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Inactivation of Listeria monocytogenes during drying and storage of peach slices treated with acidic or sodium metabisulfite solutions
ARTICLE IN PRESS FOOD MICROBIOLOGY Food Microbiology 21 (2004) 641–648
www.elsevier.nl/locate/jnlabr/yfmic
Inactivation of Listeria monocytogenes during drying and storage of peach slices treated with acidic or sodium metabisulfite solutions Patricia A. DiPersioa, Patricia A. Kendalla,*, John N. Sofosb a
Department of Food Science and Human Nutrition, Colorado State University, Fort Collins,Colorado 80523, USA b Department of Animal Sciences, Colorado State University, Fort Collins, Colorado 80523, USA Accepted 14 March 2004
Abstract This study evaluated whether treating inoculated peach slices with metabisulfite or acidic solutions enhanced inactivation of Listeria monocytogenes during dehydration and storage. Inoculated (five strain mixture of L. monocytogenes, 7.9 log cfu/g) peach slices were treated, dried for 6 h at 60 C and stored aerobically at 25 C for 14 d. Predrying treatments of inoculated peach slices included: (1) no treatment (control); or 10 min immersion in: (2) sterile water, (3) 4.18% sodium metabisulfite, (4) 3.40% ascorbic acid, or (5) 0.21% citric acid solutions. Samples were plated on tryptic soy agar with 0.1% pyruvate (TSAP) and PALCAM agar for enumeration of surviving bacteria. Immersion in sterile water reduced bacterial populations on peach slices by 0.7 log cfu/g (TSAP and PALCAM). Immersion in the sodium metabisulfite solution reduced populations by 1.5–2.0 log cfu/g, while acidic pretreatments reduced populations by 0.5–0.8 log cfu/g. After 6 h of dehydration, populations on control or water immersed slices were reduced by 3.2–3.4 log cfu/g, whereas populations on slices treated with sodium metabisulfite or acidic solutions were reduced by 4.3–5.1 log cfu/g (TSAP) and 5.3–6.2 log cfu/g (PALCAM), respectively. Bacteria were detectable by direct plating at 14 d of storage, except on acid treated slices. Immersion in acidic or metabisulfite solutions, before dehydration, should enhance inactivation of L. monocytogenes contamination on peach slices during dehydration and storage. r 2004 Elsevier Ltd. All rights reserved. Keywords: Listeria; Dried peaches; Ascorbic acid; Citric acid; Sodium metabisulfite; Storage
1. Introduction Produce is at risk of coming into contact with foodborne pathogens before, during and after harvesting. Sources of micro-organisms include soil, feces, irrigation water, wild and domestic animals, harvesting equipment, human handling and transport containers (Burnett and Beuchat, 2000; DeRoever, 1998; Janisiewicz et al., 1999; Tauxe et al., 1997). Produce implicated in listeriosis outbreaks have included coleslaw, celery, lettuce and tomatoes (Heisick et al., 1989; Ho et al., 1986; Schlech et al., 1983). Furthermore, Listeria monocytogenes has been observed to grow on fresh or processed asparagus, broccoli, cucumbers, onions and potatoes (Sizmur and Walker, 1988; Berrang et al., *Corresponding author. Tel.: +1-970-491-1945; fax: +1-970-4917252. E-mail address: [email protected] (P.A. Kendall). 0740-0020/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2004.03.011
1989). The L. monocytogenes serotypes 1/2a, 1/2b and 4b, which have been isolated from fresh coleslaw, cucumbers, potatoes and radishes (Heisick et al., 1989), are of special concern; they account for up to 96% of human listeriosis cases throughout the world (Leverentz et al., 2003; Tompkin, 2002). Dehydration is the oldest and most common form of food preservation (Salunkhe and Kadam, 1995). Today, drying is used extensively in cultures where electrical energy is expensive or non-existent in order to eliminate the need for long-term, low-temperature storage. Dehydrated products are also important staples for military troops in need of low-cost, low-weight non-perishable foods. For the modern North American home food preserver, drying is a way to produce special foods for snacks, lunches, backpacking and gifts (VanGarde and Woodburn, 1994). There have been no documented cases of L. monocytogenes infection associated with home-dried fruits to
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date. In fact, the Food and Drug Administration (FDA) Center for Food Safety and Applied Nutrition (CFSAN), estimated the risk of developing listeriosis through the consumption of raw or dried fruit as low (FDA/CFSAN, 2003). Nevertheless, L. monocytogenes has been isolated from market samples of fresh strawberries and unpasteurized fruit juices (Johannessen et al., 2002; Sado et al., 1998). Little work has been done concerning the ability of fruit (and dried fruit) to support the survival of L. monocytogenes (Conway et al., 2000; Leverentz et al., 2003). The objective of this study was to evaluate the inactivation of L. monocytogenes on inoculated peach slices during drying and to determine whether pretreating inoculated slices with metabisulfite or acidic solutions altered inactivation during dehydration and storage.
2. Material and methods 2.1. Preparation of inoculum The L. monocytogenes inoculum consisted of strains 101 m and 103 m (sausage isolates, serotype 4b and 1a, respectively), strains N-7144 and N-7143 (sausage isolates, serotype 1/2a and 3a, respectively) and strain TB00 (a turkey breast isolate, serotype not known). The five isolates were stored at 4 C on tryptic soy agar slants (TSA, Difco Laboratories, Sparks, Maryland, USA) and subcultured monthly. Isolates were activated by aerobically transferring 0.01 ml of stock culture into 9 ml of tryptic soy broth (TSB) at 30 C overnight. Strains were subcultured twice (30 C, 24 h) in TSB before use in experiments. The cell suspensions of each strain were centrifuged (4629 g; 4 C, 15 min) (Eppendorf, model 5402) and the pellet of each strain was suspended in 10 ml of phosphate buffered saline (PBS, Difco Laboratories) (pH 7.4). The five suspensions of cells were combined, re-centrifuged, and resuspended in 100 ml of PBS. Cell populations (7.9 log cfu/ml) in the composite inocula were determined by plating on tryptic soy agar (TSA; Difco) with 0.1% sodium pyruvate (Fisher Scientific) (TSAP) and incubating for 48 h at 30 C. 2.2. Preparation and inoculation of peach slices In July–August of 2001, locally grown peaches were obtained from the Colorado State University Western Colorado Research Center (Grand Junction, Colorado, USA). Redhaven and Glohaven peaches were pooled such that samples from both varieties were used in each replicate. The peaches were pitted, halved, and sliced into 0.6 cm (1/4-in) thick slices using a tomato slicer (Progressive International Corp., Kent, Washington,
USA). This process yielded slices that weighed approximately 10 g each. Peach slices were placed on trays under a biohazard hood and 0.25 ml of the L. monocytogenes inoculum (five-stain mixture) were placed onto the top surface of each slice and allowed to attached for 15 min at ambient temperature (approximately 2572 C). Slices were turned over and the other side was inoculated following the same procedure. The mean inoculation level of peach slices was 7.8170.11 log cfu/g as determined from bacterial counts on inoculated, un-immersed peach slices. 2.3. Predrying treatments Inoculated peach slices (45 slices weighing approximately 450 g per treatment) were: (1) left untreated, or 15 slices at a time were placed in metal strainers and immersed for 10 min in 1000 ml of: (2) distilled sterile water (pH 6.9770.20), (3) 4.18% sodium metabisulfite solution (pH 4.1970.15) (Fisher Scientific, Fair Lawn, New Jersey), (4) 3.40% ascorbic acid solution (pH 2.3670.20) (Fisher Scientific, Fair Lawn, New Jersey), or (5) 0.21% citric acid solution (pH 2.4870.21) (Fisher Scientific, Fair Lawn, New Jersey), at ambient temperature (2572 C). Slices were removed from the solutions and drained for 2 min before drying (not rinsed). 2.4. Dehydration The inoculated fruit slices were dehydrated for 6 h at 60 C (140 F) using two American Harvest Gardenmaster dehydrators (model FD-1000, Nesco, Chaska, Minnesota, USA) simultaneously such that each of the three trays of each dehydrator contained samples of all five treatments. The dehydrators were preheated to 60 C (140 F) for approximately 30 min, trays were loaded with inoculated, pretreated peach slices, and the internal temperature of the slices was monitored throughout drying using thermocouples (Pico Technology, Cambridge, UK). Probes were inserted into each of six peach slices; one slice on each tray of each dehydrator, and temperatures were recorded using real-time data recording software (Pico Technology). Circulating air temperature within the dehydrators was also monitored and recorded over the 6 h drying period using two thermocouple probes (Pico Technology) inserted through the center opening of each dehydrator. 2.5. Sampling for analysis For each treatment, one sample consisted of two peach slices, one slice randomly selected from within the treatment slices from each of the two dehydrators. Two samples per treatment were taken immediately after inoculation, after immersion in pretreatment solutions
