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Efficacy of chlorine dioxide gas against Alicyclobacillus acidoterrestris spores on apple surfaces
International Journal of Food Microbiology 108 (2006) 364 – 368 www.elsevier.com/locate/ijfoodmicro
Efficacy of chlorine dioxide gas against Alicyclobacillus acidoterrestris spores on apple surfaces Sun-Young Lee a,1 , Genisis Iris Dancer a , Su-sen Chang a , Min-Suk Rhee b,⁎, Dong-Hyun Kang a a
b
Department of Food Science and Human Nutrition, Washington State University, Pullman, WA 99164-6376, USA College of Life and Environmental Sciences, Division of Food Science, Korea University, 1, 5-Ka, Anam-dong, Sungbuk-ku, Seoul 136-701, South Korea Received 2 August 2005; received in revised form 10 October 2005; accepted 21 November 2005
Abstract Alicyclobacillus acidoterrestris is a thermophilic spore-forming bacterium that spoils acidic juices. In the orchard, apples may be contaminated with spores which can potentially grow in the resulting juice and cause spoilage. This study was undertaken to evaluate the efficacy of gaseous chlorine dioxide against A. acidoterrestris spores on apple surfaces. A. acidoterrestris spores were inoculated onto apple surfaces and were placed at room temperature, in a tightly sealed chamber containing a chlorine dioxide generating sachet, low, medium, or high release, for 30 min, 1, 2, and 3 h. After exposure, surviving spores were enumerated on K agar. Chlorine dioxide treated apples were stored at 4 °C for 7 days to assess the effect on visual quality. Inoculated, untreated apples served as the visual quality control. After exposure to high and medium release sachets for 1 h, spores were reduced to an undetectable level, a 5 log10 reduction; however, visual quality was compromised. After 1, 2, and 3 h of exposure to low release sachets, spore reductions were 2.7, 3.7, and 4.5 log10, respectively. And, after 7 days of storage, there were no significant visual quality differences between the apples exposed to low release sachet for all treatment times when compared to the control. Gaseous chlorine dioxide can effectively reduce viable A. acidoterrestris spores on apple surfaces. Due to the efficacy and easy of use, chlorine dioxide gas sachets may be useful to maintain apple quality during storage and shipping. © 2006 Elsevier B.V. All rights reserved. Keywords: Chlorine dioxide gas; Alicyclobacillus; Spore; Apple
1. Introduction Alicyclobacillus acidoterrestris is a thermoacidophilic sporeforming microorganism reported to grow at pH values between 2.5 and 6.0 (Yamazaki et al., 1996). A. acidoterrestris spores survive conventional juice pasteurization procedures, germinate, grow, and cause spoilage (Yamazaki et al., 1996). Spoilage is indicated by medicinal or phenolic off-flavors or odors, and the juice may appear normal or have slight sediment. A. acidoterrestris has been implicated in fruit juice spoilage in a variety of countries including the United Kingdom, Germany, Australia, Japan, and the United States (Chang and Kang, 2004), and is considered one of the major targets for quality control of
⁎ Corresponding author. Tel.: +82 2 3290 3058; fax: +82 2 925 1970. E-mail address: [email protected] (M.-S. Rhee). 1 Present address: Department of Food and Nutrition, Chung-Ang University, 72-1 Nae-ri Daedeok-myeon, Anseong-si, Gyeoggi-do, 456-756, South Korea. 0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2005.11.023
fruit juices and concentrates. Several recent articles have suggested that apples can become contaminated with spores in the orchard as this is a soil-based organism (Orr and Beuchat, 2000; Splittstoesser et al., 1994; Walls and Chuyate, 2000; Wisse and Parish, 1998). To reduce the possibility of contamination of juice, apples must be washed prior to processing. Traditionally, aqueous chlorine at concentrations between 50 and 200 ppm is used to wash fruits and vegetables, which results in a microbial reduction of less than 2 log10 on fresh fruits and vegetables (Beuchat, 1992; Brackett, 1992). Brackett (1992) observed that there was no significant difference between chlorinated water and tap water for eliminating Listeria monocytogenes from fresh produce. A variety of commercial cleansers are available for washing apples, but one study (Kenney and Beuchat, 2002) found that when used according to the manufacturer's instructions, the maximum reduction achieved by commercial apple washing solutions was 3 log10. Sanitizers are even less effective against A. acidoterrestris spores, as the resistance of spores to chemical
S.-Y. Lee et al. / International Journal of Food Microbiology 108 (2006) 364–368
