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Control of Listeria monocytogenes by low-dose irradiation in combination with refrigeration in the soft whey cheese ‘Anthotyros’
Food Microbiology, 2002, 19, 117^126 Available online at http://www.idealibrary.com on
doi:10.1006/fmic.2001.0469
ORIGINAL ARTICLE
Control of Listeria monocytogenes by low-dose irradiation in combination with refrigeration in the soft whey cheese ‘Anthotyros’ A. Tsiotsias1, I. Savvaidis1, A.Vassila1, M. Kontominas1 and P. Kotzekidou2; *
Soft whey cheese‘Anthotyros’ has the following characteristics: moisture content 65%, protein content 9?6%, fat content 16?6%, salt concentration in the aqueous phase below 1% and pH 6?4. The average counts of the product were 4?54, 3?80, and 1?2 log cfu g 1 for aerobic mesophilic bacteria, yeasts and Enterobacteriaceae, respectively.The feasibility of gamma radiation for eliminating Listeria monocytogenes Scott A inoculated into the freshly produced product, and following its counts during refrigerated storage at 4 and 101C under vacuum packaging was investigated. Cheese samples were exposed to doses of 0?5, 2 and 4 kGy of gamma irradiation at 41C. Irradiation at 0?5 kGy slightly reduced the aerobic mesophilic bacteria counts, while irradiation doses at 2 and 4 kGy reduced the microbial load by approximately 1^2 log cycles. Enterobacteriaceae could not be detected in irradiated samples. Irradiation decreased the yeast population which was detected during later stages of storage. Molds were not detected in any of the samples. The calculated D10-value for L. monocytogenes was 1.38 kGy. Surviving microbial cells could be detected throughout the entire period of 42 days storage. Sensory scores indicated that irradiation doses up to 4 kGy do not adversely a¡ect ‘Anthotyros’ sensory properties. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction Listeria monocytogenes as an agent of foodborne disease has become of major concern in recent years to the food industry as it is one of the few foodborne pathogens that are capable of growth at refrigeration temperatures under anaerobic or microaerophilic conditions. The incidence of periodic outbreaks of foodborne listeriosis with considerable mortality is of particular concern in those products that may *Corresponding author. Fax: +30 -31- 099 -87-89; E-mail: [email protected] 0740 -0020/02/2^30117 +10 $35.00/0
be consumed without any heat treatment. This concern caused the US Food and Drug Administration to establish zero tolerance for L. monocytogenes in ready-to-eat foods (Thayer and Boyd 1995). L. monocytogenes has been shown to be capable of growing readily in milk (Rosenow and Marth 1987). Although the organism is destroyed by normal pasteurization procedures, pasteurized milk was probably the vehicle for a foodborne infection caused in Massachusetts (Fleming et al. 1985) as L. monocytogenes may survive pasteurization if the bacteria were located inside leukocytes (Doyle et al. 1987). r 2002 Elsevier Science Ltd. All rights reserved.
Received: 6 November 2001 1 Laboratory of Food Chemistry and Technology, Department of Chemistry, University of Ioannina, 45110 Greece 2 Laboratory of Food Microbiology and Hygiene, Department of Food Science and Technology, Faculty of Agriculture, Aristotle University of Thessaloniki, P.B. 250, 540 06 Thessaloniki, Greece
118 A. Tsiotsias et al.
Several cases of human listeriosis have been associated with the consumption of soft cheeses (Linnan et al. 1999, Bille 1990, McLaughlin et al. 1990, Morgan et al. 2001).The survival or growth of L. monocytogenes depends on the conditions during manufacture, packaging and storage of cheeses (Pitt et al. 1999).The main technological parameter that a¡ects the behavior of this microorganism in soft whey cheeses is the e¡ectiveness of the thermal processing for inactivation of L. monocytogenes present in milk at the time of cheesemaking. Also, Listeria can easily contaminate the ¢nished cheese product during packaging and/or storage. Cheeses with high moisture (50^70%), low acid (pH6) and low to moderate levels of salt in the moisture phase are of primary public health importance regarding Listeria as a post-processing contaminant (Genigeorgis et al. 1991). A possible alternative procedure for the destruction of harmful organisms in food products, with minimal e¡ect on their physical properties is irradiation using low-dose gamma radiation which is approved for a wide variety of food products (meat, fruits, spices, etc.). In 1980, both the US Food and Drug Administration and the World Health Organization of the United Nations accepted foods irradiated with an average dose of 10 kGy as neither presenting any toxicological hazard nor introducing any special nutritional or microbiological problems and thus being ‘safe for human consumption’ (Urbain 1989). Recently, doses greater than 10 kGy are considered safe by the World Health Organization, if they do not lead to changes in the composition, nutrient content and sensory properties of foods. The objective of the present study was to determine the feasibility of gamma radiation for controlling L. monocytogenes inoculated into the freshly produced soft whey cheese ‘Anthotyros’ and following its counts during refrigerated storage.
the technological scheme described in Fig. 1. Samples were transported to the laboratory in ice inside styrofoam boxes. Two hundred gram samples of cheese (dimensions: 8 6 1?5) were placed into ¢lm pouches composed of coextruded LDPE/PA/LDPE structures 70 mm in thickness having an O2 transmission rate of 67?64 cm3 m 2 day atm and a water vapor transmission rate of 1?85 g m 2 day measured using the Oxtran 2/20 and Permatran 3/31
Materials and Methods Samples Two batches of the soft whey cheese ‘Anthotyros’ were produced in Dodoni SA, a local dairy plant (Ioannina, Greece) according to
Figure 1. Technological scheme for the manufacture of the soft whey cheese ‘Anthotyros’.
Radiosensitivity of Listeria in soft whey cheese 119
permeability testers (MOCON, MN, USA). Ninety-eight of the total 112 samples from each batch were irradiated (14 uninoculated control samples and 84 samples inoculated with L. monocytogenes) while further 14 samples of unirradiated cheese were inoculated with L. monocytogenes. All samples were vacuum sealed (vacuum level: 1 bar) using a Weromatic, modelStar, vaccum sealer (Werner Bonk, Bochum, Germany) and divided into two groups, stored at 4 and 101C, respectively. The microbiological and physicochemical analysis of the cheese samples were carried out at 0, 7, 14, 21, 28, 35 and 42 days of storage.
being within 710% of the target dose. To minimize variations in radiation dose absorption, the boxes were turned 1801 halfway through the irradiation procedure. After irradiation, the iced samples were transported within 1 h to the laboratory, divided into two groups and maintained at 41C and 101C, respectively, until microbial analysis.