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(control samples were not immersed) (0 h), at 1.5, 3, 4.5 and 6 h of drying, and on days 0, 7 and 14 of storage at 2572 C and 3076% relative humidity (Digital Relative Humidity Meter, Control Company, Friendswood, Texas, USA). An extra slice from each treatment was analysed immediately for water activity (at each sampling time including after inoculation but before treatment, after inoculation and treatment but before drying, at 1.5, 3, 4.5 and 6 h of drying and on days 0, 7 and 14 of storage). Each 2-slice sample was aseptically transferred to an 18 oz sterile plastic bag (Nasco, Modesto, California, USA) at each sampling interval. Samples collected at 0, 1.5, 3, 4.5 and 6 h of drying were stored overnight (approximately 1872 h) at 4 C. Additional samples collected at 6 h of drying were stored for 0, 7 or 14 d at 2572 C and 3076% relative humidity. After storage, the weights of the samples were recorded, 25 g of 0.1% sterile buffered peptone-water (BPW, Difco) was added to each sample, and the bags were pummeled (IUL Instruments, Barcelona, Spain) for 120 s at ambient temperature (2572 C). 2.6. Microbial analysis Decimal dilutions were made using 9 ml of 0.1% sterile BPW; 0.1 ml of each sample was spread on tryptic soy agar plus 0.1% pyruvate (TSAP, Difco) and PALCAM (Difco) agar plates, which were inverted and incubated at 30 C for 48 h (only PALCAM used during storage). Numbers of colonies from duplicate plates were used to determine the average colony forming units (cfu) per gram of peach slice (cfu/g), which were converted into log values. When zero colonies were detected, one colony on each of the duplicate plates was assumed as present for calculating the lowest detection limit of 1.1 log cfu/g. When bacterial counts dropped below 1.1 log cfu/g, the Listeria enrichment, isolation, and identification methods outlined in the FDA Bacteriological and Analytical Manual (FDA, 2001) were followed. Two randomly selected typical colonies from each day of experimentation were confirmed using the Listeria Micro-IDs kit (Remel, Lenexa, Kansas, USA). 2.7. Other analyses The pH values of samples from all treatments and times of testing were determined by measuring the homogenized sample, which was also used for microbial analysis, with a pH meter and a glass electrode (Denver Instruments, Arvada, Colorado, USA). The water activity values of samples from all treatments at all sampling times were determined with a Rotronic water activity meter (Rotronic Instrument Corp., model AwQUICK, Huntington, New York, USA).
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2.8. Statistical analysis The drying experiment was replicated four times and samples from two of the replicates were also evaluated during storage. Drying data were analysed using a 5 5 2 4 factorial design with 5 (number of treatments, including controls) 5 (number of time intervals when samples were analysed, i.e. after treatment (or 0 h), and at 1.5, 3, 4.5, and 6 h) 2 (number of agar media) 4 (number of replicates) factors. Storage data were analysed separately using a 5 3 2 factorial design with 5 (number of treatments, including controls) 3 (number of time intervals when samples were analysed, i.e. after treatment and 6 h of drying (0 d), and at 7 and 14 d of storage at 2572 C and 3076% relative humidity) 2 (number of replicates) factors. For each replicate, the mean represented the average of two samples converted into log cfu/g. All data analyses were conducted with the Statistical Analysis System (SAS Institute version 6.1, Cary, North Carolina, USA) for analysis of variance of main (fixed) effects and all interactions between fixed effects. When F -values were significant (Po0:05), least significant differences (LSD) in surviving bacterial population counts between treatments were determined using the ANOVA mixed model procedure of SAS.
3. Results and discussion 3.1. Selection of predrying treatments The quantities of sodium metabisulfite and ascorbic acid used were based on maximum amounts listed in Cooperative Extension Service home drying materials collected through an electronic request to Food and Nutrition Extension Specialists in the United States (DiPersio, 2002). Home drying recommendations were collected from 27 states and the highest amounts recommended (2 tablespoons sodium metabisulfite and 2 12 tablespoons ascorbic acid per quart/liter of water) (Brennand, 1994; Kendall and Allen, 1994) were used in the present study. These amounts were found to be equivalent to 41.84 g reagent grade sodium metabisulfite (pH 4.19) and 34 g reagent grade ascorbic acid (pH 2.36) (calculated from the average of 10 independent measurements) per 1000 ml water. The concentration of the citric acid solution used was chosen to result in a pH similar to that of the ascorbic acid solution. A solution of 2.1 g reagent grade citric acid per 1000 ml water (0.21%) had a pH of 2.4870.21 (mean7standard deviation of five independent measurements). The addition of a single granule of citric acid dramatically reduced the pH of the solution; therefore, the 0.21% citric acid solution was used in order to result in a
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solution with a pH similar to that of the ascorbic acid solution. 3.2. Dehydrator air and peach temperature Insertion of the preloaded trays into the preheated (60 C) dehydrators reduced the mean circulating air temperature in the two dehydrators to 43.773.1 C at 0 h of dehydration; the average temperature of peach slices at 0 h was 30.672.3 C. From 1.5 to 6 h of dehydration, the average temperature of circulating air ranged from 59.2 C to 61.7 C and the average temperature of peach slices ranged from 52.7 C to 60.5 C. At 6 h of dehydration, the average temperatures of the circulating air and peach slices were 59.272.1 C and 57.676.3 C, respectively. Although average temperatures at 6 h of drying were below 60 C, these results were due to single outliers and do not represent a trend. Excluding outliers, the average air and peach slice temperatures at 6 h of drying were 59.971.5 C and 59.671.5 C, respectively (data not shown). 3.3. Changes in pH and water activity during pretreatment, dehydration and storage The initial pH of peach slices used in this study was 4.2170.27 based on 8 independent measurements. For inoculated un-immersed peach slices, the pH ranged from 4.0470.22 to 4.3970.22 throughout dehydration (6 h, 60 C) and 14 d of storage (Fig. 1). Immersion in sterile water did not significantly (P > 0:05) affect pH values, whereas the pH of peach slices significantly (Po0:05) decreased following immersion in the acidic solutions (Fig. 1). The pH of peach slices decreased (Po0:05) to 3.7970.18 immediately following treat-
5.0
pH of peach slices
4.5
4.0
3.5
ment with sodium metabisulfite, then increased (Po0:05) after 1.5 h of drying to 4.0970.27 and remained higher (Po0:05) than the initial pH of peaches, as well as that of all other treatments throughout the remainder of dehydration and storage (Fig. 1). These results may be due to the dissipation of sulfur dioxide throughout the 6 h dehydration period. The water activity of peach slices was 0.9770.01 for all treatments at 0 h. After 4.5 h of dehydration, all treatments reached a water activity of o0.60, a level considered inhibitory to microbial growth (Chirife and Buera, 1996). Once the water activity level declined below 0.93 (B3 h of drying), L. monocytogenes populations on all treatments began to die off quickly. Although many bacteria are unable to grow at a water activity of less than 0.93, Miller (1991) demonstrated that L. monocytogenes was able to grow in glycerol with a water activity of 0.90, while Shahamat et al. (1980) found that L. monocytogenes (serotype 1/2a) survived longer than 100 d in a salt solution with an approximate water activity of 0.80. Water activity values of peach slices ranged from 0.34 to 0.37 immediately after 6 h of drying, equilibrated to 0.35–0.38 after cooling to room temperature, and increased to 0.41–0.50 by day 14 of storage (data not shown). Samples were stored in plastic bags (aerobically) at 2572 C and 3076% relative humidity; these conditions may have allowed the samples to gain some moisture throughout storage. Still, all samples had a water activity well below 0.60 throughout storage and, therefore, would be unlikely to support microbial growth (Jay, 2000). 3.4. Effect of agar media on bacterial recovery During drying, bacterial population counts recovered on TSAP were often higher than counts recovered on PALCAM agar (Table 1). However, differences were only significant (Po0:05) on acid and sodium metabisulfite pretreated slices after 1.5 h of drying. Expectedly, the TSAP agar recovered higher numbers of cells than the PALCAM agar. PALCAM is a selective media that inhibits the recovery and growth of injured cells (Curtis and Lee, 1995).
