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Inactivation by chlorine dioxide gas (ClO2) of Listeria monocytogenes spotted onto different apple surfaces
Food Microbiology, 2002, 19, 481^490 Available online at http://www.idealibrary.com on
doi:10.1006/yfmic.501
ORIGINAL ARTICLE
Inactivation by chlorine dioxide gas (ClO2) of Listeria monocytogenes spotted onto di¡erent apple surfacesw Jinhua Du,Y. Han and R. H. Linton*
The bactericidal e¡ects of chlorine dioxide (ClO2) gas treatments on a mixture of three strains of Listeria monocytogenes (Scott A, F5069 and LCDC 81-886) spotted on to the calyx, stem cavity and pulp surface of apples were investigated at 211C and 90% relative humidity (RH). ClO2 gas was more e¡ective for inactivating the bacteria attached to pulp skin than those attached to the calyx or stem cavity at a ClO2 level of 4?0 mg l 1 and a treatment time of 10 or more minutes. After treatments of 1?0^4?0 mg l 1 ClO2 for10 min, a 2^3 log reduction of L. monocytogenes were observed on both cavities. There were 4?370?2 and 4?371?1 log reductions of L. monocytogenes achieved on the calyx and stem cavities, respectively, by a 4?0 mg l 1 ClO2 gas treatment for 30 min. After treatment of 8?0 mg l 1 ClO2 for 30 min, no survivors were detected using an end point method for 3?6^5?3 log cfu spotted site 1 on the calyx cavity and 3?5^5?0 log cfu spotted site 1 on the stem cavity. No signi¢cant di¡erences (P40?05) were found between bacterial log reductions on pulp skin at 1?0 or 3?0 mg l 1 ClO2 gas treatment for 10 min. When the ClO2 level was increased to 4?0 mg l 1 for 10 min, a 5?571?0 log cfu spotted site 1 bacteria on pulp skin was inactivated. After 4.0 mg l 1 ClO2 gas for 30 min, L. monocytogenes levels on the pulp skin could be decreased by 3?970?0 to 6.570.1log cfu spotted site 1 using an end point determination method. ClO2 gas was a potentially powerful sanitizer for inactivating L. monocytogenes on each apple surface, especially those attached to the pulp skin. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction Listeria monocytogenes is a Gram-positive foodborne bacterium, which has been isolated from soil, silage, and several other environmental sources. It is able to grow at refrigerated temperatures down to 01C (Berrang et al. 1989, Beuchat and Brackett 1990, Walker et al. 1990, *Corresponding author. Fax: (765)- 494 -7953; E-mail: [email protected] w This paper is journal article #16675 of the Purdue University Agricultural Research Program. 0740 -0020/02/050481+10 $35.00/0
Bajard et al. 1996), on dry surfaces (Wong 1998), can tolerate high acidity (Ahamad and Marth 1989, 1990, Sorrells et al. 1989, Farber and Pagotto 1992, Schaik et al. 1999) or alkalized conditions (Taormina and Beuchat 2001), and high heat (Doyle et al. 2001). These characteristics make L. monocytogenes well adapted to food and food processing environments where the growth of other pathogens may be restricted. Incidences of L. monocytogenes infections have been associated with fruits and vegetables, such as coleslaw and cabbage (Schlech r 2002 Elsevier Science Ltd. All rights reserved.
Received: 30 November 2001 Department of Food Science, Purdue University,1160 Food Science Building, West Lafayette, Indiana 47907-1160, USA
482 J. Du et al.
et al. 1983), lettuce, celery, tomatoes (Ho et al. 1986), cabbage, cucumbers, potatoes, radishes (Heisick et al. 1989), and other vegetables (Porto and Uboldi 2001). These outbreaks were likely caused by bacterial contamination or by cross-contamination during handling. Raw apple cider has been identi¢ed as a source of bacterial contamination in the U.S. leading to foodborne illness and death.Three L. monocytogenes isolates have been detected in two kinds of retail unpasteurized apple juices (pH 3?75) (Sado et al. 1998). L. monocytogenes could also survive and grow on freshly cut apple (cv. Delicious) slices and in apple juice (Conway et al. 2000). Beuchat et al. (1998) reported that L. monocytogenes on apple surfaces had greater resistance to chlorine compared to Escherichia coli O157:H7 and Salmonella. Mazzotta (2001) showed that the heat resistance of L. monocytogenes increased signi¢cantly (Po0?05) after acid adaptation in single-strength apple, orange and white grape juices adjusted to pH 3?9. Therefore, L. monocytogenes should be considered as a potential threat to the safety of apple cider or juices. More recently, the FDA (Food and Drug Administration 2001) has established requirements to increase the safety of fruit and vegetable juices. Here, processors are required to use processes that achieve a 5 log reduction in the numbers of ‘the most resistant pathogen’ in their ¢nished products compared with levels that may be present in untreated juice. Juice processors may use microbial reduction methods other than pasteurization, including approved alternative technologies such as the recently approved ultraviolet (UV) irradiation technology or a combination of these technologies. In the apple beverage production, heat pasteurization is very e¡ective in achieving a 5 log reduction if used as a ¢nal processing step, but this process may a¡ect the taste and appearance, or add to the cost of the ¢nished products. Current methods used to preserve fresh apple beverages include refrigeration and addition of preservatives such as potassium sorbate and sodium benzoate, which are usually used to prolong the shelf life of the beverages. Researchers have reported that UV light can result in a 3?81 log reduction of E.
O157:H7 in unpasteurized cider (Wright et al. 2000a). There are many studies concentrated on reducing pathogen load of fresh fruit juices. One approach is the direct treatment of fresh juices with non-thermal methods such as pulsed electric ¢elds (Lu et al. 2001) and carbon dioxide pressure (Erkmen 2000). Other approaches include direct addition of new antimicrobials such as peptides (Appendin and Hotchkiss 2000). Chemical sanitization of whole apples before grinding is an alternative method to reduce microbial load. However, many conventional sanitizers or their combinations, such as chlorine (Beuchat et al. 1998), hydrogen peroxide and trisodium phosphate (Sapers et al. 1999, Liao and Sapers 2000, Annous et al. 2001), peroxyacetic acid and chlorine-phosphate bu¡er (Wisniewsky et al. 2000), acetic acid and peroxyacetic acid (Wright et al. 2000b), and allyl isothiocyanate (Lin et al. 2000 a, b) were not able to reduce L. monocytogenes, E. coli O157:H7, or Salmonella populations on apple surfaces by 5 logs at the recommended concentration under the test conditions. Therefore, the development of highly e¡ective sanitation technology for the reduction of pathogens on apples is needed (Annous et al. 2001). As a potential sanitizer for decontamination of fruits and vegetables, ClO2 gas has many advantages over traditional chlorinated water, such as 1?5 times more than the oxidation capacity of chlorine, less reactive than chlorine with organic compounds, e¡ectiveness at low concentration, non-conversion to chlorophenols that result in residual smells and £avors, and inability to react with ammonia or humic acid to produce harmful chloramines and trihalomethanes (Elphick 1998). ClO2 gas also has a strong penetrability and high dissolubility. It was highly e¡ective in reducing foodborne pathogens such as L. monocytogenes and E. coli O157:H7 on green pepper surfaces (Han et al. 2000a, b, 2001) and food spoilage microorganisms on epoxy-coated juice storage tank surface (Han et al. 1999). Furthermore, ClO2 has the ability to reduce fungicide residues, mancozeb and ethylenethiourea, in apples (Hwang et al. 2001). Therefore, ClO2 gas may be an e¡ective alternative sanitizer to reduce microbial population on apple surfaces.
Inactivation of L. monocytogenes on apples 483
As the National Advisory Committee on Microbiological Criteria for Foods recommended, L. monocytogenes should be used as target organism in the absence of known speci¢c pathogen product associations, as appropriate (Food and Drug Administration 1998). In this research, the e⁄cacy of ClO2 gas to inactive the L. monocytogenes cells spotted on to the calyx and, stem cavities and the pulp surface of apples was investigated. The objective of this study was to determine the ClO2 gas concentration and exposure time needed to achieve a three or more log reduction of L. monocytogenes on these three di¡erent apple surfaces using di¡erent ClO2 gas treatments.
