Growth Kinetics of Listeria monocytogenes and Salmonella enterica on Dehydrated Vegetables during Rehydration and Subsequent Storage

Dehydrated vegetables have low water activities and do not support the proliferation of pathogenic bacteria. Once rehydrated, vegetables can be incorporated into other foods or held for later use. The aim of this study was to examine the survival and proliferation of Listeria monocytogenes and Salmonella enterica on dehydrated vegetables during rehydration and subsequent storage. Carrots, corn, onion, bell peppers, and potatoes were heat dehydrated, inoculated at 4 log CFU/g, and rehydrated at either 5 or 25 °C for 24 h. Following rehydration, vegetables were stored at 5, 10, or 25 °C for 7 d. Both L. monocytogenes and S. enterica survived on all vegetables under all conditions examined. After 24 h of rehydration at 5 °C, pathogen populations on the vegetables were generally <1.70 log CFU/g, whereas rehydration at 25 °C resulted in populations of 2.28 to 6.25 log CFU/g. The highest growth rates during storage were observed by L. monocytogenes on potatoes and S. enterica on carrots (2.37 ± 0.61 and 1.63 ± 0.18 log CFU/g/d, respectively) at 25 °C when rehydration occurred at 5 °C. Results indicate that pathogen proliferation on the vegetables is both rehydration temperature and matrix dependent and highlight the importance of holding rehydrated vegetables at refrigeration temperatures to hinder pathogen proliferation. Results from this study inform time and temperature controls for the safety of these food products.


Introduction
Plant foods, including vegetables, are popular around the world because of their nutritional value and abundance. However, the high moisture content and high water activity (a w ) of these food commodities translates to a short shelf life. Various techniques are therefore employed to preserve these foods for longer periods, including desiccation methods. Dehydration using heat is a common method to desiccate vegetables, resulting in the reduction of a w (to <0.70) and also reducing the weight, which is convenient for both transportation and storage. To minimize any organoleptic changes of the vegetables during dehydration, treatments are usually mild and use low heat (<80 • C) [1]. Vegetables are often heat dehydrated using a tunnel of hot air, where the internal product temperature does not often exceed 35-45 • C [2]. Therefore, heat dehydration is generally not an effective method for reducing the microbial load or for the inactivation of foodborne bacterial pathogens, including Listeria monocytogenes and Salmonella enterica.

Inoculation of Dehydrated Vegetables
Dehydrated vegetables (400 g) were transferred to 3-L stomacher bags and inoculated with either the L. monocytogenes or S. enterica cocktail at 4 log CFU/g. The stomacher bags were hand shaken for 5 min to uniformly distribute the inoculum. Inoculated vegetables were arranged onto foil pans and dried at ambient temperature for 24 h in a biosafety cabinet with the blower on. After 24 h, triplicate 10-g samples were used for pathogen enumeration (see Section 2.7).

Rehydration of Dehydrated Vegetables
After 24 h of drying, the inoculated dehydrated vegetables were rehydrated 1:10 in water in an 8 L metal bowl at either 5 or 25 • C for 24 h. Both the water and air temperature were maintained at either 5 or 25 • C. During rehydration, vegetable samples were removed from the water at 2.5, 5, and 30 min, and 1, 2, 4, 6, and 24 h. At each of these timepoints, the vegetables were stirred and approximately 50 g were removed from the water and strained for 10 min using a 10 cm diameter strainer. Triplicate 10 g samples were used for pathogen enumeration (see Section 2.7) and duplicate 10 g samples were used for moisture content analysis (see Section 2.6). After 24 h of rehydration, the remaining vegetables were strained for 10 min using an 8 L colander.

Storage of Rehydrated Vegetables
The remaining strained rehydrated vegetables were portioned into 8 oz deli containers with lids (40 g each). Deli containers were stored at 5, 10, or 25 • C for 7 d. After 0, 1, 3, 5, and 7 d, pathogens were enumerated from the samples (see Section 2.7). Triplicate independent trials were conducted for each vegetable, temperature, and pathogen combination (dehydration through storage).

Measurement of pH, Water Activity (a w ), and Moisture Content
For pH, a 10 g sample of vegetable was stomached for 1 min with 10 mL of distilled autoclaved water in a stomacher (400 Circulator Lab Blender, Seward, UK) and the pH of the homogenate was measured with a pH meter (MP220 pH meter, Mettler Toledo, Columbus, OH, USA). To measure a w , a 1 g sample of vegetable was measured using an a w meter (Aqualab 4TE, Meter Group, Pullman, WA, USA). For moisture content, a 10 g sample of vegetable was placed into an oven at 100 • C for 24 h. The solid weight after 24 h was measured and the moisture content was then calculated.