Control
3.5. Changes in bacterial populations resulting from solution immersion
Water Sodium metabisulfite
3.0
Ascorbic acid Citric acid 2.5 AI
0
1.5
3
4.5
6
Drying Time (h) Fig. 1. Effect of peach slice preparation procedure [inoculated control (30 min, 25 C), or immersed (10 min, 25 C) in sterile water, 4.18% sodium metabisulfite, 3.40% ascorbic acid, or 0.21% citric acid] and dehydration at 60 C for 6 h, on mean pH values of L. monocytogenes inoculated peach slices (AI: after inoculation).
Immersion of peach slices in sterile water for 10 min before dehydration (0 h) produced non-significant (P > 0:05) bacterial reductions (0.7 log cfu/g) compared to inoculated un-immersed controls. Immersion in the ascorbic and citric acid solutions produced similar nonsignificant (P > 0:05) reductions of 0.5 and 0.8 log cfu/g (Table 1). Given the lack of differences seen between bacterial populations on peach slices treated with sterile
Table 1 Mean (log cfu/ga) bacterial (TSAP: tryptic soy agar with 0.1% pyruvate; PALCAM) populations (SDb) on peach slices inoculated with L. monocytogenes, exposed to various predrying treatments and dried for 6 h at 60 C (140 F) Processing steps
Dehydration (3 h) Dehydration (4.5 h) Dehydration (6 h)
Sodium metabisulfitee
Ascorbic acidf
Citric acidg
TSAP
PALCAM
TSAP
PALCAM
TSAP
PALCAM
TSAP
PALCAM
TSAP
PALCAM
7.81Aax (0.11) 7.81Abx (0.11) 7.01Acx (0.55) 6.48ABcx (0.76) 5.69Bcx (0.43) 4.66Cbx (0.52)
7.74Aax (0.09) 7.74Abx (0.09) 6.63Bcx (0.58) 6.09BCcx (0.82) 5.36Ccx (0.35) 4.40Dcx (0.66)
7.81Aax (0.11) 7.09ABabx (0.24) 6.50BCcx (0.51) 6.26Ccx (0.97) 5.01Dbcx (0.34) 4.66Dbx (0.25)
7.74Aax (0.09) 7.09ABbx (0.30) 6.26BCbx (0.58) 5.86Ccx (1.07) 4.75Dcx (0.45) 4.37Dcx (0.30)
7.81Aax (0.11) 6.28Bax (0.25) 4.93Cay (0.49) 5.04Cby (0.59) 3.57Day (0.64) 3.53Day (0.78)
7.74Aax (0.09) 5.71Bax (0.36) 3.92Cax (0.58) 4.06Cbx (0.94) 2.42Dax (0.30) 2.31Dabx (0.68)
7.81Aax (0.11) 7.13Aabx (0.28) 5.57Babx (0.27) 3.98Cay (0.96) 3.22Day (0.87) 2.72Day (0.42)
7.74Aax (0.09) 6.97Abx (0.37) 5.38Bbx (0.12) 3.02Cax (1.13) 1.73Dax (0.49) 1.59Dax (0.32)
7.81Aax (0.11) 7.29Abx (0.22) 6.13Bbcx (0.11) 4.98Cbx (1.10) 4.75Cby (1.11) 3.29Dax (0.28)
7.74Aax (0.09) 6.97Abx (0.37) 5.83Bbx (0.12) 4.54Cbx (1.29) 3.83Cbx (1.22) 2.49Dbx (0.55)
a Means represent two samples in each of four replications (n ¼ 8) (standard deviation of the replicates) of log colony forming units (cfu/g): lowest detection limit by plating, 1.1 log cfu/g (LSD: 0.88 log cfu/g). b Standard deviation. c Control, inoculated w/no pretreatment (30 min, 25 C). d Inoculated and immersed (10 min, 25 C) before drying in sterile water. e Inoculated and immersed (10 min, 25 C) before drying in sodium metabisulfite solution (4.18%, pH 4.19). f Inoculated and immersed (10 min, 25 C) before drying in ascorbic acid solution (3.40%, pH 2.36). g Inoculated and immersed (10 min, 25 C) before drying in citric acid solution (0.21%, pH 2.48). h Following inoculation (30 min attachment time, 25 C). A–D means with different letters within a column are significantly different (Po0:05). a–d means with different letters within the same medium in a row are significantly different (Po0:05). x–y means with different letters between the two media of each treatment in a row are significantly different (Po0:05).
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Following pretreatment (0 h) Dehydration (1.5 h)
Waterd
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Following inoculationh
Controlc
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water and acidic solutions, initial bacterial reductions may have been largely due to a cell washing effect. Immersion of peach slices in the sodium metabisulfite solution (pH 4.1970.15) produced significant (Po0:05) bacterial reductions of 1.5 and 2.0 log cfu/g on TSAP and PALCAM, respectively. Sodium metabisulfite (Na2S2O5) releases sulfur dioxide (SO2) at a 67.4% theoretical yield when dissolved in water. In foods, free SO2 (not bound to water, sugars or enzymes) is responsible for the antimicrobial action of sulfites. Free SO2 is more available at pHo5.0 than at a higher pH. The increased effectiveness of sulfites at low pH is likely due to the ability of un-ionized sulfur dioxide to pass across the cell membrane and disrupt the normal metabolic activity of bacterial cells (Rose and Pilkington, 1989; Barnett, 1985). 3.6. Changes in bacterial populations during dehydration The inoculated un-immersed control received no predrying treatment and, therefore, bacterial inactivation may have been caused by the heat of dehydration combined with low water activity. After 3 h of drying, bacterial populations on control peach slices decreased (Po0:05) by 1.3 (TSAP) and 1.7 (PALCAM) log cfu/g and after 6 h of drying, populations decreased (Po0:05) by 3.2 (TSAP) and 3.3 (PALCAM) log cfu/g. Dehydration of peach slices after immersion in sterile water produced similar reductions in bacterial populations, at 3 and 6 h of dehydration, to those seen with the control slices (Table 1). Dehydration of peach slices for up to 6 h, after immersion in the sodium metabisulfite solution, resulted in greater (Po0:05) reductions of bacterial populations compared to control and water treated peach slices (Table 1). While sulfur compounds are excellent preservatives and antimicrobials, there are many reports of sulfite-induced adverse health reactions, including pulmonary edema and visceral congestion, in humans suffering from asthma or sulfur sensitivities (Davidson and Branen, 1993). For peach slices immersed in the citric acid solution, bacterial populations declined by 2.8 and 3.2 log cfu/g after 3 h dehydration, and by 4.5 and 5.3 log cfu/g after 6 h dehydration, as determined with TSAP and PALCAM agar, respectively. These decreases were significantly (Po0:05) greater than decreases seen on inoculated un-immersed control and water treated slices, and similar to those found on slices immersed in the sodium metabisulfite solution (Table 1). The antimicrobial activity of citric acid is thought to be due, in part, to its pH lowering ability (Booth and Kroll, 1998; Chien, 1992) and its ability to inhibit essential metabolic reactions (Booth and Kroll, 1998). O’Driscoll et al. (1996) found that exposing L. monocytogenes to mild acid (pH 5.5) for 1 h provided cross-protection against
subsequent stresses including osmotic stress and heat shock. In the present study, bacterial inactivation detected on citric acid treated peach slices was most likely due to the combined effects of immersion (cell washing effect), the antimicrobial activity of citric acid, low pH of the solution (2.48), heat of dehydration and low water activity. The ascorbic acid treatment, plus dehydration for 6 h, caused the highest extent of bacterial inactivation compared to all other treatments; this combination of treatments induced a 5.09–6.15 log cfu/g reduction of L. monocytogenes after 6 h of drying at 60 C as detected on TSAP and PALCAM, respectively (Table 1). The ascorbic acid treatment may have caused a high extent of bacterial inactivation because it has the ability to generate free radicals, accumulate within the cellular cytoplasm and lower the pH of a solution (Gould, 1989). The pH of the ascorbic acid solution was 2.36 and that of the citric acid solution was 2.48. The pH values (3.76– 3.96) of the ascorbic acid treated peach slices were generally lower than the values (3.91–4.03) of the citric acid treated slices throughout 6 h of drying period. Therefore, the bacterial inactivation detected on ascorbic acid treated peach slices was most likely due to the combined effect of immersion (cell washing effect), acid accumulation within the pathogen cell and subsequent disruption of cellular mechanisms, low pH of the chemical solution, heat of dehydration and reduced water activity. In a similar study, Derrickson-Tharrington (2001) evaluated the effect of acidic predrying treatments and drying (6 h, 62.8 C) on the inactivation of E. coli O157:H7 on apple slices. Inoculated slices received no predrying treatment (control), or a 10 min immersion in sterile water, 2.80% ascorbic acid (2.50 pH), 1.70% citric acid (2.20 pH), or 50% commercial lemon juice. The higher drying temperature (62.8 C), higher concentration of acid (1.70%) and lower pH (2.20) of the citric acid solution used in the Derrickson-Tharrington study induced a greater inactivation of bacteria than the drying temperature (60 C) and citric acid solution (0.21%, 2.48 pH) used in the study reported here. Burnham et al. (2001) found that dehydration (57.2 C and 62.8 C) alone caused a 2.9 log cfu/g reduction of E. coli O157:H7 on apple slices, whereas immersion of inoculated slices in 3.40% ascorbic acid prior to 6 h of dehydration induced a 5-log reduction of bacteria. Similarly, in the present study, dehydration (6 h, 60 C) alone caused a 3.2–3.3 log cfu/g reduction of L. monocytogenes on peach slices, whereas immersion of inoculated slices in 3.40% ascorbic acid prior to drying induced a 5.09–6.15 log cfu/g reduction of bacteria. In the above studies, the combination of heat and low pH had a greater effect on the destruction of pathogens than did heat alone.