Materials and Methods Bacterial strains Three L. monocytogenes strains were chosen and used as a mixture. L. monocytogenes Scott A was provided by Dr L. R. Beuchat (University of Georgia, Gri⁄n, Georgia, USA). L. monocytogenes F5069 and LCDC 81^886 were obtained from Dr. Arun Bhunia of Purdue University, West Lafayette, Indiana, USA. This mixture of strains was chosen to account for potential differences in resistance to ClO2 gas treatments. All cultures were maintained on slants containing tryptic soy agar (TSA, Difco Laboratories, Division of Becton Dickinson and Company, Detroit, Michigan, USA) plus 0?6% (wt vol 1 ) yeast extract (YE, Difco Laboratories) at 41C.
ium base with Oxford antimicrobiotic supplement (MOA, Difco Laboratories, Division of Becton Dickinson and Company, Spark, Maryland, USA) to determine the population (cfu ml 1 ) of the culture.The mixed suspension was serially diluted with 0?1% peptone solution. Then 0?1 ml diluents were surface-spread on to MOA plates in duplicate. The plates were incubated at 371C for approximately 24^48 h before counting.
Inoculation of apples Unwaxed Red Delicious apples were obtained from a local supermarket. Apples free of physical damage and debris were selected, rinsed with tap water for 1 min and dried with a paper towel. As shown in Fig. 1, three parts of an apple, including the calyx and stem cavities, and the skin or surface of pulp were inoculated with 100 ml droplets of the mixed culture suspension in a class II bio-safety cabinet. The populations inoculated were approximately 106 ^ 108 cfu per inoculation spotted site (approx. 2 cm in diameter). The inoculated cultures on apple surfaces were air-dried at 211C for 2^3 h in the bio-safety cabinet prior to treatment. The levels of indigenous microorganisms on the calyx and stem cavities, and the pulp skin surface of apples were approximately 105, 104, and o103 cfu 5 g 1, respectively. No indigenous L. monocytogenes cells were detected.
Growth conditions and viable counts Each test strain was aseptically transferred into 10 ml tryptic soy broth with 0?6% (wt vol 1 ) yeast extract (TSB-YE, Difco Laboratories, Division of Becton Dickinson and Company, Detroit, Michigan, USA) and incubated at 371C for 24 h, followed by a minimum of two successive transfers to gain a stationary-phase culture. To make a composite of the three strains, an equal volume of each culture was mixed together. Before mixing, each culture was enumerated by surface-plating duplicate samples on to tryptic soy agar and Oxford med-
Figure 1. Inoculation sites (calyx and stem cavities, and pulp skin) for L. monocytogenes on apple surfaces.
484 J. Du et al.
Chlorine dioxide (ClO2) gas treatment ClO2 gas treatment was carried out in a 10 l Irvine Plexiglass cylinder with a stainlesssteel shelf, where four apples were placed on the ¢rst mesh screen (Fig. 2). A Thermo-Hydro recorder (Control Company, Friendwood, Texas, USA) was used to monitor relative humidity (RH) and temperature inside the treatment cylinder. Using a diaphragm vacuum pump (KNF Neuberger, Inc., Treton, New Jersey, USA), a 90^95% RH was achieved by circulating the air inside the cylinder through a 125 ml washing bottle with 100 ml deionized water for 10^15 min. ClO2 gas with a concentration of 160 ^180 mg l 1 was produced based on the reaction of chlorine gas (4% in nitrogen gas) with sodium hypochlorite in a CDG laboratory generator (CDG Technology, Inc., New York, USA). A 60 ml plastic gas-sampling syringe was used to deliver speci¢c volumes of ClO2 gas into the cylinder. During the treatment, the ClO2 gas inside the cylinder was circulated by the diaphragm vacuum pump, which entered from the top of the cylinder and £owed out from the bottom.The cylinder was placed in the dark to prevent light-decomposition of the ClO2. During treatment, relative humidity and temperature were maintained at 90^95% and 211C inside the treatment cylinder, respectively.
Measurement of ClO2 gas concentration A DPD (N, N-diethyl-r-phenylenediamine) colorimetric analysis kit (CHEMetrics, Inc., Cal-
Figure 2. A 10 l ClO2 gas treatment system used to treat the apples inoculated with L. monocytogenes.
verton,Virgina, USA) was used to measure the ClO2 gas concentration. Using a 25 ml gas-sampling syringe, 15 ml ClO2 gas from the treatment cylinder was immediately dissolved in 15 ml neutralized deionized water. Before injecting the gas into the water, some water was repeatedly drawn in and out the syringe to dissolved the gas completely. The initial ClO2 gas concentration was measured in duplicate and recorded as mg ClO2 l 1 .
Enumeration or detection methods of L. monocytogenes on apple surfaces A sterile fruit knife was used to cut each inoculated area to obtain a 5 -g sample size. For the control samples untreated with ClO2, each cut piece was mixed with 95 ml sterile 0?1% peptone solution in a 400 ml sterile stomacher bag (Fisher Scienti¢c Inc.). For ClO2 -treated samples, the cut piece was mixed with 95 ml neutralizing bu¡er (Difco Laboratories, Division of Becton Dickinson and Company, Detroit, Michigan, USA) to neutralize residual ClO2 on the sample, and then blended for 2 min at 260 rpm using a Seward Stomacher 400 Circulator (Steward Limited, London, UK). The blended sample was serially diluted with 0?1% peptone and surface-plated in duplicate on to MOA plates. These plates were incubated at 371C for 24^48 h. For each plate, typical colonies were chosen and identi¢ed as L. monocytogenes by an API Listeria Test (BioMerieux Vitec, Hazelwood, Missouri, USA). All the populations of L. monocytogenes were recorded as colony forming units per spotted site (cfu spotted site 1 ). To resuscitate and enumerate ClO2 -injured L. monocytogenes cells, a membrane-transferring plating method was used (Han et al. 2001). One hundred microliters of the stomached sample suspension or its diluents were spread over a sterile polycarbonate ¢lter membrane (0?4 mm pore size, track-etched, 90 mm diameter) (Osmonic Co.,Westboro, Massachusetts, USA) previously placed on the surface of a TSA plate. Plates were incubated at 371C for 4 h to repair injured cells. Then the membranes were gently and aseptically transferred onto MOA plates using sterile forceps. The membrane^ MOA plates were further incubated at 371C
Inactivation of L. monocytogenes on apples 485
for 24^36 h. L. monocytogenes colonies were counted after the same API Listeria procedure as described above. Because only Z102 cfu ml 1 level bacteria could be counted using the membrane-transferring plating method, a 3 -tube most probable number (MPN) method was used to estimate or detect o102 cfu ml 1 level L. monocytogenes in ClO2 -treated samples. At least three serial diluents (1 ml each diluent) of a blended sample were inoculated in 10 ml modi¢ed Listeria enrichment broth (LEBM) (Difco Laboratories, Division of Becton Dickinson and Company, Spark, USA) tubes, respectively. The growth of bacteria in each test tube was examined after incubation for 48 h at 371C. Positive (turbid) tube suspensions were streaked on to MOA plates and incubated at 371C for 24 h. Colonies were con¢rmed with the API Listeria procedure as described above. The L. monocytogenes populations were estimated using a ThreeTube MPN table (Swanson et al. 2001). An end point determination method was used to detect if inoculated L. monocytogenes were completely inactivated after the ClO2 gas treatment. A 100 ml blended sample was mixed with 100 ml double strength LEBM in a sterile stomacher bag, incubated at 371C for 1^2 days. Positive samples, which became turbid after incubation, were streaked on to MOA plates and
incubated at 371C for 24 h, followed by an API con¢rmation test. Negative samples that were clear after incubation were recorded as ‘none detected’.
Statistical analysis All experiments were completed as three or more replicates. The mean values of bacterial populations were calculated and reported with a 95% con¢dence interval. Data were subjected to analysis of variance and Student Newman Keuls’ (SNK) multiple range tests (SAS Inc., Cary, North Carolina, USA) to determine if signi¢cant di¡erences (Po0?05) in populations of L. monocytogenes existed between mean values.