Chemical analyses Moisture, protein, fat, salt in the aqueous phase and pH values were determined according to AOAC procedures (AOAC 1995).
Microbiological analyses Inoculation L. monocytogenes Scott A was maintained on tryptic soy agar (TSA) at 41C and subcultured in trypticase soy broth (TSB) at 371C for 18 h to achieve 109 cfu ml 1 stationary phase cells for inoculum preparation. For con¢rmation of cell concentrations, samples were enumerated by spread plating serial dilutions onto TSA plates that were incubated at 371C for 24^48 h and counted according to standard procedures (APHA 1992). The inoculation was carried out using a Volac micropipetter (John Poulten Ltd., Barking, UK) distributing the inoculum (volume: 800 ml) dropwise on the cheese surface and gently massaging the sample so as to achieve a homogeneous distribution of the inoculum on the sample’s surface. The resulting population of L. monocytogenes was approximately 105 ^106 cfu g 1 of cheese sample. Following inoculation, samples were vacuum sealed in plastic pouches.
Irradiation Iced samples in styrofoam boxes were irradiated at the EL.VIO.NI plant using a Cobalt60 radiation source. The strength of the source was 150 kCi with a dose rate capacity of 1 kGy h 1 . Dosimetry was performed using a polymethyl methacrylate dosimeter (PMMA Instruments, Harwell, UK) placed on the cheese surface.The absorbance signal was measured using a Campesc M 201 UV spectrophotometer at 640 nm. The applied doses in this study were 0?5, 2 and 4 kGy with actual doses
To determine the microbiological quality of the control samples, two duplicate 20 g samples of cheese were taken aseptically, transferred to sterile plastic pouches and homogenized for 60 s with 180 ml of sterile diluent containing peptone (1 g l 1 ) and Tween 80 (1 ml l 1 ) using a Stomacher Lab-Blender 400 (Seward Medical, London, UK). Appropriate dilutions of the sample homogenates were prepared in sterile peptone water (0?1%) and inoculated in duplicate in growth media to estimate microbial counts. The aerobic plate count was determined on plate count agar (Merck) at 301C for 3 days, Enterobacteriaceae were determined on violet red bile dextrose agar (Merck) at 371C for 24 h and the counts of yeasts and molds were determined on potato dextrose agar (Merck) at 251C for 2^7 days according to APHA (1992). Testing of the control as well as the inoculated samples for L. monocytogenes was carried out according to the IDF procedure (IDF 1995b). For enrichment, a 25 -g sample was homogenized with 225 ml Listeria enrichment broth (according to IDF) and incubated at 301C for 24 and 48 h for revival of injured cells. The enrichment broth was subcultured by surface plating on Palcam agar (Merck) incubated at 371C for 24 and 48 h and then examined for typical colonies. Five typical colonies were subcultured on blood agar for further characterization as suggested by IDF. Direct counts of Listeria were performed by spreading 0?1 ml of the homogenized sample in Listeria enrichment broth, before incubation,
120 A. Tsiotsias et al.
onto the surface of Palcam agar (Merck). After incubation at 371C for 48 h, pre-sumptive Listeria colonies were counted and con¢rmed as above.
D10-value determination Radiation sensitivity of L. monocytogenes Scott A in inoculated soft whey cheese ‘Anthotyros’at 41C was investigated. The applied radiation absorbed doses ranged from 0 to 4 kGy. Postirradiation survivors were enumerated as described above. The percentage survival values were plotted against administered dose to calculate D10 -values. Irradiation D10 -values (1 log cycle inactivation) of the cells were determined from the linear portion of the survival curve using standard linear regression analysis (Urbain 1986).
Sensory analysis The sensory characteristics of uninoculated, irradiated and non-irradiated cheese samples were evaluated according to the IDF standards (IDF Std 99A, 1995a). A trained panel composed of seven judges, members of the Laboratory of Food Chemistry and Technology was used to evaluate cheeses for appearance (color, surface), texture (consistency, body) and £avor (taste, odor) using a 0^5 point scale (where a score of 5^4 corresponds to quality class I, 3?99^3?5 corresponds to quality class II and a score o3?5 corresponds to quality class III).
Results and Discussion Values for physico-chemical parameters of the unirradiated (day 0) and irradiated (day 42) soft whey cheese ‘Anthotyros’ are presented in Table 1. Moisture content ranged between 64?5 and 65?0%, fat ranged between 16?6 and 16?8%, protein ranged between 9?5 and 9?7%, pH ranged between 6?0 and 6?4 while percentage of salt in the aqueous phase did not exceed 1% during refrigerated storage for 42 days. The microbial counts of control and irradiated samples of Anthotyros at doses of 0?5, 2 and 4 kGy at 41C are given in Fig. 2. Molds were not detected in control as well as in irradiated samples during the refrigerated storage of the product at both 4 and 101C for 42 days under vacuum. The average initial counts of the product were 4?54, 3?80, and 1?2 log g 1 for aerobic mesophilic bacteria, yeasts and Enterobacteriaceae, respectively. Irradiation to 0?5 kGy slightly reduced the aerobic mesophilic bacteria counts, while irradiation doses of 2 and 4 kGy reduced the respective microbial load by approximately 1 and 2 log cycles. Observing the micro£ora of the irradiated samples, it can be concluded that low-dose irradiation, like any other non-sterilizing treatment, has a selective e¡ect on the natural micro£ora of the soft whey cheese ‘Anthotyros’. The problems of the surviving micro£ora vary according to the nature of the food and its associated microorganisms (Farkas 1989). The more resistant species surviving low radiation doses are lactic
Figure 2. E¡ect of irradiation on (a) aerobic mesophilic bacteria; (b) yeasts; and (c) Enterobacteriaceae during refrigerated storage under vacuum of the soft whey cheese ‘Anthotyros’ at 41C. Control, (*); 0?5 kGy, (&); 2 kGy, (~); 4 kGy, (^).