Enumeration of L. monocytogenes and S. enterica
Vegetable samples (10 g) were homogenized in a stomacher at 1:10 with either Buffered Listeria Enrichment Broth (BLEB; Becton, Dickinson and Co., Sparks, MD, USA) or BPB for L. monocytogenes or S. enterica, respectively. Homogenates were serially diluted and plated onto Brain Heart Infusion Agar (BHIA; Becton, Dickinson and Co., Sparks, MD, USA) with rifampicin (BHIA rif ) or onto TSAYE with Xylose Lysine Deoxycholate (XLD; Becton, Dickinson and Co., Sparks, MD, USA) agar overlay for enumeration of L. monocytogenes or S. enterica, respectively. Agar plates were incubated at 37 • C for 24-48 h. Data were expressed as log CFU/g. The limit of enumeration of the plate count assay was 1.70 log CFU/g.

Modeling of Growth Kinetics and Statistical Analysis
Growth kinetics (i.e., growth rates and lag phases) of both L. monocytogenes and S. enterica during 7 d storage at 5, 10, or 25 • C were determined using the DMFit v 3.0 addin for Excel (Baranyi and Roberts, 1994). Differences between growth rates were statistically analyzed using ANCOVA with Tukey's post hoc test (α = 0.05). Differences in pH, a w , moisture contents, and populations were statistically analyzed using ANOVA with Tukey's post hoc test (α = 0.05).

Characteristics of the Fresh, Dehydrated, and Rehydrated Vegetables
The pH and a w values of the fresh vegetables used in this study are presented in Table 1. The pH values of the fresh vegetables ranged from 6.15 ± 0.06 (for bell pepper) to 7.00 ± 0.10 (for carrot). The pH of the bell pepper was significantly lower than all the other vegetables. The a w of all the fresh vegetables ranged from 0.951 ± 0.015 (for corn) to 0.960 ± 0.008 (for potato). There was no significant difference in the a w values between vegetables. After dehydration of the vegetables at 60 • C for 24 h, the pH values ranged from 5.73 ± 0.07 (for onion) to 6.77 ± 0.02 (for corn). When comparing the pH values of the five vegetables after dehydration, all were significantly different from each other, with the exception of carrot and potato. For a w, the values after dehydration ranged from 0.221 ± 0.025 (for potato) to 0.262 ± 0.050 (for bell pepper) and were not significantly different. Compared to their fresh counterparts, all a w and pH values for the dehydrated vegetables were significantly lower. The moisture contents of the fresh, dehydrated, and rehydrated vegetables were also measured in this study and are presented in Table 2 and Figures S1 and S2. The moisture contents of the fresh vegetables ranged from 78.00 ± 0.15 (for potato) to 93.69 ± 1.28% (for bell pepper); all values were significantly different. The moisture contents for all vegetables significantly decreased after dehydration and ranged from 4.50 ± 0.68 (for potato) to 15.58 ± 0.94% (for onion). The moisture contents of both onion and bell pepper were significantly higher than the other three vegetables, whereas the moisture content of potato was significantly lower than the four other vegetables. After 24 h of rehydration at either 5 or 25 • C, the moisture contents of all the dehydrated vegetables significantly increased. The rehydrated moisture contents ranged from 68.00 ± 4.39 (for potato rehydrated at 5 • C) to 94.19 ± 0.91% (for onion rehydrated at 25 • C). When comparing the differences between the two rehydration temperatures, the moisture contents after 24 h were not significantly different for all vegetables, with the exception of corn and bell pepper. Rehydration at 25 • C resulted in significantly higher moisture contents of both corn and bell pepper (83.36 ± 1.13 and 92.08 ± 1.41%, respectively) compared to 5 • C (79.39 ± 0.92 and 90.38 ± 0.26%, respectively). Not all vegetables rehydrated to the same moisture content levels as their fresh counterparts. Most notably, the moisture contents of potato after rehydration at 5 and 25 • C (68.00 ± 4.39 and 68.42 ± 4.27%, respectively) were significantly lower than the moisture content when fresh (78.00 ± 0.15%).