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Table 2 Mean (log cfu/ga) bacterial (PALCAM) populations (SDb) on peach slices inoculated with L. monocytogenes, exposed to various pre-drying treatments, dried for 6 h at 60 C (140 F) and stored for up to 14 d at 2572 C Processing steps
Controlc
Waterd
Sodium metabisulfitee
Ascorbic acidf
Citric acidg
Following inoculationh Following dehydrationI (0 d) Following storage (7 d) Following storage (14 d)
7.74Aa (0.09)
7.74Aa (0.09)
7.74Aa (0.09)
7.74Aa (0.09)
7.74Aa (0.09)
4.82Bb (0.46)
4.21Bb (0.36)
2.37Ba (0.89)
1.34Ba (0.16)
2.13Ba (0.39)
3.80Bb (0.56)
2.66Cab (1.63)
2.14Ba (1.14)
1.38Ba (0.09)
2.65Bab (1.64)
1.84Ca (0.65)
1.81Ca (0.54)
1.44Ba (0.05)
o1.10j
o1.10j
a Means represent two samples in each of two replications (n ¼ 4) (standard deviation of the replicates) of log colony forming units (cfu/g): lowest detection limit by plating, 1.1 log cfu/g (LSD: 1.34 log cfu/g). b Standard deviation. c Control, inoculated w/ no pre-treatment (30 min, 25 C). d Inoculated and immersed (10 min, 25 C) before drying in sterile water. e Inoculated and immersed (10 min, 25 C) before drying in sodium metabisulfite solution (4.18%, pH 4.19). f Inoculated and immersed (10 min, 25 C) before drying in ascorbic acid solution (3.40%, pH 2.36). g Inoculated and immersed (10 min, 25 C) before drying in citric acid solution (0.21%, pH 2.48). h Following inoculation (30 min attachment time, 25 C). i Following dehydration (6 h, 60 C). A–D means with different letters within a column are significantly different (Po0:05). a–c means with different letters within a row are significantly different (Po0:05). j Detectable by enrichment only.
3.7. Changes in bacterial counts during storage Steady declines in bacterial populations occurred over the 14 d storage period for all treatments (Table 2). Reductions of bacterial populations during storage were higher on treatments that had more survivors after drying (i.e. control, water and sodium metabisulfite treated slices) compared to acid treated slices. The modest reductions of bacterial populations on acid treated peach slices were expected since populations were already close to the detection limit (1.1 log cfu/g) at 0 d of storage. At 14 d of storage, bacterial populations on the ascorbic acid and citric acid pretreated slices were detectable only after enrichment. Calicioglu et al. (2002) found similar results when they examined the inactivation of L. monocytogenes during drying and storage of beef jerky. Inoculated beef slices were subjected to traditional or modified marinades prior to drying at 60 C for 10 h and aerobic storage at 25 C for 60 d. Pretreatment with traditional marinade and 60 d storage, or pretreatment with modified marinades (traditional marinade with added sodium lactate, acetic acid, soy sauce, ethanol and/or Tween 20) and 15 d storage, inactivated 6–7 logs of the pathogen. Under the conditions of this study, a mild heat treatment such as drying, combined with organic acids or sulfites, together were more effective than the heat treatment alone at inactivating L. monocytogenes inoculated onto peach slices. Immersion in 4.18% sodium metabisulfite, 3.40% ascorbic acid or 0.21% citric acid solutions, prior to dehydration of peach slices,
should be useful in enhancing inactivation of potential L. monocytogenes contamination. Dehydration at 60 C, following the ascorbic acid treatment, achieved a 5.09– 6.15 log cfu/g reduction of bacterial populations after 6 h of dehydration and resulted in undetectable bacterial populations by direct plating after 14 d of storage. Studies are currently being conducted to examine the sensory qualities of the dried product.
Acknowledgements This project was supported by USDA-CSREES National Integrated Food Safety Initiative Project x0051110-9747 and by the Colorado Agricultural Experiment Station.
References Barnett, D., 1985. Sulphites in foods: their chemistry and analysis. Food Technol. Aust. 37, 503–505. Berrang, M.E., Brackett, R.E., Beuchat, L.R., 1989. Growth of Listeria monocytogenes on fresh vegetables stored under controlled atmosphere. J. Food Prot. 52, 702–706. Booth, I.R., Kroll, R.G., 1998. The preservation of foods by low pH. In: Gould, G.W. (Ed.), Mechanism of Action of Food Preservative Procedures. Elsevier Science Publishers Ltd., London, pp. 119–160. Brennand, C.P., 1994. Home Drying of Food, FN-330. Cooperative Extension Service, Utah State University, Logan, UT, USA. Burnett, S.L., Beuchat, L.R., 2000. Human pathogens associated with raw produce and unpasteurized juices, and difficulties in decontamination. J. Ind. Microbiol. Biotech. 25, 281–287.