Results and Discussion Inactivation of L. monocytogenes after treatment with 1^4 mg l 1 ClO2 gas for 10 min For an initial level of approximately 8 log cfu spotted site 1 spotted on to di¡erent apple surfaces, a 1?0, 3?0 or 4?0 mg l 1 ClO2 gas treatment for 10 min was studied at 211C and 90% relative humidity (RH) (Table 1). For 1?0^4?0 mg l 1 ClO2 gas concentration, there was no signi¢cant
Table 1. Populations and log reductionsa of L. monocytogenes spotted onto di¡erent apple surfaces after di¡erent ClO2 gas treatments for 10 minb;c Site on apple
ClO2 gas (mg l 1 )
Population after air-drying (log cfu spotted site 1 )d
Population after ClO2 treatment (log cfu spotted site 1 )d
Log reduction after ClO2 treatment (log cfu spotted site 1 )d
Calyx cavity
1?0 3?0 4?0
7?070?6AX 7?070?2AX 6?970?3AX
4?270?4AX 4?070?4AX 3?770?3AX
2?870?8AX 2?970?4AX 3?270?3AY
Stem cavity
1?0 3?0 4?0
6?670?6BX 7?170?4AX 6?870?3BXY
4?570?7AX 4?070?5AX 3?270?4BX
2?270?5BY 3?170?3AX 3?670?3AY
Pulp surface
1?0 3?0 4?0
7?270?1AX 7?270?1AX 6?570?2BY
4?070?7AX 3?970?6AX 0?971?0BY
3?270?7BX 3?370?6BX 5?571?0AX
a
Log reduction = population after air drying population after CO2 treatment. Values = means7standard deviations (n = 6). c Initial spotting cell level was approximately 8 log cfu spotted area 1 . d Values in the same column with di¡erent uppercase bold letters (A and B) are signi¢cantly di¡erent (Po0?05) for an identical apple site under di¡erent treatment conditions. Values in the same column with di¡erent uppercase bold letters (X and Y) are signi¢cantly di¡erent (Po0?05) for di¡erent sites of apples under the same treatment conditions. b
486 J. Du et al.
di¡erence (Po0?05) in log reductions for L. monocytogenes on the calyx cavities, resulting in a log reduction ranging from 2?870?8 to 3?270?3. This indicated that a certain level of the bacteria might be protected by the calyx cavities or ClO2 gas might not expose to and affect those deeply harboring bacteria in such a short exposure time (10 min). On the stem cavities, the 1?0 mg l 1 ClO2 gas treatment resulted in a lower log reduction (2?270?5) compared with the 3?0 or 4?0 mg l 1 treatment, which resulted in a 3?170?3 and a 3?670?4 log reduction, respectively. On pulp skins, the di¡erence between 1?0 and 3?0 mg l 1 ClO2 treatment was not signi¢cant (P40?05), more than 2 log reductions were obtained. However a 5?571?0 log reduction was observed on pulp skins after a 4?0 mg l 1 treatment, suggesting that bacteria attached to the pulp skin were more easily inactivated as ClO2 concentration was increased to 4?0 mg l 1 . The e¡ects of ClO2 concentration on di¡erent apple surfaces were also compared. At the 1?0 mg l 1 level, log reductions of L. monocytogenes were similar between the calyx cavity and pulp surface, which were slightly higher than those observed on the stem cavity. The numbers of inactivated bacteria on three sites
of apples showed no signi¢cant di¡erence (P40?05) after a 3?0 mg l 1 ClO2 gas treatment. However, when the concentration was increased to 4?0 mg l 1 , the ClO2 gas treatment led to a log reduction of 3?270?3 on calyx cavity and 3?670?4 on stem cavity and a 5?571?0 log reduction on pulp surface. The former two were not signi¢cantly di¡erent (P40?05). Therefore, 4?0 mg l 1 ClO2 gas for 10 min treatment matched our objective to inactive greater than a 3 log reduction for L. monocytogenes attached to calyx cavity, stem cavity and pulp surface of apples at 211C and 90% RH condition.
Inactivation of L. monocytogenes after treatment with 4?0 mg l 1 ClO2 gas for 30 min L. monocytogenes suspensions at an initial level of 6, 7, and 8 log (cfu spotted site 1 ) were separately inoculated on to each of the three parts of apples. The bacterial populations recovered after air-drying and ClO2 treatment are shown in Table 2. At an initial level of 6 log cfu spotted site 1, the recovered bacterial populations after airdrying were approximate 4 logs on the calyx
Table 2. Populations and log reductionsa of the L. monocytogenes spotted onto di¡erent apple surfaces after a 4?0 mg l 1 ClO2 gas treatment for 30 minb Site on apple
Calyx cavity Stem cavity Pulp skind
a
Population (log cfu spotted site 1 ) Inoculums spotted
After air drying
After ClO2 treatmentc
6?070?0 7?070?2 8?070?0 6?070?0 7?070?2 8?070?0 6?070?0 7?070?2 8?070?0
4?270?2 6?670?1 7?270?1 4?070?0 6?570?2 7?470?0 3?970?0 5?570?2 6?570?1
0?670?0B 2?770?0A 2?970?3A 0?770?2B 2?970?2A 3?071?0A None detected None detected None detected
Log reduction after ClO2 treatment (log cfu spotted site )c
3?670?2A 3?970?0A 4?370?2A 3?370?2A 3?670?4A 4?371?0A 43?970?0 45?570?2 46?570?1
Log reduction = population after air drying population after ClO2 treatment Values are means7standard deviations (n = 3). c Values in the same column for identical apples sites with di¡erent bold uppercase letters are signi¢cantly di¡erent (Po0?05). d After ClO2 gas treatment, no viable bacteria were detected on pulp skin by an end point method, indicating that the e¡ectiveness of ClO2 gas was higher than detection level. b
Inactivation of L. monocytogenes on apples 487
and stem cavities and the pulp skin of apples, which were not signi¢cantly di¡erent (P40?05). The bacteria populations with 6?670?1 and 6?570?2 logs on the calyx and stem cavities at an initial inoculation level of 7 log cfu spotted site 1 were recovered, which were approximate 1 log more than those to pulp skin (5?570?2 log cfu spotted site 1 ). At an initial level of 8 log cfu spotted site 1 , the recovered L. monocytogenes on pulp skins were 6?570?1 logs, while 7?270?1 logs on calyx cavities and 7?470?0 logs on stem cavities (Po0?05) were detected, respectively. The lower the inoculation level of bacteria was, the lower the percentage of populations could be recovered. In most cases, the recovered L. monocytogenes cells on pulp skin surface of apples after air-drying were less than those on two cavities at the same inoculation level. A possible explanation for lower recovery of L. monocytogenes on apple surface may be due to micro-pore structure of apple surface. When bacteria attach to the surface, they may be sheltered within the surface micro-pores (Foschino et al. 1998, Marcy et al. 2000). A recent study on the location of E. coli O157:H7 on and in apples using confocal scanning laser microscopy (CSLM) (Kenney et al. 2001) detected inoculated bacteria at depths up to 30 mm below the surface. Cells appeared to be sealed within naturally occurring cracks and crevices in waxy cutin platelets that may protect the former from sanitization. When the bacteria on calyx cavities at three di¡erent inoculated levels (6, 7, and 8 log cfu spotted site 1 ) were treated with 4?0 mg l 1 ClO2 gas for 30 min, no signi¢cant di¡erences (P40?05) in log reductions were found within the three inoculation levels. After the same treatment, 3?370?2, 3?670?4 and 4?371?1 log reductions were found on stem cavities in sequence (Table 2), which was very similar to the results on calyx cavities. Comparing the data of the calyx cavity with those of the stem cavity (Table 2) showed that no signi¢cant di¡erences (P40?05) were found in log reductions at the same inoculation level in our experiments. However, using an end point method, no viable bacteria were detected on pulp skin in all of the samples under the same test condition (Table 2). A level of 3?9^6?5 logs cells recovered after
air-drying was completely inactivated by ClO2 gas. In summary, 4?0 mg l 1 ClO2 gas for 30 min treatment achieved greater than a 3 log reduction for L. monocytogenes cells on both cavities and a 6?570?1 log reductions on pulp surfaces of apples.