Survival of L. monocytogenes and S. enterica on Dehydrated Vegetables during Rehydration
The populations of L. monocytogenes and S. enterica on the vegetables prior to and after rehydration are depicted in Tables 3 and 4, respectively. Prior to rehydration, the population of L. monocytogenes on the dehydrated vegetables after inoculation and drying for 24 h ranged from 1.99 ± 0.18 (on corn) to 2.81 ± 0.17 log CFU/g (on bell pepper). The pathogen population on corn was significantly lower than on carrot, bell pepper, or potato. Drying of the vegetables for 24 h resulted in an approximate 1.19-2.01 log CFU/g reduction of L. monocytogenes. After rehydration at 5 • C for 24 h, L. monocytogenes was below the limit of enumeration (1.70 log CFU/g) on all vegetables with the exception of potato, where the population was 1.85 ± 0.16 log CFU/g. After rehydration at 25 • C for 24 h, the L. monocytogenes populations ranged from 2.28 ± 0.20 (on corn) to 3.42 ± 0.35 log CFU/g (on potato). Similar to the initial populations, the levels on corn were significantly lower than on carrot, bell pepper, or potato. Table 3. L. monocytogenes population dynamics on dehydrated vegetables prior to and after 24 h rehydration at 5 or 25 • C.

Vegetable
Initial Population 1 (log CFU/g ± SD 2 ) Population after Rehydration 3 (log CFU/g ± SD)  Prior to rehydration, the population of S. enterica on the dehydrated vegetables after inoculation and drying for 24 h ranged from 1.82 ± 0.51 (on carrot) to 2.83 ± 0.09 log CFU/g (on potato). The pathogen population on carrot was significantly lower than on all the other vegetables. Drying of the vegetables for 24 h resulted in an approximate 1.17-2.18 log CFU/g reduction of S. enterica. After rehydration at 5 • C for 24 h, S. enterica was below the limit of enumeration on all vegetables with the exception of onion, where the population was 1.99 ± 0.15 log CFU/g. After rehydration at 25 • C for 24 h, the S. enterica populations ranged from 2.32 ± 0.50 (on onion) to 6.25 ± 0.10 log CFU/g (on potato). The pathogen populations on onion and corn were significantly lower than on the other three vegetables. Compared to L. monocytogenes, the S. enterica populations on the vegetables after rehydration at 25 • C were all significantly higher; the only exception was on onion, where the two pathogen populations were not significantly different.