Radiosensitivity of Listeria in soft whey cheese 121
acid bacteria (especially fecal streptococci) and yeasts (ICMSF 1980). The predominant micro£ora of Anthotyros consists of lactic acid bacteria and Enterococcus faecalis as reported by Kalogridou-Vassiliadou et al. (1994). Thus, log counts of 2?25 cfu g 1 of the aerobic mesophilic microorganisms in samples irradiated at doses of 4 kGy could be determined. Enterobacteriaceae have low resistance to irradiation (Farkas 1989), so they could not be detected in irradiated samples. On the other hand, low-dose irradiation allows survival of aerobic mesophilic bacteria and yeasts. Radiation damage of microbial cells is due to scission of single or double strands of DNA, which essentially is caused by the free radicals formed in the suspending food medium and is in£uenced by food composition (Diehl 1995). Anthotyros is a soft whey cheese containing approximately 65% moisture and 0?6% salt, as indicated in Table 1. Under the above conditions, a great percentage of the typical micro£ora of the product consisting of mesophilic aerobic bacteria could survive low-dose irradiation (o4 kGy). L. monocytogenes was not detected in any of the control cheese samples examined due to the thermal treatment of milk and the inhibition of possible contamination via the production of antagonistic components by the lactic acid bacteria occurring in high percentage in cheese micro£ora (Pitt et al. 1999). Fig. 3 depicts the radiation survival curve of L. monocytogenes Scott A in the soft whey cheese Anthotyros at 41C under vacuum packaging, after irradiation. L. monocytogenes showed an exponential pattern of survival.
The D10 -value determined on the ¢rst day post-irradiation was 1?38 kGy. This value is signi¢cantly higher than values between 0?4 and 0?8 kGy determined for a variety of foodstu¡s at temperatures between 0 and 251C (Farag et al. 1990). The above di¡erences in D10 values may be due to di¡erences in composition (i.e. fat content) of foodstu¡s studied. On the other hand, the present D10 -value is in good agreement with a D10 -value of 1?4 kGy for L. monocytogenes Scott A in mozzarella cheese at 781C reported by Hashisaka et al. (1989) even though in the frozen state, D10 -values are expected to be signi¢cantly higher than at refrigeration temperatures. The radiosensitivity of bacteria varies with the medium in which irradiation occurs. Optimum conditions for irradiation are provided by media of high water activity (Aw 40?95) and lack of competitive chemical or radiochemical activity from solid particles (Urbain 1989). As Anthotyros contains
Figure 3. Radiation sensitivity of inoculated Listeria monocytogenes Scott A at 41C under vacuum packaging in soft whey cheese ‘Anthotyros’.
Table 1. Physico-chemical parameter values of the control and irradiated samples of the soft whey cheese ‘Anthotyros’ Dose/time
Control (M) Irradiation at 0?5 kGy Irradiation at 2 kGy Irradiation at 4 kGy
Moisture (%)
Fat (%)
Protein (%)
NaCl (%)
pH
Day 0
Day 42
Day 0
Day 42
Day 0
Day 42
Day 0
Day 42
Day 0
Day 42
65?0* 65?0 64?7 64?5
66?5 64?0 63?5 62?8
16?6 16?6 16?7 16?6
16?5 16?6 16?5 16?5
9?6 9?6 9?5 9?6
9?7 9?5 9?6 9?7
0?6 0?6 0?6 0?6
0?5 0?6 0?5 0?5
6?4 6?0 6?1 6?1
6?3 6?2 6?2 6?2
*Each value is the mean of four measurements corresponding to two batches of cheese (n = 2 2).
122 A. Tsiotsias et al.
approximately 17% fat and 10% protein, the comparatively high D10 -value may be explained by a double protective e¡ect of the complex protein and fat matrix of the product in combination with low temperatures as reported by Andrews et al. (1995). The relative irradiation resistance of Listeria may also be due to the increased cell wall peptidoglycan content and the ability to produce catalase and superoxide dismutase, which may readily block toxic radiolytic products (H2O2 and OH? ) responsible for most of the indirect e¡ects of ionization (Urbain 1986). Irradiated food product D10 -values have proven to be higher than those irradiated in liquid culture media (Huhtanen et al. 1989). Of special interest concerning inactivation of food pathogens by irradiation is the cellular damage which occurs via both the direct action of photons on cellular target sites and the indirect actions of free radicals and other radiolytic products to a di¡usion site (Silverman 1983), as well as damage to the membrane and other structures causing sub-lethal injury (Diehl 1995). This injury is characterized by decreased resistance to selective agents or by increased nutritional requirements (ICMSF 1980). As indicated in Table 2, log counts of injured cells (injured cells represent the di¡erence between the populations of cells able to form colonies after enrichment procedure for 24 h and those determined after direct plating on selective medium) decrease and the extent of lethality increases during refrigerated storage at both 4 and 101C for 42 days. Cells
Table 2. E¡ect of irradiation dose (0?5 and 2 kGy) on sublethal injury of Listeria monocytogenes Scott A cells during refrigerated storage under vacuum packaging for 42 days of the soft whey cheese ‘Anthotyros’at 4 and 101C, respectively L. monocytogenes after 24 henrichment (log cfu g 1 )
Irradiation at 0?5 kGy Irradiation at 2 kGy
L. monocytogenes without enrichment (log cfu g 1 )
41C
101C
41C
101C
7?65
5?83
4?32
3?52
5?96
4?30
3?30
3?12
subjected to a mild stress (radiation dose of 0?5 kGy) may represent injury that is easily repaired. Injury is important to food safety. If injured cells are mistakenly classi¢ed as dead during the determination of radiation sensitivity of L. monocytogenes, the resultant D10 -values will be errantly low. Injured cells may repair prior to food consumption especially in environments excluding oxygen as in vacuum-packaged foods and temperature abuse. The e¡ect of irradiation on inoculated L. monocytogenes Scott A in the soft whey cheese Anthotyros during refrigerated storage under vacuum packaging at 4 and 101C is presented in Fig. 4. Following the counts of inoculated L. monocytogenes in unirradiated control samples, an increase of about 3 log counts g 1 could be observed under the above packaging and storage conditions. The pH-values during the storage of Anthotyros cheese as indicated in Table 1 are optimum for the growth of the inoculated L. monocytogenes cells. In a study concerning the fate of L. monocytogenes as a post-processing contaminant in Anthotyros cheese, approximately 500 cfu g 1 L. monocytogenes Scott A or CA were inoculated and the pathogen grew rapidly and attained maximum populations of 107 ^108 cfu g 1 after 24^30 days and 5^ 12 days of storage at 5 and 121C, respectively, as reported by Papageorgiou et al. (1996).The high susceptibility of L. monocytogenes to irradiation o¡ers good possibilities for its control in foods by low-dose irradiation, if the contamination counts are low. The surviving cells can, however, substantially increase in number during refrigerated storage under vacuum packaging as indicated in Fig. 4. Sensory scores for unirradiated and irradiated cheese samples at 41C and 101C are given in Figure 5 (A^I). The attributes, appearance, texture, odor and taste as well as overall acceptance were evaluated. Regarding the cheese appearance at 41C (Fig. 5(a)) two observations can be made: (1) at the higher doses (2 and 4 kGy), cheese color turned bright white while unirradiated and cheese samples irradiated at 0?5 kGy retained their original o¡-white color and (2) at the higher doses, a small amount of moisture was expelled from the cheese body being localized on its surface. Appearance
Radiosensitivity of Listeria in soft whey cheese 123
Figure 4. E¡ect of irradiation on inoculated Listeria monocytogenes Scott A in the soft whey cheese ‘Anthotyros’ during refrigerated storage under vacuum packaging (a) at 41C; and (b) at 101C. unirradiated, (*); 0?5 kGy, (&); 2 kGy (~); 4 kGy, (^).
