conclusion

In conclusion, the present study indicated that a low-doseirradiation (less than 3 kGy) can improve the microbial qualityand reduce further the risk caused by the food-borne pathogens ofsliced and pizza cheeses. The radiation sensitivities of pathogensand irradiation effect of radiation source were different. There-fore, the level of irradiation dose for target pathogen and theradiation source should be considered to obtain satisfactoryresults.

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