Log reductions of L. monocytogenes after 8?0 mg l 1 ClO2 gas treatment Using a 6^8 log initial inoculation level, a 4?0 mg l 1 ClO2 gas treatment for 30 min inactivated 3^4 log cfu spotted area 1 on calyx and stem cavities. However, the same treatment condition could not completely inactivate all the 3^4 log attached L. monocytogenes cells on both cavities (Table 2). Therefore, 8 mg l 1 ClO2 gas treatment for 30 min at RH 90% and 211C was carried out in ¢ve replicate experiments and the results are shown in Table 3. At the recovery levels of 3?6^5?3 logs after air-drying on calyx cavities, 3?5^5?0 logs on stem cavities and 3?0^4?2 logs on pulp surfaces, no surviving bacteria harbored were detected using the end point method. It was suggested that the 8?0 mg l 1 ClO2 gas treatment for 30 min was highly e¡ective in inactivating L. monocytogenes; especially those harbored on calyx and stem cavities. Overall, ClO2 gas was a highly e¡ective sanitizer for the inactivation of L. monocytogenes on apple surfaces. In particular, it had a superior advantage in killing the bacteria attached to the calyx and stem cavities of apples. The ClO2 gas treatment with 4?0 mg l 1 for 10 min inactivated a 5?571?0 log cfu spotted site 1 L. monocytogenes on the pulp skin and about 3 log on both cavities. This supported our previous work (Han et al. 2001), in which more than 6 log cfu 5 g 1 L. monocytogenes on uninjured green pepper surfaces and about 3?5 log cfu 5 g 1 on injured surfaces were inactivated by 3 mg l 1 ClO2 gas treatment at 201C under 90^95% RH. Except for ClO2 gas and allyl isothiocyanate vapor (Lin et al. 2000b), no chemical treatments have been reported to achieve 5 logs reduction of bacteria on apple surfaces. Liao and Sapers (2000) reported that 6% hydrogen peroxide, which reduced the population of Salmonella Chester on apple skin by 3^4 logs
488 J. Du et al.
Table 3. Log reductionsa of the L. monocytogenes spotted onto di¡erent apple surfaces after a 8.0 mg l 1 ClO2 gas treatment for 30 minb Experiment
Log reduction (log cfu spotted site 1 )c
Initial inoculation (log cfu spotted site 1 )
1 2 3 4 5
Calyx cavity
Stem cavity
Pulp surface
45?3 44?6 44?5 43?8 43?6
45?0 44?7 44?0 43?8 43?5
44?2 43?6 43?4 43?1 43?0
7?0 6?8 6?7 6?0 5?8
a The data of log reduction listed in the table was the recovered populations of L. monocytogenes after airdrying detected by a surface plating method. b Data in the table were the average of duplicate measurements from two samples. No viable bacteria were detected after ClO2 gas treatment in all the experiments using an end point method. c The e¡ectiveness of ClO2 gas for inactivation of L. monocytogenes was higher than the detected level.
and the population of bacteria on stem or calyx by 1^2 logs, was the most e¡ective compared to 2% trisodium phosphate, 0?36% calcium hypochlorite, and 1?76% sodium hypochlorite. Sapers et al. (1999) also demonstrated that hydrogen peroxide could e¡ectively reduce 3^4 logs E. coli on apple surfaces while 200 ppm chlorinated water achieved only 2 logs reduction. Wisniewsky et al. (2000) reported that peroxyacetic acid, chlorine-phosphate buffer solution, and chlorine dioxide solution, when used at the recommended concentrations, could not reduce the recovered E. coli population by 5 logs under the test conditions. Under the conditions of this study, ClO2 gas treatment was an e¡ective sanitizer and should be considered as an alternative technology to decontaminate whole apples. Further investigations should be done to evaluate ClO2 gas treatment on other pathogens, such as E. coli O157:H7 and Salmonella, on apple surfaces and how the treatment a¡ects product quality.
Acknowledgements This research was supported by the USDA/ CSREES Grant No. 00 -51110 -9749.
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Ahamad, N. and E. H. Marth. (1990) Acid-injury of Listeria monocytogenes. J. Food Prot. 53, 26^29. Annous, B. A., Sapers, G. M., Mattrazzo, A. M. and Riordan, D. C. R. (2001) E⁄cacy of washing with a commercial £atbed brush washer, using conventional and experimental washing agents, in reducing populations of Escherichia coli on arti¢cially inoculated apples. J. Food Prot. 64, 159^163. Appendin, P. and Hotchkiss, J. H. (2000) Antimicrobial activity of a 14 -residue synthetic peptide against foodborne microorganisms. J. Food Prot. 63, 889^893. Bajard, S., Rosso, L., Fardel, G. and Flandrois, J. P. (1996) The particular behavior of Listeria monocytogenes under sub-optimal condition. Int. J. Food Microbiol. 29, 201^211. Berrang, M. E., Brackett, R. E. and Beuchat, L. R. (1989) Growth of Listeria monocytogenes on fresh vegetables stored under controlled atmosphere. J. Food Prot. 52, 702^705. Beuchat, L. R. and Brackett, R. E. (1990) Survival and growth of Listeria monocytogenes on lettuce as in£uenced by shredding, chlorine treatment, modi¢ed atmosphere packaging and temperature. J. Food Sci. 55, 755^758. Beuchat, L. R., Nail, B. V., Adler, B. B. and Clavero, M. R. S. (1998) E⁄cacy of spray application of chlorinated water in killing pathogenic bacteria on raw apples, tomatoes, and lettuce. J. Food Prot. 61, 1305^1311. Conway, W. S., Leverentz, B., Saftner, R. A., Janisiewicz,W. J., Sams, C. E. and Leblance E. (2000) Survival and growth of Listeria monocytogenes on fresh-cut apple slices and its interaction with Glomerella cingulata and Penicillium expansum. Plant Dis. 84, 177^181. Doyle, M. E., Mazzotta, A. S., Wang, T., Wiseman, D. W. and Scott,V. N. (2001) Heat resistance of Listeria monocytogenes. J. Food Prot. 64, 410^429.
Inactivation of L. monocytogenes on apples 489
Elphick, A. (1998) The growing use of chlorine dioxide. Processing. Erkmen, O. (2000) E¡ect of carbon dioxide pressure on Listeria monocytogenes in physiological saline and foods. Food Microbiol. 17, 589^596. Farber, J. M. and Pagotto, F. (1992) The e¡ect of acid shock on the heat resistance of Listeria monocytogenes. Lett. Appl. Microbiol. 15, 197^201. Food and Drug Administration (FDA). (1998) Hazard analysis and critical control points (HACCP); procedures for the safe and sanitary processing and importing of juice. Fed. Regist. 63, 20 450^ 20 486. Food and Drug Administration (FDA). (2001) FDA publishes ¢nal rule to increase safety of fruit and vegetable juices. HHS News. Jan. 18, 2001. http://vm.cfsan.fda.gov/Blrd/hhsjuic4.html Foschino, P. M., Nervegna, I., Motta, A. and Galli, A. (1998) Bactericidal activity of chlorine dioxide against Escherichia coli in water and hard surfaces. J. Food Prot. 61, 668^672. Han, Y., Guentert, A. M., Smith, R. S., Linton, R. H. and Nelson, P. E. (1999) E⁄cacy 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. and Nelson, P. E. (2000b) Inactivation of Escherichia coli O157:H7 on surface-uninjured and -injured green pepper (Capsicum annuum L.) by chlorine dioxide gas as demonstrated by confocal laser scanning microscopy. Food Microbiol. 17, 643^655. Han,Y., Linton, R. H., Nielsen, S. S. and 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 71C. J. Food Prot. 64, 1730^1738. Han,Y., Sherman, D. M., Linton, R. H., Nielsen, S. S. and Nelson, P. E. (2000a) The e¡ects of washing and chlorine dioxide gas on survival and attachment of Escherichia coli O157:H7 to green pepper surfaces. Food Microbiol. 17, 521^533. Heisick, J. E.,Wagner, D. E., Nierman, M. L. and Peeler, J. T. (1989) Listeria spp. found on fresh market produce. Appl. Environ. Microbiol. 55, 1925^1927. Ho, J. L., Shands, K. N., Friedland, G., Eckind, P. and Fraser, D.W. (1986) An outbreak of type 4b Listeria monocytogenes infection involving patients from eight Boston hospitals. Arch. Int. Med. 146, 520^524. Hwang, E. S., Cash, J. N. and Zabik, M. J. (2001) Postharvest treatments for the reduction of mancozeb in fresh apples. J. Agri. Food Chem. 49, 3127^3132. Kenney, S. J., Burnett, S. L. and Beuchat, L. R. (2001) Location of Escherichia coli O157:H7 on and in apples as a¡ected by bruising, washing, and rubbing. J. Food Prot. 64, 1328^1333. Liao, C. and Sapers, G. M. (2000) Attachment and growth of Salmonella Chester on apple fruits and in vivo response of attached bacteria to sanitizer treatment. J. Food Prot. 63, 876^883.