scores for all samples were above 3?5, during the ¢rst 28 days of storage corresponding to classes I and II products. At the same temperature (41C), the texture of all the samples (Fig. 5(b)) irradiated and non-irradiated was similar. Odor and taste (Figs. 5(c) and (d)) were negatively a¡ected during the ¢rst 3^4 days after irradiation (foreign odor and taste) whose e¡ect, however, was practically eliminated by day 7 of storage. The same trend in o¡-taste development was reported for irradiated trout by Ehlermann and Munzner (1972). In contrast, Ennahar et al. (1994) reported development of o¡ £avors and color changes in soft and red smear cheeses irradiated at a dose 2 kGy using X-rays. Between day 17 and day 25 of storage, all irradiated cheese samples scored higher (3?5) an the non-irradiated samples. As shown in Fig. 5(e), the overall acceptance score was higher for the control as compared to the irradiated samples during the ¢rst 4 days of storage. Between day 4 and day 21 of storage, all samples were of comparable quality while between day 21 and day 28 of storage the sample irradiated at 4 kGy scored higher than the rest of the three samples. Between day 28 and day 35 of storage, the sample irradiated at 4 kGy received a score of approximately 3?5 while all other samples received a score below 3?5. If we consider a score of 3?5 as corresponding to the
end of the product’s shelf-life, it can be stated that samples irradiated at doses of 0?5 and 2 kGy achieve a shelf-life of 28 days, while samples irradiated at a dose of 4 kGy achieve a shelf-life of 35 days (41C). Regarding the cheese appearance at 101C (Fig. 5(f)), besides the remarks made for cheeses tested at 41C an additional observation is that all irradiated cheese samples developed a pink color by day 14 of storage, which persisted throughout the experiment. Texture (Fig. 5(g)) was not signi¢cantly affected after irradiation. Odor and taste (Figs. 5(h) and (i)) were negatively a¡ected during the ¢rst 2 days of storage after irradiation (foreign odor and taste) whose e¡ect, however, was eliminated by day 4 of storage. By day 7 of storage, the control sample had an odor (yeasty) and taste (slightly sour) score in the vicinity of 3 while all irradiated samples scored between 4 and 4?5 being characterized by a creamy odor and a foreign taste. After day 14 of storage, all samples received odor and taste scores below 3?5. As shown in Fig. 5(j) the overall acceptance score (101C) was higher for the cheese sample irradiated at 0.5 kGy as compared to all the rest during the ¢rst 7 days of storage. At day 14 of storage, all samples received approximately the same score (between 3?5 and 4), while beyond day 14 of storage all samples received an overall acceptance score below 3?5.
124 A. Tsiotsias et al.
Figure 5. E¡ect of irradiation on sensory properties of ‘Anthotyros cheese’. (a) appearance, (b) texture, (c) odor, (d) taste, (e) overall acceptance at 41C. (f) appearance, (g) texture, (h) odor, (i) taste, and (j) overall acceptance at 101C. unirradiated, (*); 0?5 kGy, (&); 2 kGy, (~); 4 kGy, (^).
Radiosensitivity of Listeria in soft whey cheese 125
Conclusions Although L. monocytogenes is completely inactivated during manufacture of the soft whey cheese Anthotyros, the potential still exists for contamination after thermal processing as well as during packaging. The results demonstrate that if the organism contaminates the product and/or survives after irradiation treatment, it may be detected throughout the entire period of storage. In addition, due to the fact that L. monocytogenes is psychrotrophic and can grow at refrigeration temperatures, contamination by this organism could lead to a high risk factor. Irradiation of refrigerated packaged cheese at doses up to 4 kGy may be used for the control of L. monocytogenes in the ready-to-eat product, with no adverse e¡ects (excluding the ¢rst 4 days of storage of ‘Anthotyros’ cheese for a storage period of 28 days.