sanitizers is well documented (Gorman et al., 1984; Orr and Beuchat, 2000). The FDA approved the use of aqueous chlorine dioxide for washing fruits and vegetables in 1998 (FDA et al., 1998). Chlorine dioxide is a strong oxidizing agent, having approximately 3.5 times the oxidation capacity of chlorine (Bernarde et al., 1965). Aqueous chlorine dioxide has been shown to be a relatively effective sanitizer for fruits and vegetables (Brown and Wardowski, 1986; Costilow et al., 1984), however, it is possible for microorganisms to be bound to fruit surfaces or protected by the natural surface variation (Han et al., 2001). It has been reported that E. coli O157:H7 cells may become trapped in the floral tube wall, lenticels, and damaged cuticle surrounding puncture wounds on apple surfaces, which protects against aqueous disinfection (Burnett and Beuchat, 2002; Kenney et al., 2001). Therefore, it would be advantageous to develop a sanitizing method with greater penetration power. Gaseous chlorine dioxide has been known as a potent sanitizer/disinfectant for over 30 years (Bernarde et al., 1965). Recently, its use in food processing environments has been investigated. Han et al. (1999) reported that 10ppm gaseous chlorine dioxide could reduce spoilage organisms by 6 log10 on juice tank surfaces. The use of chlorine dioxide has also been tested on foods, including lettuce (Lee et al., 2004). Gaseous chlorine dioxide has been shown to be more effective than aqueous chlorine dioxide at the same concentration against L. monocytogenes on the surface of green peppers (Han et al., 2001). Recent publications also indicate that chlorine dioxide gas is effective against the spores of Bacillus thuringiensis on a wide variety of surfaces (Han et al., 2003). In this study, the efficacy of easy-to-use chlorine dioxide gas generating sachets against spores of A. acidoterrestris on the surface of apples was investigated.
365
suspension onto apple skin in 10 locations using a micropipette. Apples were dried for 30min with the fan running. 2.3. Comparison of spore recovery with buffered peptone water and D/E neutralizing broth Inoculated apples were subjected to chlorine dioxide gas treatment using low release chlorine dioxide gas sachets ((ICA TriNova, LLC). Sachets were activated with 10 mL sterile water, added to the bucket with an inoculated apple, bucket sealed, and the fan started. Controls were added to the bucket and sealed without a sachet. All treatments were performed at room temperature (22 ± 2 °C). Control and gas treated apples were aseptically removed from buckets at 5, 10, 15, and 30 min. Apples were placed in sterile stomacher bags containing 50 mL of buffered peptone water or D/E neutralizing broth (Difco, Sparks, MD) and massaged vigorously by hand for 1 min to remove spores from the surface. Ten-fold serial dilutions were performed and plated onto K agar composed of yeast extract (2.5 g), peptone (5.0 g), glucose (1.0 g), tween-80 (1.0 g), and agar (15.0 g) per liter and adjusted to pH 3.7 with filter-sterilized 10% malic acid (Orr and Beuchat, 2000). Plates were incubated in an inverted position at 43 °C for 48 h prior to enumeration. 2.4. Chlorine dioxide gas treatment and storage conditions A 20 L polypropylene bucket was used as a model gas treatment chamber (Fig. 1). A small electric fan (Hankscraft Motors, Inc., Reedsburg, WI) was installed on the lid to facilitate circulation of gas in the chamber. Three types of chlorine dioxide gas sachets (ICA TriNova, LLC) were used: low, medium, and high release. Sachets were activated as previously described, and treatment was performed at room temperature. Treated apples were placed in UV sterilized plastic zip lock bags
2. Materials and methods 2.1. Bacterial cultures, growth conditions, and spore suspension preparation Two strains of A. acidoterrestris provided by the National Food Processor's Association (NFPA1013 and NFPA1101) were used to produce spores. These strains were isolated from spoiled apple juice. Cells were spread onto Potato Dextrose Agar (PDA, pH 5.6) and incubated at 43°C until at least 80% of cells had sporulated (6 days) by microscopic examination. Spores of each strain were individually harvested from agar surface by adding 1 mL aliquots of sterile water, swabbing gently with a sterile swab, and collecting the fluid, 3 times per plate. The resulting suspension was centrifuged at 4000 ×g for 30 min and washed four times with sterile water. The final suspension was stored at − 20 °C until needed. 2.2. Inoculation of apples Unwaxed Fuji apples were purchased from a local grocery store (Pullman, WA). Apples were inoculated in a biosafety cabinet by depositing a total of 100 μL of a 2 strain spore
Fan
Apple inoculated with A. acidoterrestris spores
10 ml sterile water
ClO 2 gas pack
Fig. 1. Diagram of experimental gas cabinet made using a 20 L polypropylene bucket (this figure was adapted from Lee et al., 2004 and revised).