Lin, C., Kim, J., Du,W. and Wei, C. (2000a) Bactericidal activity of isothiocyanate against pathogens on fresh produce. J. Food Prot. 63, 25^30. Lin, C., Preston III, J. F. and Wei, C. (2000b) Antibacterial mechanism of allyl isothiocyanate. J. Food Prot. 63, 727^734. Lu, J., Mittal, G. S. and Gri⁄ths, M.W. (2001) Reduction in levels of Escherichia coli O157:H7 in apple cider by pulsed electric ¢elds. J. Food Prot. 64, 964^969. Marcy, A. W., Bonatta, A. G., Mark, L. G. and Cheryll, A. R. (2000) Reduction of Escherichia coli O157:H7 on whole fresh apples by treatment with sanitizers. J. Food Prot. 63, 703^708. Mazzotta, A. S. (2001) Thermal inactivation of stationary-phase and acid-adapted Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in fruit juices. J. Food Prot. 64, 315^320. Porto, E. and Uboldi, E. M. N. (2001) Occurrence of Listeria monocytogenes in vegetables. Dairy Food Environ. Sanitat. 21, 282^286. Sado, P. N., Jinneman, K. C., Husby, G. J., Sorg, S. M. and Omiecinski, C. J. (1998) Identi¢cation of Listeria monocytogenes from unpasturized apple juice using rapid test kits. J. Food Prot. 61, 1199^1202. Sapers, G. M., Miller, R. L. and Mattrazzo, A. M. (1999) E¡ectiveness of sanitizing agents in inactivating Escherichia coli in golden delicious apples. J. Food Sci. 64, 734^737. Schaik, W. van, Gahan, C. G. M. and Hill, C. (1999) Acid-adapted Listeria monocytogenes displays enhanced tolerance against the antibiotics nisin and lacticin 3147. J. Food Prot. 62, 536^539. Schlech, W. F., Lavigne, P. M., Bortolussi, R. A., Allen, A. C., Haldene, E.V.,Wort, A. J., Hightower, A. W., Johnston, S. E., King, S. H., Nicholls, E. S. and Broome, C. V. (1983) Epidemic listeriosis F evidence for transmission by food. N. Engl. J. Med. 308, 203^206. Sorrells, K. M., Enigl, D. C. and Hat¢ed, J. R. (1989) E¡ect of pH, acidulant, time and temperature on the growth and survival of Listeria monocytogenes. J. Food Prot. 52, 571^573. Swanson, K. M. J., Petran, R. L. and Hanlin, J. H. (2001) Culture methods for enumeration of microorganisms. In Compendium of Methods for the Microbiological Examination of Foods 4th edn (Eds F. P. Downes and K. Ito).Washington, DC, American Public Health Association. Taormina, P. J. and Beuchat, L. R. (2001) Survival and heat resistance of Listeria monocytogenes after exposure to alkali and chlorine. Appl. Environ. Microbiol. 67, 2555^2563. Walker, S. J., Archer, P. and Banks, J. G. 1990. Growth of Listeria monocytogenes at refrigeration temperature. J. Appl. Bacteriol. 68, 157^162. Wright, J. R., Sumner, S. S., Hackney, C. R., Pierson, M. D., and Zoecklein, B. W. (2000a) E⁄cacy of ultraviolet light for reducing Escherichia coli
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O157:H7 in unpasteurized apple cider. J. Food Prot. 63, 563^567. Wright, J. R., Sumner, S. S., Hackney, C. R., Pierson, M. D. and Zoecklein, B. W. (2000b) Reduction of Escherichia coli O157:H7 on apples using wash and chemical sanitizer treatments. Dairy Food Environ. Sanitat. 20, 120^126.
Wisniewsky, M. A., Glatz, B. A., Gleason, M. L. and Reitmeier, C. A. (2000) Reduction of Escherichia coli O157:H7 counts on whole fresh apples by treatment with sanitizers. J. Food Prot. 63, 703^ 708. Wong, A. C. L. (1998) Bio¢lms in food processing environments. J. Dairy Sci. 81, 2765^2770.
doi:10.1006/yfmic.501
ORIGINAL ARTICLE
Inactivation by chlorine dioxide gas (ClO2) of Listeria monocytogenes spotted onto di¡erent apple surfacesw Jinhua Du,Y. Han and R. H. Linton*
The bactericidal e¡ects of chlorine dioxide (ClO2) gas treatments on a mixture of three strains of Listeria monocytogenes (Scott A, F5069 and LCDC 81-886) spotted on to the calyx, stem cavity and pulp surface of apples were investigated at 211C and 90% relative humidity (RH). ClO2 gas was more e¡ective for inactivating the bacteria attached to pulp skin than those attached to the calyx or stem cavity at a ClO2 level of 4?0 mg l 1 and a treatment time of 10 or more minutes. After treatments of 1?0^4?0 mg l 1 ClO2 for10 min, a 2^3 log reduction of L. monocytogenes were observed on both cavities. There were 4?370?2 and 4?371?1 log reductions of L. monocytogenes achieved on the calyx and stem cavities, respectively, by a 4?0 mg l 1 ClO2 gas treatment for 30 min. After treatment of 8?0 mg l 1 ClO2 for 30 min, no survivors were detected using an end point method for 3?6^5?3 log cfu spotted site 1 on the calyx cavity and 3?5^5?0 log cfu spotted site 1 on the stem cavity. No signi¢cant di¡erences (P40?05) were found between bacterial log reductions on pulp skin at 1?0 or 3?0 mg l 1 ClO2 gas treatment for 10 min. When the ClO2 level was increased to 4?0 mg l 1 for 10 min, a 5?571?0 log cfu spotted site 1 bacteria on pulp skin was inactivated. After 4.0 mg l 1 ClO2 gas for 30 min, L. monocytogenes levels on the pulp skin could be decreased by 3?970?0 to 6.570.1log cfu spotted site 1 using an end point determination method. ClO2 gas was a potentially powerful sanitizer for inactivating L. monocytogenes on each apple surface, especially those attached to the pulp skin. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction Listeria monocytogenes is a Gram-positive foodborne bacterium, which has been isolated from soil, silage, and several other environmental sources. It is able to grow at refrigerated temperatures down to 01C (Berrang et al. 1989, Beuchat and Brackett 1990, Walker et al. 1990, *Corresponding author. Fax: (765)- 494 -7953; E-mail: [email protected] w This paper is journal article #16675 of the Purdue University Agricultural Research Program. 0740 -0020/02/050481+10 $35.00/0
Bajard et al. 1996), on dry surfaces (Wong 1998), can tolerate high acidity (Ahamad and Marth 1989, 1990, Sorrells et al. 1989, Farber and Pagotto 1992, Schaik et al. 1999) or alkalized conditions (Taormina and Beuchat 2001), and high heat (Doyle et al. 2001). These characteristics make L. monocytogenes well adapted to food and food processing environments where the growth of other pathogens may be restricted. Incidences of L. monocytogenes infections have been associated with fruits and vegetables, such as coleslaw and cabbage (Schlech r 2002 Elsevier Science Ltd. All rights reserved.