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Burnham, J.A., Kendall, P.A., Sofos, J.N., 2001. Ascorbic acid enhances destruction of Escherichia coli O157:H7 during hometype drying of apple slices. J. Food Prot. 64, 1244–1248. Calicioglu, M., Sofos, J.N., Samelis, J., Kendall, P.A., Smith, G.C., 2002. Destruction of acid- and non-adapted Listeria monocytogenes during drying and storage of beef jerky. Food Microbiol. 19, 545–559. Chien, M.S., 1992. Food preservatives—organic acids and esters. Food Ind. 29, 5–9. Chirife, J., Buera, M.D., 1996. Water activity, water glass dynamics, and the control of microbiological growth in foods. Crit. Rev. Food Sci. Nutr. 36, 465–513. Conway, W.S., Leverentz, B., Saftner, R.A., Janisiewicz, W.J., Sams, C.E., Leblanc, 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. Curtis, G.D.W., Lee, W.H., 1995. Culture media and methods for the isolation of Listeria monocytogenes. Int. J. Food Microbiol. 26, 1–13. Davidson, P.M., Branen, A.L., 1993. Antimicrobials in Foods 2nd Edition. Marcel Dekker Inc., New York. DeRoever, C., 1998. Microbiological safety evaluations and recommendations on fresh produce. Food Control 9, 321–347. Derrickson-Tharrington, E.L., 2001. Evaluation of common acidulants for enhancement of destruction of E. coli O157:H7 during drying of Gala apple slices and whole muscle beef jerky. M.S. Thesis, Colorado State University, Fort Collins, CO. DiPersio, P.A., 2002. Inactivation of Salmonella and Listeria monocytogenes during drying and storage of apple and peach slices treated with acidic or sodium metabisulfite solutions. M.S. Thesis, Colorado State University, Fort Collins, CO. Food and Drug Administration, 2001. Bacteriological and Analytical Manual: URL: http://www.cfsan.fda.gov/Bebam/bam-5.html Food and Drug Administration-Center for Food Safety and Applied Nutrition, 2003. Quantitative assessment of relative risk to public health from foodborne Listeria monocytogenes among selected categories of ready-to-eat foods: URL: http://www.cfsan.fda.gov/ Bdms/lmr2-su.html Gould, G.W., 1989. Mechanisms of Action of Food Preservation Procedures. Elsevier Science Publishers Ltd., New York. Heisick, J.E., Wagner, D.E., Neirman, M.L., 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., Ecklind, P., Fraser, D.W., 1986. An outbreak of type 4b Listeria monocytogenes infection involving patients from eight Boston hospitals. Arch. Intern. Med. 146, 520–524. Janisiewicz, W.J., Conway, W.S., Brown, M.W., Sapers, G.M., Fratamico, P., Buchanan, R.L., 1999. Fate of E. coli O157:H7 on
fresh-cut apple tissue and its potential for transmission by fruit flies. Appl. Environ. Microbiol. 65, 1–5. Jay, J.M., 2000. Modern Food Microbiology 6th Edition. Aspen Publishers Inc., Gaithersburg. Johannessen, G.S., Loncarevic, S., Kruse, H., 2002. Bacteriological analysis of fresh produce in Norway. Int. J. Food Mircobiol. 77, 199–204. Kendall, P., Allen, L., 1994. Drying Fruits. Service in Action 9.309. Cooperative Extension, Colorado State University, Fort Collins, CO, USA. Leverentz, B., Conway, W.S., Camp, M.J., Janisiewicz, W.J., Abuladze, T., Yang, M., Saftner, R., Sulakvelidze, A., 2003. Biocontrol of Listeria monocytogenes on fresh-cut produce by treatment with lytic bacteriophages and a bacteriocin. Appl. Environ. Microbiol. 69, 4519–4526. Miller, A.J., 1991. Combined water activity and solute effects on growth and survival of Listeria monocytogenes Scott A. J. Food Prot. 55, 414–418. O’Driscoll, B., Gahan, C.G.M., Hill, C., 1996. Adaptive acid tolerance response in Listeria monocytogenes: isolation of an acid-tolerant mutant which demonstrates increased virulence. Appl. Environ. Microbiol. 62, 1693–1698. Rose, A.H., Pilkington, B.J., 1989. Sulfite. In: Gould, G.W. (Ed.), Mechanisms of Action of Food Preservation Procedures. Elsevier Science Publishers Ltd., New York, pp. 201–224. Sado, P.N., Jinneman, K.C., Husby, G.J., Sorg, S.M., Omiecinski, C.J., 1998. Identification of Listeria monocytogenes from unpasteurized apple juice using rapid test kits. J. Food Prot. 61, 1199–1202. Salunkhe, D.K., Kadam, S.S., 1995. Handbook of Fruit Science and Technology. Marcel Dekker Inc., New York. Schlech, W.F., Lavigne, P.M., Bortolussi, R.A., Allen, A.C., Haldane, E.V., Wort, A.J., Hightower, A.W., Johnson, S.E., King, S.H., Nichols, E.S., Broome, V.C., 1983. Epidemic listeriosis evidence for transmission by food. N. Engl. J. Med. 308, 203–206. Shahamat, M., Seaman, A., Woodbine, M., 1980. Survival of Listeria monocytogenes in high salt concentrations. Zentrabl. Bakeriol. Hyg. I. Abt. Orig. A 246, 506–511. Sizmur, K., Walker, C.W., 1988. Listeria in prepackaged salads. Lancet I, 1167. Tauxe, R., Kruse, H., Hedberg, C., Potter, M., Madden, J., Wachsmuth, K., 1997. Microbial hazards and emerging issues associated with produce-a preliminary report to the national advisory committee on microbiologic criteria for foods. J. Food Prot. 60, 1400–1408. Tompkin, R.B., 2002. Control of Listeria monocytogenes in the foodprocessing environment. J. Food Prot. 65, 709–7025. VanGarde, S.J., Woodburn, M., 1994. Food Preservation and Safety Principles and Practice. Iowa State University Press, Ames, IA, USA.
www.elsevier.nl/locate/jnlabr/yfmic
Inactivation of Listeria monocytogenes during drying and storage of peach slices treated with acidic or sodium metabisulfite solutions Patricia A. DiPersioa, Patricia A. Kendalla,*, John N. Sofosb a
Department of Food Science and Human Nutrition, Colorado State University, Fort Collins,Colorado 80523, USA b Department of Animal Sciences, Colorado State University, Fort Collins, Colorado 80523, USA Accepted 14 March 2004
Abstract This study evaluated whether treating inoculated peach slices with metabisulfite or acidic solutions enhanced inactivation of Listeria monocytogenes during dehydration and storage. Inoculated (five strain mixture of L. monocytogenes, 7.9 log cfu/g) peach slices were treated, dried for 6 h at 60 C and stored aerobically at 25 C for 14 d. Predrying treatments of inoculated peach slices included: (1) no treatment (control); or 10 min immersion in: (2) sterile water, (3) 4.18% sodium metabisulfite, (4) 3.40% ascorbic acid, or (5) 0.21% citric acid solutions. Samples were plated on tryptic soy agar with 0.1% pyruvate (TSAP) and PALCAM agar for enumeration of surviving bacteria. Immersion in sterile water reduced bacterial populations on peach slices by 0.7 log cfu/g (TSAP and PALCAM). Immersion in the sodium metabisulfite solution reduced populations by 1.5–2.0 log cfu/g, while acidic pretreatments reduced populations by 0.5–0.8 log cfu/g. After 6 h of dehydration, populations on control or water immersed slices were reduced by 3.2–3.4 log cfu/g, whereas populations on slices treated with sodium metabisulfite or acidic solutions were reduced by 4.3–5.1 log cfu/g (TSAP) and 5.3–6.2 log cfu/g (PALCAM), respectively. Bacteria were detectable by direct plating at 14 d of storage, except on acid treated slices. Immersion in acidic or metabisulfite solutions, before dehydration, should enhance inactivation of L. monocytogenes contamination on peach slices during dehydration and storage. r 2004 Elsevier Ltd. All rights reserved. Keywords: Listeria; Dried peaches; Ascorbic acid; Citric acid; Sodium metabisulfite; Storage
1. Introduction Produce is at risk of coming into contact with foodborne pathogens before, during and after harvesting. Sources of micro-organisms include soil, feces, irrigation water, wild and domestic animals, harvesting equipment, human handling and transport containers (Burnett and Beuchat, 2000; DeRoever, 1998; Janisiewicz et al., 1999; Tauxe et al., 1997). Produce implicated in listeriosis outbreaks have included coleslaw, celery, lettuce and tomatoes (Heisick et al., 1989; Ho et al., 1986; Schlech et al., 1983). Furthermore, Listeria monocytogenes has been observed to grow on fresh or processed asparagus, broccoli, cucumbers, onions and potatoes (Sizmur and Walker, 1988; Berrang et al., *Corresponding author. Tel.: +1-970-491-1945; fax: +1-970-4917252. E-mail address: [email protected] (P.A. Kendall). 0740-0020/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2004.03.011
1989). The L. monocytogenes serotypes 1/2a, 1/2b and 4b, which have been isolated from fresh coleslaw, cucumbers, potatoes and radishes (Heisick et al., 1989), are of special concern; they account for up to 96% of human listeriosis cases throughout the world (Leverentz et al., 2003; Tompkin, 2002). Dehydration is the oldest and most common form of food preservation (Salunkhe and Kadam, 1995). Today, drying is used extensively in cultures where electrical energy is expensive or non-existent in order to eliminate the need for long-term, low-temperature storage. Dehydrated products are also important staples for military troops in need of low-cost, low-weight non-perishable foods. For the modern North American home food preserver, drying is a way to produce special foods for snacks, lunches, backpacking and gifts (VanGarde and Woodburn, 1994). There have been no documented cases of L. monocytogenes infection associated with home-dried fruits to
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date. In fact, the Food and Drug Administration (FDA) Center for Food Safety and Applied Nutrition (CFSAN), estimated the risk of developing listeriosis through the consumption of raw or dried fruit as low (FDA/CFSAN, 2003). Nevertheless, L. monocytogenes has been isolated from market samples of fresh strawberries and unpasteurized fruit juices (Johannessen et al., 2002; Sado et al., 1998). Little work has been done concerning the ability of fruit (and dried fruit) to support the survival of L. monocytogenes (Conway et al., 2000; Leverentz et al., 2003). The objective of this study was to evaluate the inactivation of L. monocytogenes on inoculated peach slices during drying and to determine whether pretreating inoculated slices with metabisulfite or acidic solutions altered inactivation during dehydration and storage.