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randomized design (treatment, storage time, and treatment × storage). Where effect was statistically significant (P b 0.05), means were separated using Duncan's multiple range test.
5
3. Results and discussion
4.5
3.1. Comparison of spore recovery with buffered peptone water and D/E neutralizing broth
4 Buffered peptone water
3.5 D/E neutralizing broth
3 0
10
20
30
40
Time(m) Fig. 2. Effect of different buffers for the recovery of Alicyclobacillus acidoterrestris spores exposed to chlorine dioxide gas at room temperature (22 ± 2°C). The error bars indicate 95% confidence intervals.
(G.T. Bag Company, Novato, CA) and stored at 4°C for 7 days. Evaluation of apple visual quality was performed daily.
Buffered peptone water and D/E neutralizing broth were compared for their ability to recover spores from inoculated apples treated with chlorine dioxide gas. As illustrated in Fig. 2, spore recovery rates were similar between the two broths and no significant differences were detected (P N 0.05) for up to 30 min of treatment. Though aqueous chlorine dioxide should be neutralized prior to microorganism enumeration, our results indicate that this is not necessary when chlorine dioxide is used in gas form. Therefore buffered peptone water was used as the selected buffer in following experiments. 3.2. Chlorine dioxide generation and final concentration
2.5. Sampling and spore enumeration Control and gas treated apples were aseptically removed from buckets at 30min, 1, 2, and 3 h. Apples were placed in sterile stomacher bags containing 50mL of the previously determined buffer and massaged vigorously by hand for 1min to remove spores from surface. Serial dilutions and conventional plating were performed as described in Section 2.3. Plates were inverted and incubated at 43°C for 48 h prior to enumeration.
Low, medium, and high release gas sachets (ICA TriNova, LLC) were evaluated in this study. Table 1 shows the peak concentration of chlorine dioxide gas generated each hour for 3h for each type of sachet. High release sachets released a total of 86 mg of chlorine dioxide over 1 h, resulting in a peak gas concentration of 4.32mg/L in the bucket. The medium release sachets released 36mg chlorine dioxide over 1 h, resulting in a peak gas concentration of 1.78 mg/L.
2.6. Measurement of chlorine dioxide gas generation 3.3. Effect of treatments on A. acidoterrestris spores Gas sachets (low, medium, and high release) were activated and suspended 1 to 2 cm above 50mL of 10% (w / w) KI solution in a 200 mL glass jar, sealed with a rubber gasket and a screw on lid. After 30min, the sachet was transferred to a new jar containing fresh KI solution. The transfer was repeated at 1, 2 and 3 h. Chlorine dioxide produced in each jar was quantified by iodometric titration, and total chlorine dioxide generation is represented as a sum of the titration results at 1, 2, and 3 h.
After 1 h of treatment with either high release or medium release sachets, more than a 5 log10 reduction was achieved, reducing the spores to undetectable levels (Fig. 3). The low 6 5
All experiments were repeated three times with duplicate samples. Analysis of variance was performed using the ANOVA procedure of SAS (SAS Institute, Cary, NC) for a completely
Log 10 CFU/ml
4
2.7. Experimental design and statistical analysis
Control (non-treated) Low release Medium release High release
3 2 1
Table 1 Peak concentration of chorine dioxide gas generated by high, medium, and low release sachets after each reaction time Reaction time (h) 0 1 2 3
0
Peak concentration (mg/L) Low 0.00 0.39 0.50 0.60
Medium 0.00 1.78 2.42 2.69
High 0.00 4.32 5.95 6.55
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Treatment time (h)
Fig. 3. Survival curves for Alicyclobacillus acidoterrestris spores on apple surfaces exposed to chlorine dioxide gas at room temperature (22 ± 2°C). The error bars indicate 95% confidence intervals.