References Andrews, L. S., Marshall, D. L. and Grodner, R. M. (1995) Radiosensitivity of Listeria monocytogenes at various temperatures and cell concentrations. J. Food Prot. 58, 748^751. AOAC (1995) AOAC O⁄cial Methods 16th edn. Methods 948.12, 935.43, 971.19, 955.30, 933.05. APHA (1992) Compendium of Methods for the Microbiological Examination of Foods, 3rd edn.Washington, American Public Health Association. Bille, J. (1990) Epidemiology of human listeriosis in Europe, with special reference to the Swiss outbreak. In Foodborne Listeriosis, (Eds A. L. Miller, J. L. Smith, and G. A. Somkouti) pp. 71^74. Amsterdam, Elsevier. Diehl, J.F. (1995) Safety of Irradiated Foods. NewYork, Marcel Dekker. Doyle, M. P., Glass, K. A., Beery, J. T., Garcia, G. A., Pollard, D. J. and Schultz, R. D. (1987) Survival of Listeria monocytogenes in milk during high-temperature, short-time pasteurisation. Appl. Environ. Microbiol. 53, 1433^1438. Ehlermann, D. and Munzner, R. (1972) Radurization of fresh-water ¢sh in the Federal Republic of Germany, In Preservation of Fish by Irradiation pp. 65^ 74. International Atomic Energy Agency,Vienna. Ennahar, S., Kuntz, F., Strasser, A., Bergaentzle, M., Hasselmann, C. and Stahl, V. (1994) Elimination of Listeria monocytogenes in soft and red smear cheeses by irradiation with low energy electrons. Int. J. Food Sci. Technol. 29, 395^403.
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doi:10.1006/fmic.2001.0469
ORIGINAL ARTICLE
Control of Listeria monocytogenes by low-dose irradiation in combination with refrigeration in the soft whey cheese ‘Anthotyros’ A. Tsiotsias1, I. Savvaidis1, A.Vassila1, M. Kontominas1 and P. Kotzekidou2; *
Soft whey cheese‘Anthotyros’ has the following characteristics: moisture content 65%, protein content 9?6%, fat content 16?6%, salt concentration in the aqueous phase below 1% and pH 6?4. The average counts of the product were 4?54, 3?80, and 1?2 log cfu g 1 for aerobic mesophilic bacteria, yeasts and Enterobacteriaceae, respectively.The feasibility of gamma radiation for eliminating Listeria monocytogenes Scott A inoculated into the freshly produced product, and following its counts during refrigerated storage at 4 and 101C under vacuum packaging was investigated. Cheese samples were exposed to doses of 0?5, 2 and 4 kGy of gamma irradiation at 41C. Irradiation at 0?5 kGy slightly reduced the aerobic mesophilic bacteria counts, while irradiation doses at 2 and 4 kGy reduced the microbial load by approximately 1^2 log cycles. Enterobacteriaceae could not be detected in irradiated samples. Irradiation decreased the yeast population which was detected during later stages of storage. Molds were not detected in any of the samples. The calculated D10-value for L. monocytogenes was 1.38 kGy. Surviving microbial cells could be detected throughout the entire period of 42 days storage. Sensory scores indicated that irradiation doses up to 4 kGy do not adversely a¡ect ‘Anthotyros’ sensory properties. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction Listeria monocytogenes as an agent of foodborne disease has become of major concern in recent years to the food industry as it is one of the few foodborne pathogens that are capable of growth at refrigeration temperatures under anaerobic or microaerophilic conditions. The incidence of periodic outbreaks of foodborne listeriosis with considerable mortality is of particular concern in those products that may *Corresponding author. Fax: +30 -31- 099 -87-89; E-mail: [email protected] 0740 -0020/02/2^30117 +10 $35.00/0
be consumed without any heat treatment. This concern caused the US Food and Drug Administration to establish zero tolerance for L. monocytogenes in ready-to-eat foods (Thayer and Boyd 1995). L. monocytogenes has been shown to be capable of growing readily in milk (Rosenow and Marth 1987). Although the organism is destroyed by normal pasteurization procedures, pasteurized milk was probably the vehicle for a foodborne infection caused in Massachusetts (Fleming et al. 1985) as L. monocytogenes may survive pasteurization if the bacteria were located inside leukocytes (Doyle et al. 1987). r 2002 Elsevier Science Ltd. All rights reserved.
Received: 6 November 2001 1 Laboratory of Food Chemistry and Technology, Department of Chemistry, University of Ioannina, 45110 Greece 2 Laboratory of Food Microbiology and Hygiene, Department of Food Science and Technology, Faculty of Agriculture, Aristotle University of Thessaloniki, P.B. 250, 540 06 Thessaloniki, Greece
118 A. Tsiotsias et al.
Several cases of human listeriosis have been associated with the consumption of soft cheeses (Linnan et al. 1999, Bille 1990, McLaughlin et al. 1990, Morgan et al. 2001).The survival or growth of L. monocytogenes depends on the conditions during manufacture, packaging and storage of cheeses (Pitt et al. 1999).The main technological parameter that a¡ects the behavior of this microorganism in soft whey cheeses is the e¡ectiveness of the thermal processing for inactivation of L. monocytogenes present in milk at the time of cheesemaking. Also, Listeria can easily contaminate the ¢nished cheese product during packaging and/or storage. Cheeses with high moisture (50^70%), low acid (pH6) and low to moderate levels of salt in the moisture phase are of primary public health importance regarding Listeria as a post-processing contaminant (Genigeorgis et al. 1991). A possible alternative procedure for the destruction of harmful organisms in food products, with minimal e¡ect on their physical properties is irradiation using low-dose gamma radiation which is approved for a wide variety of food products (meat, fruits, spices, etc.). In 1980, both the US Food and Drug Administration and the World Health Organization of the United Nations accepted foods irradiated with an average dose of 10 kGy as neither presenting any toxicological hazard nor introducing any special nutritional or microbiological problems and thus being ‘safe for human consumption’ (Urbain 1989). Recently, doses greater than 10 kGy are considered safe by the World Health Organization, if they do not lead to changes in the composition, nutrient content and sensory properties of foods. The objective of the present study was to determine the feasibility of gamma radiation for controlling L. monocytogenes inoculated into the freshly produced soft whey cheese ‘Anthotyros’ and following its counts during refrigerated storage.
the technological scheme described in Fig. 1. Samples were transported to the laboratory in ice inside styrofoam boxes. Two hundred gram samples of cheese (dimensions: 8 6 1?5) were placed into ¢lm pouches composed of coextruded LDPE/PA/LDPE structures 70 mm in thickness having an O2 transmission rate of 67?64 cm3 m 2 day atm and a water vapor transmission rate of 1?85 g m 2 day measured using the Oxtran 2/20 and Permatran 3/31
Materials and Methods Samples Two batches of the soft whey cheese ‘Anthotyros’ were produced in Dodoni SA, a local dairy plant (Ioannina, Greece) according to
Figure 1. Technological scheme for the manufacture of the soft whey cheese ‘Anthotyros’.