2. Material and methods 2.1. Preparation of inoculum The L. monocytogenes inoculum consisted of strains 101 m and 103 m (sausage isolates, serotype 4b and 1a, respectively), strains N-7144 and N-7143 (sausage isolates, serotype 1/2a and 3a, respectively) and strain TB00 (a turkey breast isolate, serotype not known). The five isolates were stored at 4 C on tryptic soy agar slants (TSA, Difco Laboratories, Sparks, Maryland, USA) and subcultured monthly. Isolates were activated by aerobically transferring 0.01 ml of stock culture into 9 ml of tryptic soy broth (TSB) at 30 C overnight. Strains were subcultured twice (30 C, 24 h) in TSB before use in experiments. The cell suspensions of each strain were centrifuged (4629 g; 4 C, 15 min) (Eppendorf, model 5402) and the pellet of each strain was suspended in 10 ml of phosphate buffered saline (PBS, Difco Laboratories) (pH 7.4). The five suspensions of cells were combined, re-centrifuged, and resuspended in 100 ml of PBS. Cell populations (7.9 log cfu/ml) in the composite inocula were determined by plating on tryptic soy agar (TSA; Difco) with 0.1% sodium pyruvate (Fisher Scientific) (TSAP) and incubating for 48 h at 30 C. 2.2. Preparation and inoculation of peach slices In July–August of 2001, locally grown peaches were obtained from the Colorado State University Western Colorado Research Center (Grand Junction, Colorado, USA). Redhaven and Glohaven peaches were pooled such that samples from both varieties were used in each replicate. The peaches were pitted, halved, and sliced into 0.6 cm (1/4-in) thick slices using a tomato slicer (Progressive International Corp., Kent, Washington,
USA). This process yielded slices that weighed approximately 10 g each. Peach slices were placed on trays under a biohazard hood and 0.25 ml of the L. monocytogenes inoculum (five-stain mixture) were placed onto the top surface of each slice and allowed to attached for 15 min at ambient temperature (approximately 2572 C). Slices were turned over and the other side was inoculated following the same procedure. The mean inoculation level of peach slices was 7.8170.11 log cfu/g as determined from bacterial counts on inoculated, un-immersed peach slices. 2.3. Predrying treatments Inoculated peach slices (45 slices weighing approximately 450 g per treatment) were: (1) left untreated, or 15 slices at a time were placed in metal strainers and immersed for 10 min in 1000 ml of: (2) distilled sterile water (pH 6.9770.20), (3) 4.18% sodium metabisulfite solution (pH 4.1970.15) (Fisher Scientific, Fair Lawn, New Jersey), (4) 3.40% ascorbic acid solution (pH 2.3670.20) (Fisher Scientific, Fair Lawn, New Jersey), or (5) 0.21% citric acid solution (pH 2.4870.21) (Fisher Scientific, Fair Lawn, New Jersey), at ambient temperature (2572 C). Slices were removed from the solutions and drained for 2 min before drying (not rinsed). 2.4. Dehydration The inoculated fruit slices were dehydrated for 6 h at 60 C (140 F) using two American Harvest Gardenmaster dehydrators (model FD-1000, Nesco, Chaska, Minnesota, USA) simultaneously such that each of the three trays of each dehydrator contained samples of all five treatments. The dehydrators were preheated to 60 C (140 F) for approximately 30 min, trays were loaded with inoculated, pretreated peach slices, and the internal temperature of the slices was monitored throughout drying using thermocouples (Pico Technology, Cambridge, UK). Probes were inserted into each of six peach slices; one slice on each tray of each dehydrator, and temperatures were recorded using real-time data recording software (Pico Technology). Circulating air temperature within the dehydrators was also monitored and recorded over the 6 h drying period using two thermocouple probes (Pico Technology) inserted through the center opening of each dehydrator. 2.5. Sampling for analysis For each treatment, one sample consisted of two peach slices, one slice randomly selected from within the treatment slices from each of the two dehydrators. Two samples per treatment were taken immediately after inoculation, after immersion in pretreatment solutions
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(control samples were not immersed) (0 h), at 1.5, 3, 4.5 and 6 h of drying, and on days 0, 7 and 14 of storage at 2572 C and 3076% relative humidity (Digital Relative Humidity Meter, Control Company, Friendswood, Texas, USA). An extra slice from each treatment was analysed immediately for water activity (at each sampling time including after inoculation but before treatment, after inoculation and treatment but before drying, at 1.5, 3, 4.5 and 6 h of drying and on days 0, 7 and 14 of storage). Each 2-slice sample was aseptically transferred to an 18 oz sterile plastic bag (Nasco, Modesto, California, USA) at each sampling interval. Samples collected at 0, 1.5, 3, 4.5 and 6 h of drying were stored overnight (approximately 1872 h) at 4 C. Additional samples collected at 6 h of drying were stored for 0, 7 or 14 d at 2572 C and 3076% relative humidity. After storage, the weights of the samples were recorded, 25 g of 0.1% sterile buffered peptone-water (BPW, Difco) was added to each sample, and the bags were pummeled (IUL Instruments, Barcelona, Spain) for 120 s at ambient temperature (2572 C). 2.6. Microbial analysis Decimal dilutions were made using 9 ml of 0.1% sterile BPW; 0.1 ml of each sample was spread on tryptic soy agar plus 0.1% pyruvate (TSAP, Difco) and PALCAM (Difco) agar plates, which were inverted and incubated at 30 C for 48 h (only PALCAM used during storage). Numbers of colonies from duplicate plates were used to determine the average colony forming units (cfu) per gram of peach slice (cfu/g), which were converted into log values. When zero colonies were detected, one colony on each of the duplicate plates was assumed as present for calculating the lowest detection limit of 1.1 log cfu/g. When bacterial counts dropped below 1.1 log cfu/g, the Listeria enrichment, isolation, and identification methods outlined in the FDA Bacteriological and Analytical Manual (FDA, 2001) were followed. Two randomly selected typical colonies from each day of experimentation were confirmed using the Listeria Micro-IDs kit (Remel, Lenexa, Kansas, USA). 2.7. Other analyses The pH values of samples from all treatments and times of testing were determined by measuring the homogenized sample, which was also used for microbial analysis, with a pH meter and a glass electrode (Denver Instruments, Arvada, Colorado, USA). The water activity values of samples from all treatments at all sampling times were determined with a Rotronic water activity meter (Rotronic Instrument Corp., model AwQUICK, Huntington, New York, USA).
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2.8. Statistical analysis The drying experiment was replicated four times and samples from two of the replicates were also evaluated during storage. Drying data were analysed using a 5 5 2 4 factorial design with 5 (number of treatments, including controls) 5 (number of time intervals when samples were analysed, i.e. after treatment (or 0 h), and at 1.5, 3, 4.5, and 6 h) 2 (number of agar media) 4 (number of replicates) factors. Storage data were analysed separately using a 5 3 2 factorial design with 5 (number of treatments, including controls) 3 (number of time intervals when samples were analysed, i.e. after treatment and 6 h of drying (0 d), and at 7 and 14 d of storage at 2572 C and 3076% relative humidity) 2 (number of replicates) factors. For each replicate, the mean represented the average of two samples converted into log cfu/g. All data analyses were conducted with the Statistical Analysis System (SAS Institute version 6.1, Cary, North Carolina, USA) for analysis of variance of main (fixed) effects and all interactions between fixed effects. When F -values were significant (Po0:05), least significant differences (LSD) in surviving bacterial population counts between treatments were determined using the ANOVA mixed model procedure of SAS.