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release sachets released 8mg chlorine dioxide over 1h, resulting in a peak gas concentration of 0.39 mg/L and a 2.7 log10 reduction in A. acidoterrestris spores. Over 2 h, 10mg of chlorine dioxide, equivalent to 0.50mg/L chlorine dioxide, and a 3.7 log10 reduction of spores was achieved. Over 3h, 12 mg chlorine dioxide, peak concentration 0.60 mg/L, a 4.5 log10 was achieved. Han et al. (2003) reported that a 5mg/L peak gas concentration over a period of 12h only achieved 2.5 and 3.6 log10 reduction of 6 log10 B. thuringiensis spore populations on paper and wood, respectively. Paper and wood may be slightly more porous than apple skin, but the discrepancy in the results indicates that A. acidoterrestris spores may be more susceptible to chlorine dioxide than those of B. thuringiensis. Further studies are necessary to characterize the chlorine dioxide gas susceptibility of various sporeformers. 3.4. Effect of treatment on visual quality Chlorine dioxide treated apples were stored at 4°C for 7 days to assess the effect on visual quality. Although medium and high release sachets proved effective against A. acidoterrestris spores, during storage trials the skin of the apples receiving the treatment developed small black spots within 3 days. Therefore, the concentration of chlorine dioxide gas generated by these sachets was determined to be too high for apple treatment from a visual quality standpoint, and further tests were not performed. However, when low release sachets were applied, there were no significant differences in visual quality
367
after 7 days storage at 4°C between control (untreated) and the apples treated for 30min, 1, 2, or 3 h. Therefore, treatment with low release chlorine dioxide gas sachets did not affect the visual quality of apples over the 7 day period (Fig. 4). In another, Lee et al. (2004) reported that treatment with chlorine dioxide gas sachets generating 8.7mg/L for 3 h on lettuce leaves did not affect visual quality after 18 days storage at 4 °C. However, a lower concentration of chlorine dioxide gas affected visual quality of apples in this study. Therefore, the effect of chlorine dioxide gas on the visual quality of produce may differ depending on the type of produce. While the concentrations of chlorine dioxide generated by the high and medium release sachets were effective at reducing A. acidoterrestris spores on apple surfaces, the treatments caused cosmetic damage to the apple, which would not be appealing to consumers. In juice production, cosmetic defects associated with chlorine dioxide gas treatment may be acceptable when weighed against control of A. acidoterrestris spores. In contrast, lower levels of chlorine dioxide generated by low release sachets reduced A. acidoterrestris without visible damage to apple skin, which may be a more appropriate treatment for apples intended for the fresh produce market. Further studies are necessary to determine optimal gas concentrations for a particular application. Han et al. (2001) determined that higher relative humidity has a synergistic effect with chlorine dioxide gas treatment. Other environmental factors, such as temperature, presence of light and composition of treatment vessel need to be studied.
A
B
C
Fig. 4. Digital photographs of apples stored for 7 days (A) or 3 days (B, C) at 4 °C after treatment with chlorine dioxide gas. Apples treated with ClO2 gas using low release sachets, A; using medium release sachets, B; and using high release sachets, C before storage. All pictures show untreated apple (control), and apples treated for 30min, 1, and 3h from left.
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Acknowledgements The authors wish to thank ICA TriNova, LLC (Forest Park, GA) for supplying the chlorine dioxide gas sachets, and especially Joel Tenney and Bill Ernst for their technical assistance and support. References Beuchat, L.R., 1992. Surface disinfection of raw produce. Dairy Food Environ. Sanit. 12, 6–9. Bernarde, M.A., Israel, B.M., Oliveri, V.P., Granstram, M.L., 1965. Efficiency of chlorine dioxide as a bactericide. Appl. Microbiol. 13, 776–780. Brackett, R.E., 1992. Shelf stability and safety of fresh produce as influenced by sanitation and disinfection. J. Food Prot. 55, 808–814. Brown, G.E., Wardowski, W.E., 1986. Use of chlorine dioxide in Florida citrus packinghouses to reduce inoculum of decay pathogens. Citrus Ind. 67, 48–56. Burnett, S.L., Beuchat, L.R., 2002. Differentiation of viable and dead Escherichia coli O157:H7 cells on and in apple structures and tissues following chlorine treatment. J. Food Prot. 65, 251–259. Chang, S.S., Kang, D.H., 2004. Alicyclobacillus ssp. in the fruit juice industry: history, characteristics, and current isolation/detection procedures. Crit. Rev. Microbiol. 30, 55–74. Costilow, R.N., Uebersax, M.A., Waed, P.J., 1984. Use of chlorine dioxide for controlling microorganisms during handling and storage of fresh cucumbers. J. Food Sci. 49, 396–401. FDA, USDA, CDC, 1998. Guidance for industry — guide to minimize microbial food safety hazards for fresh fruits and vegetables. http://vm.cfsan. fda.gov/~DMA/prodguid.html. Gorman, S.P., Scott, E.M., Hutchinson, E.P., 1984. Hypochlorite effects on spores and spore forms of Bacillus subtilis and on a spore lytic enzyme. J. Appl. Bacteriol. 56, 295–303.