Growth Kinetics of L. monocytogenes and S. enterica on Rehydrated Vegetables during Storage
After rehydration at either 5 or 25 • C, the vegetables were stored at 5, 10, or 25 • C for 7 d. The population dynamics of L. monocytogenes and S. enterica on the rehydrated vegetables during storage are presented in Figures 1 and 2, respectively. Additionally, the growth rates, calculated times for a 1 log CFU/g increase in population, and the L. monocytogenes and S. enterica populations at the end of the 7 d storage period are depicted in Tables 5 and 6, respectively. In general, storage at 25 • C resulted in the highest growth rates and populations after 7 d storage for both pathogens. For L. monocytogenes, no significant proliferation was observed during storage at 5 • C on carrots, corn, onion, or potato when rehydrated at 5 • C or on onion when rehydrated at 25 • C. At 10 • C storage, the L. monocytogenes populations increased significantly on pepper and potato regardless of the rehydration temperature; populations after 7 d were 3.89 ± 0.30 and 4.75 ± 0.25 (on pepper) and 4.62 ± 1.00 and 6.23 ± 0.24 log CFU/g (on potato) when rehydrated at 5 and 25 • C, respectively. The pathogen also increased significantly in population on onion at 10 • C when rehydrated at 25 • C, with a population of 5.29 ± 0.06 log CFU/g after 7 d storage; no significant population increase was observed when onion was rehydrated at 5 • C and stored at 10 • C. L. monocytogenes increased significantly in population on all vegetables stored at 25 • C, regardless of the rehydration temperature, with one exception: no proliferation was observed on onions rehydrated at 5 • C. With the exception of onion, the population of L. monocytogenes on the vegetables after 7 d storage at 25 • C ranged from 6.53 ± 0.84 (on carrots rehydrated at 25 • C) to 8.56 ± 0.91 log CFU/g (on pepper rehydrated at 5 • C). stored at 10 °C. L. monocytogenes increased significantly in population on all vegetab stored at 25 °C, regardless of the rehydration temperature, with one exception: no pro eration was observed on onions rehydrated at 5 °C. With the exception of onion, the p ulation of L. monocytogenes on the vegetables after 7 d storage at 25 °C ranged from 6.5 0.84 (on carrots rehydrated at 25 °C) to 8.56 ± 0.91 log CFU/g (on pepper rehydrated °C).     The highest growth rate of L. monocytogenes during storage at 5 • C was observed on potato rehydrated at 25 • C (0.08 ± 0.08 log CFU/g/d), with a 1 log CFU/g increase in 12.50 d. At 10 • C, the highest growth rate was observed on potato rehydrated at 5 • C (1.35 ± 0.79 log CFU/g/d) where the time for a 1 log CFU/g increase was 1.74 d. At 25 • C storage, the lowest growth rate of L. monocytogenes (not including onion rehydrated at 5 • C) was observed on carrot rehydrated at 25 • C (0.39 ± 0.06 log CFU/g/d) resulting in a 1 log CFU/g increase in 2.56 d. The highest growth rate at 25 • C storage was observed on potato rehydrated at 5 • C (2.37 ± 0.61 log CFU/g/d) with an increase of 1 log CFU/g in only 0.42 d (or 10.08 h).
For S. enterica, no significant proliferation was observed on the vegetables during storage at 5 • C, with the exception of potato rehydrated at 25 • C, where the ending population after 7 d storage was 7.72 ± 0.13 log CFU/g (an increase of approximately 1.47 log CFU/g). At 10 • C, S. enterica significantly proliferated on only potato and carrot; populations after 7 d were 6.18 ± 0.90 and 8.02 ± 0.17 log CFU/g on potato when rehydrated at 5 and 25 • C, respectively, and 0.21 ± 0.06 log CFU/g on carrot when rehydrated at 25 • C. At 25 • C, S. enterica did not significantly grow on onions or peppers rehydrated at 5 • C or on corn rehydrated at 25 • C. When rehydrated at 5 • C and stored at 25 • C, the highest S. enterica populations after 7 d were observed on carrot and potato (7.26 ± 0.59 and 7.44 ± 0.76 log CFU/g, respectively). When rehydrated at 25 • C and stored at 25 • C, the highest population was observed after 7 d on pepper (10.25 ± 0.06 log CFU/g). This was also the highest population attained by either pathogen after 7 d at 25 • C. In comparison to L. monocytogenes, many of the growth rates of S. enterica during storage on the vegetables were negative. These negative rates were observed at all storage temperatures on corn rehydrated at 25 • C, on onion stored at 5 and 10 • C at both rehydration temperatures, and on pepper rehydrated at 5 • C and stored at 5 and 25 • C and rehydrated at 25 • C and stored at 5 and 10 • C. At 5 • C, the highest growth rate of S. enterica was observed on potato rehydrated at 25 • C (0.16 ± 0.03 log CFU/g/d) resulting in a 1 log CFU/g increase in 6.65 d. At 10 • C, the highest growth rate was observed on potato rehydrated at 5 • C (0.67 ± 0.06 log CFU/g/d) with a 1 log CFU/g increase in only 1.49 d. During storage at 25 • C, the highest growth rate of S. enterica was observed on carrot rehydrated at 5 • C (1.63 ± 0.18 log CFU/g/d) resulting in a 1 log CFU/g increase in only 0.61 d (or 14.64 h).