3. Results and discussion 3.1. Selection of predrying treatments The quantities of sodium metabisulfite and ascorbic acid used were based on maximum amounts listed in Cooperative Extension Service home drying materials collected through an electronic request to Food and Nutrition Extension Specialists in the United States (DiPersio, 2002). Home drying recommendations were collected from 27 states and the highest amounts recommended (2 tablespoons sodium metabisulfite and 2 12 tablespoons ascorbic acid per quart/liter of water) (Brennand, 1994; Kendall and Allen, 1994) were used in the present study. These amounts were found to be equivalent to 41.84 g reagent grade sodium metabisulfite (pH 4.19) and 34 g reagent grade ascorbic acid (pH 2.36) (calculated from the average of 10 independent measurements) per 1000 ml water. The concentration of the citric acid solution used was chosen to result in a pH similar to that of the ascorbic acid solution. A solution of 2.1 g reagent grade citric acid per 1000 ml water (0.21%) had a pH of 2.4870.21 (mean7standard deviation of five independent measurements). The addition of a single granule of citric acid dramatically reduced the pH of the solution; therefore, the 0.21% citric acid solution was used in order to result in a
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solution with a pH similar to that of the ascorbic acid solution. 3.2. Dehydrator air and peach temperature Insertion of the preloaded trays into the preheated (60 C) dehydrators reduced the mean circulating air temperature in the two dehydrators to 43.773.1 C at 0 h of dehydration; the average temperature of peach slices at 0 h was 30.672.3 C. From 1.5 to 6 h of dehydration, the average temperature of circulating air ranged from 59.2 C to 61.7 C and the average temperature of peach slices ranged from 52.7 C to 60.5 C. At 6 h of dehydration, the average temperatures of the circulating air and peach slices were 59.272.1 C and 57.676.3 C, respectively. Although average temperatures at 6 h of drying were below 60 C, these results were due to single outliers and do not represent a trend. Excluding outliers, the average air and peach slice temperatures at 6 h of drying were 59.971.5 C and 59.671.5 C, respectively (data not shown). 3.3. Changes in pH and water activity during pretreatment, dehydration and storage The initial pH of peach slices used in this study was 4.2170.27 based on 8 independent measurements. For inoculated un-immersed peach slices, the pH ranged from 4.0470.22 to 4.3970.22 throughout dehydration (6 h, 60 C) and 14 d of storage (Fig. 1). Immersion in sterile water did not significantly (P > 0:05) affect pH values, whereas the pH of peach slices significantly (Po0:05) decreased following immersion in the acidic solutions (Fig. 1). The pH of peach slices decreased (Po0:05) to 3.7970.18 immediately following treat-
5.0
pH of peach slices
4.5
4.0
3.5
ment with sodium metabisulfite, then increased (Po0:05) after 1.5 h of drying to 4.0970.27 and remained higher (Po0:05) than the initial pH of peaches, as well as that of all other treatments throughout the remainder of dehydration and storage (Fig. 1). These results may be due to the dissipation of sulfur dioxide throughout the 6 h dehydration period. The water activity of peach slices was 0.9770.01 for all treatments at 0 h. After 4.5 h of dehydration, all treatments reached a water activity of o0.60, a level considered inhibitory to microbial growth (Chirife and Buera, 1996). Once the water activity level declined below 0.93 (B3 h of drying), L. monocytogenes populations on all treatments began to die off quickly. Although many bacteria are unable to grow at a water activity of less than 0.93, Miller (1991) demonstrated that L. monocytogenes was able to grow in glycerol with a water activity of 0.90, while Shahamat et al. (1980) found that L. monocytogenes (serotype 1/2a) survived longer than 100 d in a salt solution with an approximate water activity of 0.80. Water activity values of peach slices ranged from 0.34 to 0.37 immediately after 6 h of drying, equilibrated to 0.35–0.38 after cooling to room temperature, and increased to 0.41–0.50 by day 14 of storage (data not shown). Samples were stored in plastic bags (aerobically) at 2572 C and 3076% relative humidity; these conditions may have allowed the samples to gain some moisture throughout storage. Still, all samples had a water activity well below 0.60 throughout storage and, therefore, would be unlikely to support microbial growth (Jay, 2000). 3.4. Effect of agar media on bacterial recovery During drying, bacterial population counts recovered on TSAP were often higher than counts recovered on PALCAM agar (Table 1). However, differences were only significant (Po0:05) on acid and sodium metabisulfite pretreated slices after 1.5 h of drying. Expectedly, the TSAP agar recovered higher numbers of cells than the PALCAM agar. PALCAM is a selective media that inhibits the recovery and growth of injured cells (Curtis and Lee, 1995).
Control
3.5. Changes in bacterial populations resulting from solution immersion
Water Sodium metabisulfite
3.0
Ascorbic acid Citric acid 2.5 AI
0
1.5
3
4.5
6
Drying Time (h) Fig. 1. Effect of peach slice preparation procedure [inoculated control (30 min, 25 C), or immersed (10 min, 25 C) in sterile water, 4.18% sodium metabisulfite, 3.40% ascorbic acid, or 0.21% citric acid] and dehydration at 60 C for 6 h, on mean pH values of L. monocytogenes inoculated peach slices (AI: after inoculation).
Immersion of peach slices in sterile water for 10 min before dehydration (0 h) produced non-significant (P > 0:05) bacterial reductions (0.7 log cfu/g) compared to inoculated un-immersed controls. Immersion in the ascorbic and citric acid solutions produced similar nonsignificant (P > 0:05) reductions of 0.5 and 0.8 log cfu/g (Table 1). Given the lack of differences seen between bacterial populations on peach slices treated with sterile
Table 1 Mean (log cfu/ga) bacterial (TSAP: tryptic soy agar with 0.1% pyruvate; PALCAM) populations (SDb) on peach slices inoculated with L. monocytogenes, exposed to various predrying treatments and dried for 6 h at 60 C (140 F) Processing steps
Dehydration (3 h) Dehydration (4.5 h) Dehydration (6 h)
Sodium metabisulfitee
Ascorbic acidf
Citric acidg
TSAP
PALCAM
TSAP
PALCAM
TSAP
PALCAM
TSAP
PALCAM
TSAP
PALCAM
7.81Aax (0.11) 7.81Abx (0.11) 7.01Acx (0.55) 6.48ABcx (0.76) 5.69Bcx (0.43) 4.66Cbx (0.52)
7.74Aax (0.09) 7.74Abx (0.09) 6.63Bcx (0.58) 6.09BCcx (0.82) 5.36Ccx (0.35) 4.40Dcx (0.66)
7.81Aax (0.11) 7.09ABabx (0.24) 6.50BCcx (0.51) 6.26Ccx (0.97) 5.01Dbcx (0.34) 4.66Dbx (0.25)
7.74Aax (0.09) 7.09ABbx (0.30) 6.26BCbx (0.58) 5.86Ccx (1.07) 4.75Dcx (0.45) 4.37Dcx (0.30)
7.81Aax (0.11) 6.28Bax (0.25) 4.93Cay (0.49) 5.04Cby (0.59) 3.57Day (0.64) 3.53Day (0.78)
7.74Aax (0.09) 5.71Bax (0.36) 3.92Cax (0.58) 4.06Cbx (0.94) 2.42Dax (0.30) 2.31Dabx (0.68)
7.81Aax (0.11) 7.13Aabx (0.28) 5.57Babx (0.27) 3.98Cay (0.96) 3.22Day (0.87) 2.72Day (0.42)
7.74Aax (0.09) 6.97Abx (0.37) 5.38Bbx (0.12) 3.02Cax (1.13) 1.73Dax (0.49) 1.59Dax (0.32)
7.81Aax (0.11) 7.29Abx (0.22) 6.13Bbcx (0.11) 4.98Cbx (1.10) 4.75Cby (1.11) 3.29Dax (0.28)
7.74Aax (0.09) 6.97Abx (0.37) 5.83Bbx (0.12) 4.54Cbx (1.29) 3.83Cbx (1.22) 2.49Dbx (0.55)
a Means represent two samples in each of four replications (n ¼ 8) (standard deviation of the replicates) of log colony forming units (cfu/g): lowest detection limit by plating, 1.1 log cfu/g (LSD: 0.88 log cfu/g). b Standard deviation. c Control, inoculated w/no pretreatment (30 min, 25 C). d Inoculated and immersed (10 min, 25 C) before drying in sterile water. e Inoculated and immersed (10 min, 25 C) before drying in sodium metabisulfite solution (4.18%, pH 4.19). f Inoculated and immersed (10 min, 25 C) before drying in ascorbic acid solution (3.40%, pH 2.36). g Inoculated and immersed (10 min, 25 C) before drying in citric acid solution (0.21%, pH 2.48). h Following inoculation (30 min attachment time, 25 C). A–D means with different letters within a column are significantly different (Po0:05). a–d means with different letters within the same medium in a row are significantly different (Po0:05). x–y means with different letters between the two media of each treatment in a row are significantly different (Po0:05).