Han, Y., Guentert, A.M., Smith, R.S., Linton, R.H., Nelson, P.E., 1999. Efficacy of chlorine dioxide gas as a sanitizer for tanks used for aseptic juice storage. Food Microbiol. 16, 53–61. Han, Y., Linton, R.H., Nielsen, S.S., Nelson, P.E., 2001. Reduction of Listeria monocytogenes on green peppers (Capsicum annuum L.) by gaseous and aqueous chlorine dioxide and water washing and its growth at 7°C. J. Food Prot. 64, 1730–1738. Han, Y., Applegate, B., Linton, R.H., Nelson, P.E., 2003. Decontamination of Bacillus thuringiensis spores on selected surfaces by chlorine dioxide gas. J. Environ. Health 66, 16–21. Kenney, S.J., Beuchat, L.R., 2002. Comparison of aqueous commercial cleaners for effectiveness in removing Escherichia coli O157:H7 and Salmonella muenchen from the surface of apples. Int. J. Food Microbiol. 74, 47–55. Kenney, S.J., Burnett, S.L., Beuchat, L.R., 2001. Location of E. coli O157:H7 on and in apples as affected by bruising, washing, and rubbing. J. Food Prot. 64, 1328–1333. Lee, S.Y., Costello, M., Kang, D.H., 2004. Efficacy of chlorine dioxide gas as a sanitizer of lettuce leaves. J. Food Prot. 67, 1371–1376. Orr, R.V., Beuchat, L.R., 2000. Efficacy of disinfectants in killing spores of Alicyclobacillus acidoterrestris and performance of media for supporting colony development by survivors. J. Food Prot. 63, 1117–1122. Splittstoesser, D.F., Churry, J.J., Lee, C.Y., 1994. Growth characteristics of aciduric sporeforming Bacilli isolated from fruit juices. J. Food Prot. 57, 1080–1083. Walls, I., Chuyate, R., 2000. Spoilage of fruit juices by Alicyclobacillus acidoterrestris. Food Aust. 52, 286–288. Wisse, C.A., Parish, M.E., 1998. Isolation and enumeration of spore-forming, thermoacidophilic, rod-shaped bacteria from citrus processing environments. Dairy Food Environ. Sanit. 8, 504. Yamazaki, K., Teduka, H., Shinano, H., 1996. Isolation and identification of Alicyclobacillus acidoterrestris from acidic beverages. Biosci. Biotechnol. Biochem. 60, 543–545.
Efficacy of chlorine dioxide gas against Alicyclobacillus acidoterrestris spores on apple surfaces Sun-Young Lee a,1 , Genisis Iris Dancer a , Su-sen Chang a , Min-Suk Rhee b,⁎, Dong-Hyun Kang a a
b
Department of Food Science and Human Nutrition, Washington State University, Pullman, WA 99164-6376, USA College of Life and Environmental Sciences, Division of Food Science, Korea University, 1, 5-Ka, Anam-dong, Sungbuk-ku, Seoul 136-701, South Korea Received 2 August 2005; received in revised form 10 October 2005; accepted 21 November 2005
Abstract Alicyclobacillus acidoterrestris is a thermophilic spore-forming bacterium that spoils acidic juices. In the orchard, apples may be contaminated with spores which can potentially grow in the resulting juice and cause spoilage. This study was undertaken to evaluate the efficacy of gaseous chlorine dioxide against A. acidoterrestris spores on apple surfaces. A. acidoterrestris spores were inoculated onto apple surfaces and were placed at room temperature, in a tightly sealed chamber containing a chlorine dioxide generating sachet, low, medium, or high release, for 30 min, 1, 2, and 3 h. After exposure, surviving spores were enumerated on K agar. Chlorine dioxide treated apples were stored at 4 °C for 7 days to assess the effect on visual quality. Inoculated, untreated apples served as the visual quality control. After exposure to high and medium release sachets for 1 h, spores were reduced to an undetectable level, a 5 log10 reduction; however, visual quality was compromised. After 1, 2, and 3 h of exposure to low release sachets, spore reductions were 2.7, 3.7, and 4.5 log10, respectively. And, after 7 days of storage, there were no significant visual quality differences between the apples exposed to low release sachet for all treatment times when compared to the control. Gaseous chlorine dioxide can effectively reduce viable A. acidoterrestris spores on apple surfaces. Due to the efficacy and easy of use, chlorine dioxide gas sachets may be useful to maintain apple quality during storage and shipping. © 2006 Elsevier B.V. All rights reserved. Keywords: Chlorine dioxide gas; Alicyclobacillus; Spore; Apple