Received: 30 November 2001 Department of Food Science, Purdue University,1160 Food Science Building, West Lafayette, Indiana 47907-1160, USA
482 J. Du et al.
et al. 1983), lettuce, celery, tomatoes (Ho et al. 1986), cabbage, cucumbers, potatoes, radishes (Heisick et al. 1989), and other vegetables (Porto and Uboldi 2001). These outbreaks were likely caused by bacterial contamination or by cross-contamination during handling. Raw apple cider has been identi¢ed as a source of bacterial contamination in the U.S. leading to foodborne illness and death.Three L. monocytogenes isolates have been detected in two kinds of retail unpasteurized apple juices (pH 3?75) (Sado et al. 1998). L. monocytogenes could also survive and grow on freshly cut apple (cv. Delicious) slices and in apple juice (Conway et al. 2000). Beuchat et al. (1998) reported that L. monocytogenes on apple surfaces had greater resistance to chlorine compared to Escherichia coli O157:H7 and Salmonella. Mazzotta (2001) showed that the heat resistance of L. monocytogenes increased signi¢cantly (Po0?05) after acid adaptation in single-strength apple, orange and white grape juices adjusted to pH 3?9. Therefore, L. monocytogenes should be considered as a potential threat to the safety of apple cider or juices. More recently, the FDA (Food and Drug Administration 2001) has established requirements to increase the safety of fruit and vegetable juices. Here, processors are required to use processes that achieve a 5 log reduction in the numbers of ‘the most resistant pathogen’ in their ¢nished products compared with levels that may be present in untreated juice. Juice processors may use microbial reduction methods other than pasteurization, including approved alternative technologies such as the recently approved ultraviolet (UV) irradiation technology or a combination of these technologies. In the apple beverage production, heat pasteurization is very e¡ective in achieving a 5 log reduction if used as a ¢nal processing step, but this process may a¡ect the taste and appearance, or add to the cost of the ¢nished products. Current methods used to preserve fresh apple beverages include refrigeration and addition of preservatives such as potassium sorbate and sodium benzoate, which are usually used to prolong the shelf life of the beverages. Researchers have reported that UV light can result in a 3?81 log reduction of E.
O157:H7 in unpasteurized cider (Wright et al. 2000a). There are many studies concentrated on reducing pathogen load of fresh fruit juices. One approach is the direct treatment of fresh juices with non-thermal methods such as pulsed electric ¢elds (Lu et al. 2001) and carbon dioxide pressure (Erkmen 2000). Other approaches include direct addition of new antimicrobials such as peptides (Appendin and Hotchkiss 2000). Chemical sanitization of whole apples before grinding is an alternative method to reduce microbial load. However, many conventional sanitizers or their combinations, such as chlorine (Beuchat et al. 1998), hydrogen peroxide and trisodium phosphate (Sapers et al. 1999, Liao and Sapers 2000, Annous et al. 2001), peroxyacetic acid and chlorine-phosphate bu¡er (Wisniewsky et al. 2000), acetic acid and peroxyacetic acid (Wright et al. 2000b), and allyl isothiocyanate (Lin et al. 2000 a, b) were not able to reduce L. monocytogenes, E. coli O157:H7, or Salmonella populations on apple surfaces by 5 logs at the recommended concentration under the test conditions. Therefore, the development of highly e¡ective sanitation technology for the reduction of pathogens on apples is needed (Annous et al. 2001). As a potential sanitizer for decontamination of fruits and vegetables, ClO2 gas has many advantages over traditional chlorinated water, such as 1?5 times more than the oxidation capacity of chlorine, less reactive than chlorine with organic compounds, e¡ectiveness at low concentration, non-conversion to chlorophenols that result in residual smells and £avors, and inability to react with ammonia or humic acid to produce harmful chloramines and trihalomethanes (Elphick 1998). ClO2 gas also has a strong penetrability and high dissolubility. It was highly e¡ective in reducing foodborne pathogens such as L. monocytogenes and E. coli O157:H7 on green pepper surfaces (Han et al. 2000a, b, 2001) and food spoilage microorganisms on epoxy-coated juice storage tank surface (Han et al. 1999). Furthermore, ClO2 has the ability to reduce fungicide residues, mancozeb and ethylenethiourea, in apples (Hwang et al. 2001). Therefore, ClO2 gas may be an e¡ective alternative sanitizer to reduce microbial population on apple surfaces.
Inactivation of L. monocytogenes on apples 483
As the National Advisory Committee on Microbiological Criteria for Foods recommended, L. monocytogenes should be used as target organism in the absence of known speci¢c pathogen product associations, as appropriate (Food and Drug Administration 1998). In this research, the e⁄cacy of ClO2 gas to inactive the L. monocytogenes cells spotted on to the calyx and, stem cavities and the pulp surface of apples was investigated. The objective of this study was to determine the ClO2 gas concentration and exposure time needed to achieve a three or more log reduction of L. monocytogenes on these three di¡erent apple surfaces using di¡erent ClO2 gas treatments.
Materials and Methods Bacterial strains Three L. monocytogenes strains were chosen and used as a mixture. L. monocytogenes Scott A was provided by Dr L. R. Beuchat (University of Georgia, Gri⁄n, Georgia, USA). L. monocytogenes F5069 and LCDC 81^886 were obtained from Dr. Arun Bhunia of Purdue University, West Lafayette, Indiana, USA. This mixture of strains was chosen to account for potential differences in resistance to ClO2 gas treatments. All cultures were maintained on slants containing tryptic soy agar (TSA, Difco Laboratories, Division of Becton Dickinson and Company, Detroit, Michigan, USA) plus 0?6% (wt vol 1 ) yeast extract (YE, Difco Laboratories) at 41C.
ium base with Oxford antimicrobiotic supplement (MOA, Difco Laboratories, Division of Becton Dickinson and Company, Spark, Maryland, USA) to determine the population (cfu ml 1 ) of the culture.The mixed suspension was serially diluted with 0?1% peptone solution. Then 0?1 ml diluents were surface-spread on to MOA plates in duplicate. The plates were incubated at 371C for approximately 24^48 h before counting.
Inoculation of apples Unwaxed Red Delicious apples were obtained from a local supermarket. Apples free of physical damage and debris were selected, rinsed with tap water for 1 min and dried with a paper towel. As shown in Fig. 1, three parts of an apple, including the calyx and stem cavities, and the skin or surface of pulp were inoculated with 100 ml droplets of the mixed culture suspension in a class II bio-safety cabinet. The populations inoculated were approximately 106 ^ 108 cfu per inoculation spotted site (approx. 2 cm in diameter). The inoculated cultures on apple surfaces were air-dried at 211C for 2^3 h in the bio-safety cabinet prior to treatment. The levels of indigenous microorganisms on the calyx and stem cavities, and the pulp skin surface of apples were approximately 105, 104, and o103 cfu 5 g 1, respectively. No indigenous L. monocytogenes cells were detected.
Growth conditions and viable counts Each test strain was aseptically transferred into 10 ml tryptic soy broth with 0?6% (wt vol 1 ) yeast extract (TSB-YE, Difco Laboratories, Division of Becton Dickinson and Company, Detroit, Michigan, USA) and incubated at 371C for 24 h, followed by a minimum of two successive transfers to gain a stationary-phase culture. To make a composite of the three strains, an equal volume of each culture was mixed together. Before mixing, each culture was enumerated by surface-plating duplicate samples on to tryptic soy agar and Oxford med-
Figure 1. Inoculation sites (calyx and stem cavities, and pulp skin) for L. monocytogenes on apple surfaces.
484 J. Du et al.
Chlorine dioxide (ClO2) gas treatment ClO2 gas treatment was carried out in a 10 l Irvine Plexiglass cylinder with a stainlesssteel shelf, where four apples were placed on the ¢rst mesh screen (Fig. 2). A Thermo-Hydro recorder (Control Company, Friendwood, Texas, USA) was used to monitor relative humidity (RH) and temperature inside the treatment cylinder. Using a diaphragm vacuum pump (KNF Neuberger, Inc., Treton, New Jersey, USA), a 90^95% RH was achieved by circulating the air inside the cylinder through a 125 ml washing bottle with 100 ml deionized water for 10^15 min. ClO2 gas with a concentration of 160 ^180 mg l 1 was produced based on the reaction of chlorine gas (4% in nitrogen gas) with sodium hypochlorite in a CDG laboratory generator (CDG Technology, Inc., New York, USA). A 60 ml plastic gas-sampling syringe was used to deliver speci¢c volumes of ClO2 gas into the cylinder. During the treatment, the ClO2 gas inside the cylinder was circulated by the diaphragm vacuum pump, which entered from the top of the cylinder and £owed out from the bottom.The cylinder was placed in the dark to prevent light-decomposition of the ClO2. During treatment, relative humidity and temperature were maintained at 90^95% and 211C inside the treatment cylinder, respectively.