Radiosensitivity of Listeria in soft whey cheese 119
permeability testers (MOCON, MN, USA). Ninety-eight of the total 112 samples from each batch were irradiated (14 uninoculated control samples and 84 samples inoculated with L. monocytogenes) while further 14 samples of unirradiated cheese were inoculated with L. monocytogenes. All samples were vacuum sealed (vacuum level: 1 bar) using a Weromatic, modelStar, vaccum sealer (Werner Bonk, Bochum, Germany) and divided into two groups, stored at 4 and 101C, respectively. The microbiological and physicochemical analysis of the cheese samples were carried out at 0, 7, 14, 21, 28, 35 and 42 days of storage.
being within 710% of the target dose. To minimize variations in radiation dose absorption, the boxes were turned 1801 halfway through the irradiation procedure. After irradiation, the iced samples were transported within 1 h to the laboratory, divided into two groups and maintained at 41C and 101C, respectively, until microbial analysis.
Chemical analyses Moisture, protein, fat, salt in the aqueous phase and pH values were determined according to AOAC procedures (AOAC 1995).
Microbiological analyses Inoculation L. monocytogenes Scott A was maintained on tryptic soy agar (TSA) at 41C and subcultured in trypticase soy broth (TSB) at 371C for 18 h to achieve 109 cfu ml 1 stationary phase cells for inoculum preparation. For con¢rmation of cell concentrations, samples were enumerated by spread plating serial dilutions onto TSA plates that were incubated at 371C for 24^48 h and counted according to standard procedures (APHA 1992). The inoculation was carried out using a Volac micropipetter (John Poulten Ltd., Barking, UK) distributing the inoculum (volume: 800 ml) dropwise on the cheese surface and gently massaging the sample so as to achieve a homogeneous distribution of the inoculum on the sample’s surface. The resulting population of L. monocytogenes was approximately 105 ^106 cfu g 1 of cheese sample. Following inoculation, samples were vacuum sealed in plastic pouches.
Irradiation Iced samples in styrofoam boxes were irradiated at the EL.VIO.NI plant using a Cobalt60 radiation source. The strength of the source was 150 kCi with a dose rate capacity of 1 kGy h 1 . Dosimetry was performed using a polymethyl methacrylate dosimeter (PMMA Instruments, Harwell, UK) placed on the cheese surface.The absorbance signal was measured using a Campesc M 201 UV spectrophotometer at 640 nm. The applied doses in this study were 0?5, 2 and 4 kGy with actual doses
To determine the microbiological quality of the control samples, two duplicate 20 g samples of cheese were taken aseptically, transferred to sterile plastic pouches and homogenized for 60 s with 180 ml of sterile diluent containing peptone (1 g l 1 ) and Tween 80 (1 ml l 1 ) using a Stomacher Lab-Blender 400 (Seward Medical, London, UK). Appropriate dilutions of the sample homogenates were prepared in sterile peptone water (0?1%) and inoculated in duplicate in growth media to estimate microbial counts. The aerobic plate count was determined on plate count agar (Merck) at 301C for 3 days, Enterobacteriaceae were determined on violet red bile dextrose agar (Merck) at 371C for 24 h and the counts of yeasts and molds were determined on potato dextrose agar (Merck) at 251C for 2^7 days according to APHA (1992). Testing of the control as well as the inoculated samples for L. monocytogenes was carried out according to the IDF procedure (IDF 1995b). For enrichment, a 25 -g sample was homogenized with 225 ml Listeria enrichment broth (according to IDF) and incubated at 301C for 24 and 48 h for revival of injured cells. The enrichment broth was subcultured by surface plating on Palcam agar (Merck) incubated at 371C for 24 and 48 h and then examined for typical colonies. Five typical colonies were subcultured on blood agar for further characterization as suggested by IDF. Direct counts of Listeria were performed by spreading 0?1 ml of the homogenized sample in Listeria enrichment broth, before incubation,
120 A. Tsiotsias et al.
onto the surface of Palcam agar (Merck). After incubation at 371C for 48 h, pre-sumptive Listeria colonies were counted and con¢rmed as above.
D10-value determination Radiation sensitivity of L. monocytogenes Scott A in inoculated soft whey cheese ‘Anthotyros’at 41C was investigated. The applied radiation absorbed doses ranged from 0 to 4 kGy. Postirradiation survivors were enumerated as described above. The percentage survival values were plotted against administered dose to calculate D10 -values. Irradiation D10 -values (1 log cycle inactivation) of the cells were determined from the linear portion of the survival curve using standard linear regression analysis (Urbain 1986).
Sensory analysis The sensory characteristics of uninoculated, irradiated and non-irradiated cheese samples were evaluated according to the IDF standards (IDF Std 99A, 1995a). A trained panel composed of seven judges, members of the Laboratory of Food Chemistry and Technology was used to evaluate cheeses for appearance (color, surface), texture (consistency, body) and £avor (taste, odor) using a 0^5 point scale (where a score of 5^4 corresponds to quality class I, 3?99^3?5 corresponds to quality class II and a score o3?5 corresponds to quality class III).
Results and Discussion Values for physico-chemical parameters of the unirradiated (day 0) and irradiated (day 42) soft whey cheese ‘Anthotyros’ are presented in Table 1. Moisture content ranged between 64?5 and 65?0%, fat ranged between 16?6 and 16?8%, protein ranged between 9?5 and 9?7%, pH ranged between 6?0 and 6?4 while percentage of salt in the aqueous phase did not exceed 1% during refrigerated storage for 42 days. The microbial counts of control and irradiated samples of Anthotyros at doses of 0?5, 2 and 4 kGy at 41C are given in Fig. 2. Molds were not detected in control as well as in irradiated samples during the refrigerated storage of the product at both 4 and 101C for 42 days under vacuum. The average initial counts of the product were 4?54, 3?80, and 1?2 log g 1 for aerobic mesophilic bacteria, yeasts and Enterobacteriaceae, respectively. Irradiation to 0?5 kGy slightly reduced the aerobic mesophilic bacteria counts, while irradiation doses of 2 and 4 kGy reduced the respective microbial load by approximately 1 and 2 log cycles. Observing the micro£ora of the irradiated samples, it can be concluded that low-dose irradiation, like any other non-sterilizing treatment, has a selective e¡ect on the natural micro£ora of the soft whey cheese ‘Anthotyros’. The problems of the surviving micro£ora vary according to the nature of the food and its associated microorganisms (Farkas 1989). The more resistant species surviving low radiation doses are lactic
Figure 2. E¡ect of irradiation on (a) aerobic mesophilic bacteria; (b) yeasts; and (c) Enterobacteriaceae during refrigerated storage under vacuum of the soft whey cheese ‘Anthotyros’ at 41C. Control, (*); 0?5 kGy, (&); 2 kGy, (~); 4 kGy, (^).