1. Introduction Alicyclobacillus acidoterrestris is a thermoacidophilic sporeforming microorganism reported to grow at pH values between 2.5 and 6.0 (Yamazaki et al., 1996). A. acidoterrestris spores survive conventional juice pasteurization procedures, germinate, grow, and cause spoilage (Yamazaki et al., 1996). Spoilage is indicated by medicinal or phenolic off-flavors or odors, and the juice may appear normal or have slight sediment. A. acidoterrestris has been implicated in fruit juice spoilage in a variety of countries including the United Kingdom, Germany, Australia, Japan, and the United States (Chang and Kang, 2004), and is considered one of the major targets for quality control of
⁎ Corresponding author. Tel.: +82 2 3290 3058; fax: +82 2 925 1970. E-mail address: [email protected] (M.-S. Rhee). 1 Present address: Department of Food and Nutrition, Chung-Ang University, 72-1 Nae-ri Daedeok-myeon, Anseong-si, Gyeoggi-do, 456-756, South Korea. 0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2005.11.023
fruit juices and concentrates. Several recent articles have suggested that apples can become contaminated with spores in the orchard as this is a soil-based organism (Orr and Beuchat, 2000; Splittstoesser et al., 1994; Walls and Chuyate, 2000; Wisse and Parish, 1998). To reduce the possibility of contamination of juice, apples must be washed prior to processing. Traditionally, aqueous chlorine at concentrations between 50 and 200 ppm is used to wash fruits and vegetables, which results in a microbial reduction of less than 2 log10 on fresh fruits and vegetables (Beuchat, 1992; Brackett, 1992). Brackett (1992) observed that there was no significant difference between chlorinated water and tap water for eliminating Listeria monocytogenes from fresh produce. A variety of commercial cleansers are available for washing apples, but one study (Kenney and Beuchat, 2002) found that when used according to the manufacturer's instructions, the maximum reduction achieved by commercial apple washing solutions was 3 log10. Sanitizers are even less effective against A. acidoterrestris spores, as the resistance of spores to chemical
S.-Y. Lee et al. / International Journal of Food Microbiology 108 (2006) 364–368
sanitizers is well documented (Gorman et al., 1984; Orr and Beuchat, 2000). The FDA approved the use of aqueous chlorine dioxide for washing fruits and vegetables in 1998 (FDA et al., 1998). Chlorine dioxide is a strong oxidizing agent, having approximately 3.5 times the oxidation capacity of chlorine (Bernarde et al., 1965). Aqueous chlorine dioxide has been shown to be a relatively effective sanitizer for fruits and vegetables (Brown and Wardowski, 1986; Costilow et al., 1984), however, it is possible for microorganisms to be bound to fruit surfaces or protected by the natural surface variation (Han et al., 2001). It has been reported that E. coli O157:H7 cells may become trapped in the floral tube wall, lenticels, and damaged cuticle surrounding puncture wounds on apple surfaces, which protects against aqueous disinfection (Burnett and Beuchat, 2002; Kenney et al., 2001). Therefore, it would be advantageous to develop a sanitizing method with greater penetration power. Gaseous chlorine dioxide has been known as a potent sanitizer/disinfectant for over 30 years (Bernarde et al., 1965). Recently, its use in food processing environments has been investigated. Han et al. (1999) reported that 10ppm gaseous chlorine dioxide could reduce spoilage organisms by 6 log10 on juice tank surfaces. The use of chlorine dioxide has also been tested on foods, including lettuce (Lee et al., 2004). Gaseous chlorine dioxide has been shown to be more effective than aqueous chlorine dioxide at the same concentration against L. monocytogenes on the surface of green peppers (Han et al., 2001). Recent publications also indicate that chlorine dioxide gas is effective against the spores of Bacillus thuringiensis on a wide variety of surfaces (Han et al., 2003). In this study, the efficacy of easy-to-use chlorine dioxide gas generating sachets against spores of A. acidoterrestris on the surface of apples was investigated.