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Waterd
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Following inoculationh
Controlc
645
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water and acidic solutions, initial bacterial reductions may have been largely due to a cell washing effect. Immersion of peach slices in the sodium metabisulfite solution (pH 4.1970.15) produced significant (Po0:05) bacterial reductions of 1.5 and 2.0 log cfu/g on TSAP and PALCAM, respectively. Sodium metabisulfite (Na2S2O5) releases sulfur dioxide (SO2) at a 67.4% theoretical yield when dissolved in water. In foods, free SO2 (not bound to water, sugars or enzymes) is responsible for the antimicrobial action of sulfites. Free SO2 is more available at pHo5.0 than at a higher pH. The increased effectiveness of sulfites at low pH is likely due to the ability of un-ionized sulfur dioxide to pass across the cell membrane and disrupt the normal metabolic activity of bacterial cells (Rose and Pilkington, 1989; Barnett, 1985). 3.6. Changes in bacterial populations during dehydration The inoculated un-immersed control received no predrying treatment and, therefore, bacterial inactivation may have been caused by the heat of dehydration combined with low water activity. After 3 h of drying, bacterial populations on control peach slices decreased (Po0:05) by 1.3 (TSAP) and 1.7 (PALCAM) log cfu/g and after 6 h of drying, populations decreased (Po0:05) by 3.2 (TSAP) and 3.3 (PALCAM) log cfu/g. Dehydration of peach slices after immersion in sterile water produced similar reductions in bacterial populations, at 3 and 6 h of dehydration, to those seen with the control slices (Table 1). Dehydration of peach slices for up to 6 h, after immersion in the sodium metabisulfite solution, resulted in greater (Po0:05) reductions of bacterial populations compared to control and water treated peach slices (Table 1). While sulfur compounds are excellent preservatives and antimicrobials, there are many reports of sulfite-induced adverse health reactions, including pulmonary edema and visceral congestion, in humans suffering from asthma or sulfur sensitivities (Davidson and Branen, 1993). For peach slices immersed in the citric acid solution, bacterial populations declined by 2.8 and 3.2 log cfu/g after 3 h dehydration, and by 4.5 and 5.3 log cfu/g after 6 h dehydration, as determined with TSAP and PALCAM agar, respectively. These decreases were significantly (Po0:05) greater than decreases seen on inoculated un-immersed control and water treated slices, and similar to those found on slices immersed in the sodium metabisulfite solution (Table 1). The antimicrobial activity of citric acid is thought to be due, in part, to its pH lowering ability (Booth and Kroll, 1998; Chien, 1992) and its ability to inhibit essential metabolic reactions (Booth and Kroll, 1998). O’Driscoll et al. (1996) found that exposing L. monocytogenes to mild acid (pH 5.5) for 1 h provided cross-protection against
subsequent stresses including osmotic stress and heat shock. In the present study, bacterial inactivation detected on citric acid treated peach slices was most likely due to the combined effects of immersion (cell washing effect), the antimicrobial activity of citric acid, low pH of the solution (2.48), heat of dehydration and low water activity. The ascorbic acid treatment, plus dehydration for 6 h, caused the highest extent of bacterial inactivation compared to all other treatments; this combination of treatments induced a 5.09–6.15 log cfu/g reduction of L. monocytogenes after 6 h of drying at 60 C as detected on TSAP and PALCAM, respectively (Table 1). The ascorbic acid treatment may have caused a high extent of bacterial inactivation because it has the ability to generate free radicals, accumulate within the cellular cytoplasm and lower the pH of a solution (Gould, 1989). The pH of the ascorbic acid solution was 2.36 and that of the citric acid solution was 2.48. The pH values (3.76– 3.96) of the ascorbic acid treated peach slices were generally lower than the values (3.91–4.03) of the citric acid treated slices throughout 6 h of drying period. Therefore, the bacterial inactivation detected on ascorbic acid treated peach slices was most likely due to the combined effect of immersion (cell washing effect), acid accumulation within the pathogen cell and subsequent disruption of cellular mechanisms, low pH of the chemical solution, heat of dehydration and reduced water activity. In a similar study, Derrickson-Tharrington (2001) evaluated the effect of acidic predrying treatments and drying (6 h, 62.8 C) on the inactivation of E. coli O157:H7 on apple slices. Inoculated slices received no predrying treatment (control), or a 10 min immersion in sterile water, 2.80% ascorbic acid (2.50 pH), 1.70% citric acid (2.20 pH), or 50% commercial lemon juice. The higher drying temperature (62.8 C), higher concentration of acid (1.70%) and lower pH (2.20) of the citric acid solution used in the Derrickson-Tharrington study induced a greater inactivation of bacteria than the drying temperature (60 C) and citric acid solution (0.21%, 2.48 pH) used in the study reported here. Burnham et al. (2001) found that dehydration (57.2 C and 62.8 C) alone caused a 2.9 log cfu/g reduction of E. coli O157:H7 on apple slices, whereas immersion of inoculated slices in 3.40% ascorbic acid prior to 6 h of dehydration induced a 5-log reduction of bacteria. Similarly, in the present study, dehydration (6 h, 60 C) alone caused a 3.2–3.3 log cfu/g reduction of L. monocytogenes on peach slices, whereas immersion of inoculated slices in 3.40% ascorbic acid prior to drying induced a 5.09–6.15 log cfu/g reduction of bacteria. In the above studies, the combination of heat and low pH had a greater effect on the destruction of pathogens than did heat alone.
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Table 2 Mean (log cfu/ga) bacterial (PALCAM) populations (SDb) on peach slices inoculated with L. monocytogenes, exposed to various pre-drying treatments, dried for 6 h at 60 C (140 F) and stored for up to 14 d at 2572 C Processing steps
Controlc
Waterd
Sodium metabisulfitee
Ascorbic acidf
Citric acidg
Following inoculationh Following dehydrationI (0 d) Following storage (7 d) Following storage (14 d)
7.74Aa (0.09)
7.74Aa (0.09)
7.74Aa (0.09)
7.74Aa (0.09)
7.74Aa (0.09)
4.82Bb (0.46)
4.21Bb (0.36)
2.37Ba (0.89)
1.34Ba (0.16)
2.13Ba (0.39)
3.80Bb (0.56)
2.66Cab (1.63)
2.14Ba (1.14)
1.38Ba (0.09)
2.65Bab (1.64)
1.84Ca (0.65)
1.81Ca (0.54)
1.44Ba (0.05)
o1.10j
o1.10j
a Means represent two samples in each of two replications (n ¼ 4) (standard deviation of the replicates) of log colony forming units (cfu/g): lowest detection limit by plating, 1.1 log cfu/g (LSD: 1.34 log cfu/g). b Standard deviation. c Control, inoculated w/ no pre-treatment (30 min, 25 C). d Inoculated and immersed (10 min, 25 C) before drying in sterile water. e Inoculated and immersed (10 min, 25 C) before drying in sodium metabisulfite solution (4.18%, pH 4.19). f Inoculated and immersed (10 min, 25 C) before drying in ascorbic acid solution (3.40%, pH 2.36). g Inoculated and immersed (10 min, 25 C) before drying in citric acid solution (0.21%, pH 2.48). h Following inoculation (30 min attachment time, 25 C). i Following dehydration (6 h, 60 C). A–D means with different letters within a column are significantly different (Po0:05). a–c means with different letters within a row are significantly different (Po0:05). j Detectable by enrichment only.
3.7. Changes in bacterial counts during storage Steady declines in bacterial populations occurred over the 14 d storage period for all treatments (Table 2). Reductions of bacterial populations during storage were higher on treatments that had more survivors after drying (i.e. control, water and sodium metabisulfite treated slices) compared to acid treated slices. The modest reductions of bacterial populations on acid treated peach slices were expected since populations were already close to the detection limit (1.1 log cfu/g) at 0 d of storage. At 14 d of storage, bacterial populations on the ascorbic acid and citric acid pretreated slices were detectable only after enrichment. Calicioglu et al. (2002) found similar results when they examined the inactivation of L. monocytogenes during drying and storage of beef jerky. Inoculated beef slices were subjected to traditional or modified marinades prior to drying at 60 C for 10 h and aerobic storage at 25 C for 60 d. Pretreatment with traditional marinade and 60 d storage, or pretreatment with modified marinades (traditional marinade with added sodium lactate, acetic acid, soy sauce, ethanol and/or Tween 20) and 15 d storage, inactivated 6–7 logs of the pathogen. Under the conditions of this study, a mild heat treatment such as drying, combined with organic acids or sulfites, together were more effective than the heat treatment alone at inactivating L. monocytogenes inoculated onto peach slices. Immersion in 4.18% sodium metabisulfite, 3.40% ascorbic acid or 0.21% citric acid solutions, prior to dehydration of peach slices,
should be useful in enhancing inactivation of potential L. monocytogenes contamination. Dehydration at 60 C, following the ascorbic acid treatment, achieved a 5.09– 6.15 log cfu/g reduction of bacterial populations after 6 h of dehydration and resulted in undetectable bacterial populations by direct plating after 14 d of storage. Studies are currently being conducted to examine the sensory qualities of the dried product.
Acknowledgements This project was supported by USDA-CSREES National Integrated Food Safety Initiative Project x0051110-9747 and by the Colorado Agricultural Experiment Station.
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