Radiosensitivity of Listeria in soft whey cheese 121
acid bacteria (especially fecal streptococci) and yeasts (ICMSF 1980). The predominant micro£ora of Anthotyros consists of lactic acid bacteria and Enterococcus faecalis as reported by Kalogridou-Vassiliadou et al. (1994). Thus, log counts of 2?25 cfu g 1 of the aerobic mesophilic microorganisms in samples irradiated at doses of 4 kGy could be determined. Enterobacteriaceae have low resistance to irradiation (Farkas 1989), so they could not be detected in irradiated samples. On the other hand, low-dose irradiation allows survival of aerobic mesophilic bacteria and yeasts. Radiation damage of microbial cells is due to scission of single or double strands of DNA, which essentially is caused by the free radicals formed in the suspending food medium and is in£uenced by food composition (Diehl 1995). Anthotyros is a soft whey cheese containing approximately 65% moisture and 0?6% salt, as indicated in Table 1. Under the above conditions, a great percentage of the typical micro£ora of the product consisting of mesophilic aerobic bacteria could survive low-dose irradiation (o4 kGy). L. monocytogenes was not detected in any of the control cheese samples examined due to the thermal treatment of milk and the inhibition of possible contamination via the production of antagonistic components by the lactic acid bacteria occurring in high percentage in cheese micro£ora (Pitt et al. 1999). Fig. 3 depicts the radiation survival curve of L. monocytogenes Scott A in the soft whey cheese Anthotyros at 41C under vacuum packaging, after irradiation. L. monocytogenes showed an exponential pattern of survival.
The D10 -value determined on the ¢rst day post-irradiation was 1?38 kGy. This value is signi¢cantly higher than values between 0?4 and 0?8 kGy determined for a variety of foodstu¡s at temperatures between 0 and 251C (Farag et al. 1990). The above di¡erences in D10 values may be due to di¡erences in composition (i.e. fat content) of foodstu¡s studied. On the other hand, the present D10 -value is in good agreement with a D10 -value of 1?4 kGy for L. monocytogenes Scott A in mozzarella cheese at 781C reported by Hashisaka et al. (1989) even though in the frozen state, D10 -values are expected to be signi¢cantly higher than at refrigeration temperatures. The radiosensitivity of bacteria varies with the medium in which irradiation occurs. Optimum conditions for irradiation are provided by media of high water activity (Aw 40?95) and lack of competitive chemical or radiochemical activity from solid particles (Urbain 1989). As Anthotyros contains
Figure 3. Radiation sensitivity of inoculated Listeria monocytogenes Scott A at 41C under vacuum packaging in soft whey cheese ‘Anthotyros’.
Table 1. Physico-chemical parameter values of the control and irradiated samples of the soft whey cheese ‘Anthotyros’ Dose/time
Control (M) Irradiation at 0?5 kGy Irradiation at 2 kGy Irradiation at 4 kGy
Moisture (%)
Fat (%)
Protein (%)
NaCl (%)
pH
Day 0
Day 42
Day 0
Day 42
Day 0
Day 42
Day 0
Day 42
Day 0
Day 42
65?0* 65?0 64?7 64?5
66?5 64?0 63?5 62?8
16?6 16?6 16?7 16?6
16?5 16?6 16?5 16?5
9?6 9?6 9?5 9?6
9?7 9?5 9?6 9?7
0?6 0?6 0?6 0?6
0?5 0?6 0?5 0?5
6?4 6?0 6?1 6?1
6?3 6?2 6?2 6?2
*Each value is the mean of four measurements corresponding to two batches of cheese (n = 2 2).