365
suspension onto apple skin in 10 locations using a micropipette. Apples were dried for 30min with the fan running. 2.3. Comparison of spore recovery with buffered peptone water and D/E neutralizing broth Inoculated apples were subjected to chlorine dioxide gas treatment using low release chlorine dioxide gas sachets ((ICA TriNova, LLC). Sachets were activated with 10 mL sterile water, added to the bucket with an inoculated apple, bucket sealed, and the fan started. Controls were added to the bucket and sealed without a sachet. All treatments were performed at room temperature (22 ± 2 °C). Control and gas treated apples were aseptically removed from buckets at 5, 10, 15, and 30 min. Apples were placed in sterile stomacher bags containing 50 mL of buffered peptone water or D/E neutralizing broth (Difco, Sparks, MD) and massaged vigorously by hand for 1 min to remove spores from the surface. Ten-fold serial dilutions were performed and plated onto K agar composed of yeast extract (2.5 g), peptone (5.0 g), glucose (1.0 g), tween-80 (1.0 g), and agar (15.0 g) per liter and adjusted to pH 3.7 with filter-sterilized 10% malic acid (Orr and Beuchat, 2000). Plates were incubated in an inverted position at 43 °C for 48 h prior to enumeration. 2.4. Chlorine dioxide gas treatment and storage conditions A 20 L polypropylene bucket was used as a model gas treatment chamber (Fig. 1). A small electric fan (Hankscraft Motors, Inc., Reedsburg, WI) was installed on the lid to facilitate circulation of gas in the chamber. Three types of chlorine dioxide gas sachets (ICA TriNova, LLC) were used: low, medium, and high release. Sachets were activated as previously described, and treatment was performed at room temperature. Treated apples were placed in UV sterilized plastic zip lock bags
2. Materials and methods 2.1. Bacterial cultures, growth conditions, and spore suspension preparation Two strains of A. acidoterrestris provided by the National Food Processor's Association (NFPA1013 and NFPA1101) were used to produce spores. These strains were isolated from spoiled apple juice. Cells were spread onto Potato Dextrose Agar (PDA, pH 5.6) and incubated at 43°C until at least 80% of cells had sporulated (6 days) by microscopic examination. Spores of each strain were individually harvested from agar surface by adding 1 mL aliquots of sterile water, swabbing gently with a sterile swab, and collecting the fluid, 3 times per plate. The resulting suspension was centrifuged at 4000 ×g for 30 min and washed four times with sterile water. The final suspension was stored at − 20 °C until needed. 2.2. Inoculation of apples Unwaxed Fuji apples were purchased from a local grocery store (Pullman, WA). Apples were inoculated in a biosafety cabinet by depositing a total of 100 μL of a 2 strain spore
Fan
Apple inoculated with A. acidoterrestris spores
10 ml sterile water
ClO 2 gas pack
Fig. 1. Diagram of experimental gas cabinet made using a 20 L polypropylene bucket (this figure was adapted from Lee et al., 2004 and revised).
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S.-Y. Lee et al. / International Journal of Food Microbiology 108 (2006) 364–368 5.5
randomized design (treatment, storage time, and treatment × storage). Where effect was statistically significant (P b 0.05), means were separated using Duncan's multiple range test.
5
3. Results and discussion
4.5
3.1. Comparison of spore recovery with buffered peptone water and D/E neutralizing broth
4 Buffered peptone water
3.5 D/E neutralizing broth
3 0
10
20
30
40
Time(m) Fig. 2. Effect of different buffers for the recovery of Alicyclobacillus acidoterrestris spores exposed to chlorine dioxide gas at room temperature (22 ± 2°C). The error bars indicate 95% confidence intervals.
(G.T. Bag Company, Novato, CA) and stored at 4°C for 7 days. Evaluation of apple visual quality was performed daily.
Buffered peptone water and D/E neutralizing broth were compared for their ability to recover spores from inoculated apples treated with chlorine dioxide gas. As illustrated in Fig. 2, spore recovery rates were similar between the two broths and no significant differences were detected (P N 0.05) for up to 30 min of treatment. Though aqueous chlorine dioxide should be neutralized prior to microorganism enumeration, our results indicate that this is not necessary when chlorine dioxide is used in gas form. Therefore buffered peptone water was used as the selected buffer in following experiments. 3.2. Chlorine dioxide generation and final concentration
2.5. Sampling and spore enumeration Control and gas treated apples were aseptically removed from buckets at 30min, 1, 2, and 3 h. Apples were placed in sterile stomacher bags containing 50mL of the previously determined buffer and massaged vigorously by hand for 1min to remove spores from surface. Serial dilutions and conventional plating were performed as described in Section 2.3. Plates were inverted and incubated at 43°C for 48 h prior to enumeration.
Low, medium, and high release gas sachets (ICA TriNova, LLC) were evaluated in this study. Table 1 shows the peak concentration of chlorine dioxide gas generated each hour for 3h for each type of sachet. High release sachets released a total of 86 mg of chlorine dioxide over 1 h, resulting in a peak gas concentration of 4.32mg/L in the bucket. The medium release sachets released 36mg chlorine dioxide over 1 h, resulting in a peak gas concentration of 1.78 mg/L.