Measurement of ClO2 gas concentration A DPD (N, N-diethyl-r-phenylenediamine) colorimetric analysis kit (CHEMetrics, Inc., Cal-
Figure 2. A 10 l ClO2 gas treatment system used to treat the apples inoculated with L. monocytogenes.
verton,Virgina, USA) was used to measure the ClO2 gas concentration. Using a 25 ml gas-sampling syringe, 15 ml ClO2 gas from the treatment cylinder was immediately dissolved in 15 ml neutralized deionized water. Before injecting the gas into the water, some water was repeatedly drawn in and out the syringe to dissolved the gas completely. The initial ClO2 gas concentration was measured in duplicate and recorded as mg ClO2 l 1 .
Enumeration or detection methods of L. monocytogenes on apple surfaces A sterile fruit knife was used to cut each inoculated area to obtain a 5 -g sample size. For the control samples untreated with ClO2, each cut piece was mixed with 95 ml sterile 0?1% peptone solution in a 400 ml sterile stomacher bag (Fisher Scienti¢c Inc.). For ClO2 -treated samples, the cut piece was mixed with 95 ml neutralizing bu¡er (Difco Laboratories, Division of Becton Dickinson and Company, Detroit, Michigan, USA) to neutralize residual ClO2 on the sample, and then blended for 2 min at 260 rpm using a Seward Stomacher 400 Circulator (Steward Limited, London, UK). The blended sample was serially diluted with 0?1% peptone and surface-plated in duplicate on to MOA plates. These plates were incubated at 371C for 24^48 h. For each plate, typical colonies were chosen and identi¢ed as L. monocytogenes by an API Listeria Test (BioMerieux Vitec, Hazelwood, Missouri, USA). All the populations of L. monocytogenes were recorded as colony forming units per spotted site (cfu spotted site 1 ). To resuscitate and enumerate ClO2 -injured L. monocytogenes cells, a membrane-transferring plating method was used (Han et al. 2001). One hundred microliters of the stomached sample suspension or its diluents were spread over a sterile polycarbonate ¢lter membrane (0?4 mm pore size, track-etched, 90 mm diameter) (Osmonic Co.,Westboro, Massachusetts, USA) previously placed on the surface of a TSA plate. Plates were incubated at 371C for 4 h to repair injured cells. Then the membranes were gently and aseptically transferred onto MOA plates using sterile forceps. The membrane^ MOA plates were further incubated at 371C
Inactivation of L. monocytogenes on apples 485
for 24^36 h. L. monocytogenes colonies were counted after the same API Listeria procedure as described above. Because only Z102 cfu ml 1 level bacteria could be counted using the membrane-transferring plating method, a 3 -tube most probable number (MPN) method was used to estimate or detect o102 cfu ml 1 level L. monocytogenes in ClO2 -treated samples. At least three serial diluents (1 ml each diluent) of a blended sample were inoculated in 10 ml modi¢ed Listeria enrichment broth (LEBM) (Difco Laboratories, Division of Becton Dickinson and Company, Spark, USA) tubes, respectively. The growth of bacteria in each test tube was examined after incubation for 48 h at 371C. Positive (turbid) tube suspensions were streaked on to MOA plates and incubated at 371C for 24 h. Colonies were con¢rmed with the API Listeria procedure as described above. The L. monocytogenes populations were estimated using a ThreeTube MPN table (Swanson et al. 2001). An end point determination method was used to detect if inoculated L. monocytogenes were completely inactivated after the ClO2 gas treatment. A 100 ml blended sample was mixed with 100 ml double strength LEBM in a sterile stomacher bag, incubated at 371C for 1^2 days. Positive samples, which became turbid after incubation, were streaked on to MOA plates and
incubated at 371C for 24 h, followed by an API con¢rmation test. Negative samples that were clear after incubation were recorded as ‘none detected’.
Statistical analysis All experiments were completed as three or more replicates. The mean values of bacterial populations were calculated and reported with a 95% con¢dence interval. Data were subjected to analysis of variance and Student Newman Keuls’ (SNK) multiple range tests (SAS Inc., Cary, North Carolina, USA) to determine if signi¢cant di¡erences (Po0?05) in populations of L. monocytogenes existed between mean values.
Results and Discussion Inactivation of L. monocytogenes after treatment with 1^4 mg l 1 ClO2 gas for 10 min For an initial level of approximately 8 log cfu spotted site 1 spotted on to di¡erent apple surfaces, a 1?0, 3?0 or 4?0 mg l 1 ClO2 gas treatment for 10 min was studied at 211C and 90% relative humidity (RH) (Table 1). For 1?0^4?0 mg l 1 ClO2 gas concentration, there was no signi¢cant
Table 1. Populations and log reductionsa of L. monocytogenes spotted onto di¡erent apple surfaces after di¡erent ClO2 gas treatments for 10 minb;c Site on apple
ClO2 gas (mg l 1 )
Population after air-drying (log cfu spotted site 1 )d
Population after ClO2 treatment (log cfu spotted site 1 )d
Log reduction after ClO2 treatment (log cfu spotted site 1 )d
Calyx cavity
1?0 3?0 4?0
7?070?6AX 7?070?2AX 6?970?3AX
4?270?4AX 4?070?4AX 3?770?3AX
2?870?8AX 2?970?4AX 3?270?3AY
Stem cavity
1?0 3?0 4?0
6?670?6BX 7?170?4AX 6?870?3BXY
4?570?7AX 4?070?5AX 3?270?4BX
2?270?5BY 3?170?3AX 3?670?3AY
Pulp surface
1?0 3?0 4?0
7?270?1AX 7?270?1AX 6?570?2BY
4?070?7AX 3?970?6AX 0?971?0BY
3?270?7BX 3?370?6BX 5?571?0AX
a
Log reduction = population after air drying population after CO2 treatment. Values = means7standard deviations (n = 6). c Initial spotting cell level was approximately 8 log cfu spotted area 1 . d Values in the same column with di¡erent uppercase bold letters (A and B) are signi¢cantly di¡erent (Po0?05) for an identical apple site under di¡erent treatment conditions. Values in the same column with di¡erent uppercase bold letters (X and Y) are signi¢cantly di¡erent (Po0?05) for di¡erent sites of apples under the same treatment conditions. b
486 J. Du et al.
di¡erence (Po0?05) in log reductions for L. monocytogenes on the calyx cavities, resulting in a log reduction ranging from 2?870?8 to 3?270?3. This indicated that a certain level of the bacteria might be protected by the calyx cavities or ClO2 gas might not expose to and affect those deeply harboring bacteria in such a short exposure time (10 min). On the stem cavities, the 1?0 mg l 1 ClO2 gas treatment resulted in a lower log reduction (2?270?5) compared with the 3?0 or 4?0 mg l 1 treatment, which resulted in a 3?170?3 and a 3?670?4 log reduction, respectively. On pulp skins, the di¡erence between 1?0 and 3?0 mg l 1 ClO2 treatment was not signi¢cant (P40?05), more than 2 log reductions were obtained. However a 5?571?0 log reduction was observed on pulp skins after a 4?0 mg l 1 treatment, suggesting that bacteria attached to the pulp skin were more easily inactivated as ClO2 concentration was increased to 4?0 mg l 1 . The e¡ects of ClO2 concentration on di¡erent apple surfaces were also compared. At the 1?0 mg l 1 level, log reductions of L. monocytogenes were similar between the calyx cavity and pulp surface, which were slightly higher than those observed on the stem cavity. The numbers of inactivated bacteria on three sites
of apples showed no signi¢cant di¡erence (P40?05) after a 3?0 mg l 1 ClO2 gas treatment. However, when the concentration was increased to 4?0 mg l 1 , the ClO2 gas treatment led to a log reduction of 3?270?3 on calyx cavity and 3?670?4 on stem cavity and a 5?571?0 log reduction on pulp surface. The former two were not signi¢cantly di¡erent (P40?05). Therefore, 4?0 mg l 1 ClO2 gas for 10 min treatment matched our objective to inactive greater than a 3 log reduction for L. monocytogenes attached to calyx cavity, stem cavity and pulp surface of apples at 211C and 90% RH condition.