122 A. Tsiotsias et al.
approximately 17% fat and 10% protein, the comparatively high D10 -value may be explained by a double protective e¡ect of the complex protein and fat matrix of the product in combination with low temperatures as reported by Andrews et al. (1995). The relative irradiation resistance of Listeria may also be due to the increased cell wall peptidoglycan content and the ability to produce catalase and superoxide dismutase, which may readily block toxic radiolytic products (H2O2 and OH? ) responsible for most of the indirect e¡ects of ionization (Urbain 1986). Irradiated food product D10 -values have proven to be higher than those irradiated in liquid culture media (Huhtanen et al. 1989). Of special interest concerning inactivation of food pathogens by irradiation is the cellular damage which occurs via both the direct action of photons on cellular target sites and the indirect actions of free radicals and other radiolytic products to a di¡usion site (Silverman 1983), as well as damage to the membrane and other structures causing sub-lethal injury (Diehl 1995). This injury is characterized by decreased resistance to selective agents or by increased nutritional requirements (ICMSF 1980). As indicated in Table 2, log counts of injured cells (injured cells represent the di¡erence between the populations of cells able to form colonies after enrichment procedure for 24 h and those determined after direct plating on selective medium) decrease and the extent of lethality increases during refrigerated storage at both 4 and 101C for 42 days. Cells
Table 2. E¡ect of irradiation dose (0?5 and 2 kGy) on sublethal injury of Listeria monocytogenes Scott A cells during refrigerated storage under vacuum packaging for 42 days of the soft whey cheese ‘Anthotyros’at 4 and 101C, respectively L. monocytogenes after 24 henrichment (log cfu g 1 )
Irradiation at 0?5 kGy Irradiation at 2 kGy
L. monocytogenes without enrichment (log cfu g 1 )
41C
101C
41C
101C
7?65
5?83
4?32
3?52
5?96
4?30
3?30
3?12
subjected to a mild stress (radiation dose of 0?5 kGy) may represent injury that is easily repaired. Injury is important to food safety. If injured cells are mistakenly classi¢ed as dead during the determination of radiation sensitivity of L. monocytogenes, the resultant D10 -values will be errantly low. Injured cells may repair prior to food consumption especially in environments excluding oxygen as in vacuum-packaged foods and temperature abuse. The e¡ect of irradiation on inoculated L. monocytogenes Scott A in the soft whey cheese Anthotyros during refrigerated storage under vacuum packaging at 4 and 101C is presented in Fig. 4. Following the counts of inoculated L. monocytogenes in unirradiated control samples, an increase of about 3 log counts g 1 could be observed under the above packaging and storage conditions. The pH-values during the storage of Anthotyros cheese as indicated in Table 1 are optimum for the growth of the inoculated L. monocytogenes cells. In a study concerning the fate of L. monocytogenes as a post-processing contaminant in Anthotyros cheese, approximately 500 cfu g 1 L. monocytogenes Scott A or CA were inoculated and the pathogen grew rapidly and attained maximum populations of 107 ^108 cfu g 1 after 24^30 days and 5^ 12 days of storage at 5 and 121C, respectively, as reported by Papageorgiou et al. (1996).The high susceptibility of L. monocytogenes to irradiation o¡ers good possibilities for its control in foods by low-dose irradiation, if the contamination counts are low. The surviving cells can, however, substantially increase in number during refrigerated storage under vacuum packaging as indicated in Fig. 4. Sensory scores for unirradiated and irradiated cheese samples at 41C and 101C are given in Figure 5 (A^I). The attributes, appearance, texture, odor and taste as well as overall acceptance were evaluated. Regarding the cheese appearance at 41C (Fig. 5(a)) two observations can be made: (1) at the higher doses (2 and 4 kGy), cheese color turned bright white while unirradiated and cheese samples irradiated at 0?5 kGy retained their original o¡-white color and (2) at the higher doses, a small amount of moisture was expelled from the cheese body being localized on its surface. Appearance
Radiosensitivity of Listeria in soft whey cheese 123
Figure 4. E¡ect of irradiation on inoculated Listeria monocytogenes Scott A in the soft whey cheese ‘Anthotyros’ during refrigerated storage under vacuum packaging (a) at 41C; and (b) at 101C. unirradiated, (*); 0?5 kGy, (&); 2 kGy (~); 4 kGy, (^).
scores for all samples were above 3?5, during the ¢rst 28 days of storage corresponding to classes I and II products. At the same temperature (41C), the texture of all the samples (Fig. 5(b)) irradiated and non-irradiated was similar. Odor and taste (Figs. 5(c) and (d)) were negatively a¡ected during the ¢rst 3^4 days after irradiation (foreign odor and taste) whose e¡ect, however, was practically eliminated by day 7 of storage. The same trend in o¡-taste development was reported for irradiated trout by Ehlermann and Munzner (1972). In contrast, Ennahar et al. (1994) reported development of o¡ £avors and color changes in soft and red smear cheeses irradiated at a dose 2 kGy using X-rays. Between day 17 and day 25 of storage, all irradiated cheese samples scored higher (3?5) an the non-irradiated samples. As shown in Fig. 5(e), the overall acceptance score was higher for the control as compared to the irradiated samples during the ¢rst 4 days of storage. Between day 4 and day 21 of storage, all samples were of comparable quality while between day 21 and day 28 of storage the sample irradiated at 4 kGy scored higher than the rest of the three samples. Between day 28 and day 35 of storage, the sample irradiated at 4 kGy received a score of approximately 3?5 while all other samples received a score below 3?5. If we consider a score of 3?5 as corresponding to the
end of the product’s shelf-life, it can be stated that samples irradiated at doses of 0?5 and 2 kGy achieve a shelf-life of 28 days, while samples irradiated at a dose of 4 kGy achieve a shelf-life of 35 days (41C). Regarding the cheese appearance at 101C (Fig. 5(f)), besides the remarks made for cheeses tested at 41C an additional observation is that all irradiated cheese samples developed a pink color by day 14 of storage, which persisted throughout the experiment. Texture (Fig. 5(g)) was not signi¢cantly affected after irradiation. Odor and taste (Figs. 5(h) and (i)) were negatively a¡ected during the ¢rst 2 days of storage after irradiation (foreign odor and taste) whose e¡ect, however, was eliminated by day 4 of storage. By day 7 of storage, the control sample had an odor (yeasty) and taste (slightly sour) score in the vicinity of 3 while all irradiated samples scored between 4 and 4?5 being characterized by a creamy odor and a foreign taste. After day 14 of storage, all samples received odor and taste scores below 3?5. As shown in Fig. 5(j) the overall acceptance score (101C) was higher for the cheese sample irradiated at 0.5 kGy as compared to all the rest during the ¢rst 7 days of storage. At day 14 of storage, all samples received approximately the same score (between 3?5 and 4), while beyond day 14 of storage all samples received an overall acceptance score below 3?5.
124 A. Tsiotsias et al.
Figure 5. E¡ect of irradiation on sensory properties of ‘Anthotyros cheese’. (a) appearance, (b) texture, (c) odor, (d) taste, (e) overall acceptance at 41C. (f) appearance, (g) texture, (h) odor, (i) taste, and (j) overall acceptance at 101C. unirradiated, (*); 0?5 kGy, (&); 2 kGy, (~); 4 kGy, (^).
Radiosensitivity of Listeria in soft whey cheese 125
Conclusions Although L. monocytogenes is completely inactivated during manufacture of the soft whey cheese Anthotyros, the potential still exists for contamination after thermal processing as well as during packaging. The results demonstrate that if the organism contaminates the product and/or survives after irradiation treatment, it may be detected throughout the entire period of storage. In addition, due to the fact that L. monocytogenes is psychrotrophic and can grow at refrigeration temperatures, contamination by this organism could lead to a high risk factor. Irradiation of refrigerated packaged cheese at doses up to 4 kGy may be used for the control of L. monocytogenes in the ready-to-eat product, with no adverse e¡ects (excluding the ¢rst 4 days of storage of ‘Anthotyros’ cheese for a storage period of 28 days.
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