2.6. Measurement of chlorine dioxide gas generation 3.3. Effect of treatments on A. acidoterrestris spores Gas sachets (low, medium, and high release) were activated and suspended 1 to 2 cm above 50mL of 10% (w / w) KI solution in a 200 mL glass jar, sealed with a rubber gasket and a screw on lid. After 30min, the sachet was transferred to a new jar containing fresh KI solution. The transfer was repeated at 1, 2 and 3 h. Chlorine dioxide produced in each jar was quantified by iodometric titration, and total chlorine dioxide generation is represented as a sum of the titration results at 1, 2, and 3 h.
After 1 h of treatment with either high release or medium release sachets, more than a 5 log10 reduction was achieved, reducing the spores to undetectable levels (Fig. 3). The low 6 5
All experiments were repeated three times with duplicate samples. Analysis of variance was performed using the ANOVA procedure of SAS (SAS Institute, Cary, NC) for a completely
Log 10 CFU/ml
4
2.7. Experimental design and statistical analysis
Control (non-treated) Low release Medium release High release
3 2 1
Table 1 Peak concentration of chorine dioxide gas generated by high, medium, and low release sachets after each reaction time Reaction time (h) 0 1 2 3
0
Peak concentration (mg/L) Low 0.00 0.39 0.50 0.60
Medium 0.00 1.78 2.42 2.69
High 0.00 4.32 5.95 6.55
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Treatment time (h)
Fig. 3. Survival curves for Alicyclobacillus acidoterrestris spores on apple surfaces exposed to chlorine dioxide gas at room temperature (22 ± 2°C). The error bars indicate 95% confidence intervals.
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release sachets released 8mg chlorine dioxide over 1h, resulting in a peak gas concentration of 0.39 mg/L and a 2.7 log10 reduction in A. acidoterrestris spores. Over 2 h, 10mg of chlorine dioxide, equivalent to 0.50mg/L chlorine dioxide, and a 3.7 log10 reduction of spores was achieved. Over 3h, 12 mg chlorine dioxide, peak concentration 0.60 mg/L, a 4.5 log10 was achieved. Han et al. (2003) reported that a 5mg/L peak gas concentration over a period of 12h only achieved 2.5 and 3.6 log10 reduction of 6 log10 B. thuringiensis spore populations on paper and wood, respectively. Paper and wood may be slightly more porous than apple skin, but the discrepancy in the results indicates that A. acidoterrestris spores may be more susceptible to chlorine dioxide than those of B. thuringiensis. Further studies are necessary to characterize the chlorine dioxide gas susceptibility of various sporeformers. 3.4. Effect of treatment on visual quality Chlorine dioxide treated apples were stored at 4°C for 7 days to assess the effect on visual quality. Although medium and high release sachets proved effective against A. acidoterrestris spores, during storage trials the skin of the apples receiving the treatment developed small black spots within 3 days. Therefore, the concentration of chlorine dioxide gas generated by these sachets was determined to be too high for apple treatment from a visual quality standpoint, and further tests were not performed. However, when low release sachets were applied, there were no significant differences in visual quality
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after 7 days storage at 4°C between control (untreated) and the apples treated for 30min, 1, 2, or 3 h. Therefore, treatment with low release chlorine dioxide gas sachets did not affect the visual quality of apples over the 7 day period (Fig. 4). In another, Lee et al. (2004) reported that treatment with chlorine dioxide gas sachets generating 8.7mg/L for 3 h on lettuce leaves did not affect visual quality after 18 days storage at 4 °C. However, a lower concentration of chlorine dioxide gas affected visual quality of apples in this study. Therefore, the effect of chlorine dioxide gas on the visual quality of produce may differ depending on the type of produce. While the concentrations of chlorine dioxide generated by the high and medium release sachets were effective at reducing A. acidoterrestris spores on apple surfaces, the treatments caused cosmetic damage to the apple, which would not be appealing to consumers. In juice production, cosmetic defects associated with chlorine dioxide gas treatment may be acceptable when weighed against control of A. acidoterrestris spores. In contrast, lower levels of chlorine dioxide generated by low release sachets reduced A. acidoterrestris without visible damage to apple skin, which may be a more appropriate treatment for apples intended for the fresh produce market. Further studies are necessary to determine optimal gas concentrations for a particular application. Han et al. (2001) determined that higher relative humidity has a synergistic effect with chlorine dioxide gas treatment. Other environmental factors, such as temperature, presence of light and composition of treatment vessel need to be studied.
A
B
C
Fig. 4. Digital photographs of apples stored for 7 days (A) or 3 days (B, C) at 4 °C after treatment with chlorine dioxide gas. Apples treated with ClO2 gas using low release sachets, A; using medium release sachets, B; and using high release sachets, C before storage. All pictures show untreated apple (control), and apples treated for 30min, 1, and 3h from left.
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