Inactivation of L. monocytogenes after treatment with 4?0 mg l 1 ClO2 gas for 30 min L. monocytogenes suspensions at an initial level of 6, 7, and 8 log (cfu spotted site 1 ) were separately inoculated on to each of the three parts of apples. The bacterial populations recovered after air-drying and ClO2 treatment are shown in Table 2. At an initial level of 6 log cfu spotted site 1, the recovered bacterial populations after airdrying were approximate 4 logs on the calyx
Table 2. Populations and log reductionsa of the L. monocytogenes spotted onto di¡erent apple surfaces after a 4?0 mg l 1 ClO2 gas treatment for 30 minb Site on apple
Calyx cavity Stem cavity Pulp skind
a
Population (log cfu spotted site 1 ) Inoculums spotted
After air drying
After ClO2 treatmentc
6?070?0 7?070?2 8?070?0 6?070?0 7?070?2 8?070?0 6?070?0 7?070?2 8?070?0
4?270?2 6?670?1 7?270?1 4?070?0 6?570?2 7?470?0 3?970?0 5?570?2 6?570?1
0?670?0B 2?770?0A 2?970?3A 0?770?2B 2?970?2A 3?071?0A None detected None detected None detected
Log reduction after ClO2 treatment (log cfu spotted site )c
3?670?2A 3?970?0A 4?370?2A 3?370?2A 3?670?4A 4?371?0A 43?970?0 45?570?2 46?570?1
Log reduction = population after air drying population after ClO2 treatment Values are means7standard deviations (n = 3). c Values in the same column for identical apples sites with di¡erent bold uppercase letters are signi¢cantly di¡erent (Po0?05). d After ClO2 gas treatment, no viable bacteria were detected on pulp skin by an end point method, indicating that the e¡ectiveness of ClO2 gas was higher than detection level. b
Inactivation of L. monocytogenes on apples 487
and stem cavities and the pulp skin of apples, which were not signi¢cantly di¡erent (P40?05). The bacteria populations with 6?670?1 and 6?570?2 logs on the calyx and stem cavities at an initial inoculation level of 7 log cfu spotted site 1 were recovered, which were approximate 1 log more than those to pulp skin (5?570?2 log cfu spotted site 1 ). At an initial level of 8 log cfu spotted site 1 , the recovered L. monocytogenes on pulp skins were 6?570?1 logs, while 7?270?1 logs on calyx cavities and 7?470?0 logs on stem cavities (Po0?05) were detected, respectively. The lower the inoculation level of bacteria was, the lower the percentage of populations could be recovered. In most cases, the recovered L. monocytogenes cells on pulp skin surface of apples after air-drying were less than those on two cavities at the same inoculation level. A possible explanation for lower recovery of L. monocytogenes on apple surface may be due to micro-pore structure of apple surface. When bacteria attach to the surface, they may be sheltered within the surface micro-pores (Foschino et al. 1998, Marcy et al. 2000). A recent study on the location of E. coli O157:H7 on and in apples using confocal scanning laser microscopy (CSLM) (Kenney et al. 2001) detected inoculated bacteria at depths up to 30 mm below the surface. Cells appeared to be sealed within naturally occurring cracks and crevices in waxy cutin platelets that may protect the former from sanitization. When the bacteria on calyx cavities at three di¡erent inoculated levels (6, 7, and 8 log cfu spotted site 1 ) were treated with 4?0 mg l 1 ClO2 gas for 30 min, no signi¢cant di¡erences (P40?05) in log reductions were found within the three inoculation levels. After the same treatment, 3?370?2, 3?670?4 and 4?371?1 log reductions were found on stem cavities in sequence (Table 2), which was very similar to the results on calyx cavities. Comparing the data of the calyx cavity with those of the stem cavity (Table 2) showed that no signi¢cant di¡erences (P40?05) were found in log reductions at the same inoculation level in our experiments. However, using an end point method, no viable bacteria were detected on pulp skin in all of the samples under the same test condition (Table 2). A level of 3?9^6?5 logs cells recovered after
air-drying was completely inactivated by ClO2 gas. In summary, 4?0 mg l 1 ClO2 gas for 30 min treatment achieved greater than a 3 log reduction for L. monocytogenes cells on both cavities and a 6?570?1 log reductions on pulp surfaces of apples.
Log reductions of L. monocytogenes after 8?0 mg l 1 ClO2 gas treatment Using a 6^8 log initial inoculation level, a 4?0 mg l 1 ClO2 gas treatment for 30 min inactivated 3^4 log cfu spotted area 1 on calyx and stem cavities. However, the same treatment condition could not completely inactivate all the 3^4 log attached L. monocytogenes cells on both cavities (Table 2). Therefore, 8 mg l 1 ClO2 gas treatment for 30 min at RH 90% and 211C was carried out in ¢ve replicate experiments and the results are shown in Table 3. At the recovery levels of 3?6^5?3 logs after air-drying on calyx cavities, 3?5^5?0 logs on stem cavities and 3?0^4?2 logs on pulp surfaces, no surviving bacteria harbored were detected using the end point method. It was suggested that the 8?0 mg l 1 ClO2 gas treatment for 30 min was highly e¡ective in inactivating L. monocytogenes; especially those harbored on calyx and stem cavities. Overall, ClO2 gas was a highly e¡ective sanitizer for the inactivation of L. monocytogenes on apple surfaces. In particular, it had a superior advantage in killing the bacteria attached to the calyx and stem cavities of apples. The ClO2 gas treatment with 4?0 mg l 1 for 10 min inactivated a 5?571?0 log cfu spotted site 1 L. monocytogenes on the pulp skin and about 3 log on both cavities. This supported our previous work (Han et al. 2001), in which more than 6 log cfu 5 g 1 L. monocytogenes on uninjured green pepper surfaces and about 3?5 log cfu 5 g 1 on injured surfaces were inactivated by 3 mg l 1 ClO2 gas treatment at 201C under 90^95% RH. Except for ClO2 gas and allyl isothiocyanate vapor (Lin et al. 2000b), no chemical treatments have been reported to achieve 5 logs reduction of bacteria on apple surfaces. Liao and Sapers (2000) reported that 6% hydrogen peroxide, which reduced the population of Salmonella Chester on apple skin by 3^4 logs
488 J. Du et al.
Table 3. Log reductionsa of the L. monocytogenes spotted onto di¡erent apple surfaces after a 8.0 mg l 1 ClO2 gas treatment for 30 minb Experiment
Log reduction (log cfu spotted site 1 )c
Initial inoculation (log cfu spotted site 1 )
1 2 3 4 5
Calyx cavity
Stem cavity
Pulp surface
45?3 44?6 44?5 43?8 43?6
45?0 44?7 44?0 43?8 43?5
44?2 43?6 43?4 43?1 43?0
7?0 6?8 6?7 6?0 5?8
a The data of log reduction listed in the table was the recovered populations of L. monocytogenes after airdrying detected by a surface plating method. b Data in the table were the average of duplicate measurements from two samples. No viable bacteria were detected after ClO2 gas treatment in all the experiments using an end point method. c The e¡ectiveness of ClO2 gas for inactivation of L. monocytogenes was higher than the detected level.
and the population of bacteria on stem or calyx by 1^2 logs, was the most e¡ective compared to 2% trisodium phosphate, 0?36% calcium hypochlorite, and 1?76% sodium hypochlorite. Sapers et al. (1999) also demonstrated that hydrogen peroxide could e¡ectively reduce 3^4 logs E. coli on apple surfaces while 200 ppm chlorinated water achieved only 2 logs reduction. Wisniewsky et al. (2000) reported that peroxyacetic acid, chlorine-phosphate buffer solution, and chlorine dioxide solution, when used at the recommended concentrations, could not reduce the recovered E. coli population by 5 logs under the test conditions. Under the conditions of this study, ClO2 gas treatment was an e¡ective sanitizer and should be considered as an alternative technology to decontaminate whole apples. Further investigations should be done to evaluate ClO2 gas treatment on other pathogens, such as E. coli O157:H7 and Salmonella, on apple surfaces and how the treatment a¡ects product quality.
Acknowledgements This research was supported by the USDA/ CSREES Grant No. 00 -51110 -9749.
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