Discussion
This study examined the population dynamics of two foodborne pathogens, L. monocytogenes and S. enterica, on dehydrated vegetables during rehydration and subsequent storage. Compared to their fresh state, dehydrated vegetables are a convenient option for retail establishments, restaurants, and consumers, as they have long shelf lives, are lightweight and take up minimal space when stored, and they retain much of their nutritional values [32]. Dehydrated vegetables are also versatile and can be consumed alone once rehydrated or incorporated into other foods. However, once dehydrated vegetables are rehydrated, the high a w could provide an environment suitable for the proliferation of foodborne pathogens. Since rehydrated vegetables may be stored for hours or days prior to consumption, this study evaluated the need for time and temperature control for safety for these food commodities.
Two different rehydration temperatures were employed in this study, 5 and 25 • C. Whereas no published studies have evaluated the survival of foodborne pathogens during the rehydration of dehydrated vegetables, studies have examined the rehydration kinetics of different vegetables, including those used in this study [28,30]. One of these studies used high temperatures ranging from 40 to 80 • C to rehydrate myriad vegetables, including carrot, corn, onion, pepper, and potato, with an interest in examining the rehydration kinetics, equilibrium moisture contents, and quality characteristics [28]. The authors determined that the water temperature used for rehydration influenced the rehydration rate, as higher temperatures resulted in higher rehydration rates and thus lower times to reach equilibrium moisture. While high temperatures may be used to rehydrate dehydrated vegetables, especially if they are incorporated into hot soups or stews, retail establishments and consumers may also rehydrate these products in water at ambient temperature or in the refrigerator when guides or instructions do not include specific temperatures for rehydrating dehydrated vegetables.
This study determined that rehydration of dehydrated vegetables at 5 • C for 24 h resulted in a decrease in L. monocytogenes and S. enterica populations, whereas populations of both pathogens increased on carrot, corn, and potato and of S. enterica on bell pepper when rehydration occurred at 25 • C. Compared to L. monocytogenes, higher population increases were observed for S. enterica during rehydration at 25 • C; the highest increases were observed on potato (3.42 log CFU/g) and carrot (2.56 log CFU/g). Compared to the other vegetables used in this study, potato and carrot are both root vegetables and contain the highest carbohydrate contents (16 and 10%, respectively) [33]. The higher temperature of 25 • C coupled with the nutrient contents of these two root vegetables may have played a role in the proliferation of S. enterica during rehydration. Interestingly, neither pathogen grew on onion and only L. monocytogenes proliferated on corn during rehydration. Onions are known to have antimicrobial properties [34], which may have resulted in extended lag phases during adaptation to the environment, thereby hindering the proliferation of both pathogens.
Once rehydrated, vegetables may be stored for hours or days prior to use. This study examined L. monocytogenes and S. enterica survival and growth on rehydrated vegetables at three different storage temperatures: 5 • C (refrigeration), 10 • C (temperature abuse), and 25 • C (ambient). Overall, both pathogens survived on all vegetables during storage at 5 • C regardless of the rehydration temperature; the fastest 1 log CFU/g population increases were observed by both pathogens on potato rehydrated at 25 • C (12.50 and 6.65 days for L. monocytogenes and S. enterica, respectively). Similar growth rates of L. monocytogenes have been observed for freshly chopped broccoli and cauliflower stored at 4 • C (growth rates of 0.05-0.10 log CFU/g/d with extrapolated 1 log CFU/g increases in 10.00-20.00 days) [35]. However, higher growth rates of L. monocytogenes have also been observed on freshly chopped red and green bell pepper, cucumber, and avocado pulp stored at 5 • C (growth rates of 0.016-0.071 log CFU/g/h with 1 log CFU/g increases in 14.19-63.85 h (or 0.38-1.69 days) [36]. It is possible that the process of rehydration deterred the growth of the pathogens on the vegetables stored at 5 • C, and that growth rates may be higher when inoculated directly onto freshly chopped matrices.
During storage at the temperature abuse condition of 10 • C, potato rehydrated at either 5 or 25 • C supported the proliferation of both pathogens, while bell pepper rehydrated at either 5 or 25 • C also supported the growth of L. monocytogenes. On potato stored at 10 • C, the fastest 1 log CFU/g population increases were observed for both pathogens when rehydration occurred at 5 • C (1.74 and 1.49 days for L. monocytogenes and S. enterica, respectively) as opposed to 25 • C (2.33 and 11.11 days, respectively). When rehydrated at 25 • C, the native microbiota of the potato may have also proliferated, inducing competition for nutrients by either pathogen when subsequently stored at 10 • C. These results are comparable to the literature, as L. monocytogenes was observed to increase in population by approximately 2 log CFU/g on fresh potato tuber slices stored at 8 • C for 12 days [37]. On bell pepper stored at 10 • C, 1 log CFU/g population increases of L. monocytogenes occurred after 0.83 and 4.17 days when rehydration occurred at 5 or 25 • C, respectively. Similarly, with 5 • C storage, higher growth rates have been documented on freshly chopped green bell pepper stored at 10 • C, where L. monocytogenes increased 1 log CFU/g in 51.12-53.92 h (or 0.45-0.47 days) [36].
At the ambient storage condition of 25 • C, the growth rates of both L. monocytogenes and S. enterica on carrot, corn, and potato were always higher when rehydration occurred at 5 • C, whereas growth rates were always higher on onion and pepper when rehydration occurred at 25 • C. One of the most drastic differences in the two rehydration temperatures was observed with onions, where both pathogens did not proliferate during storage at 25 • C on onions rehydrated at 5 • C, but when rehydrated at 25 • C, 1 log increases occurred in 3.35 and 0.74 days for L. monocytogenes and S. enterica, respectively. These results suggest that the proliferation of both pathogens on rehydrated vegetables at ambient temperature is both rehydration temperature and matrix dependent. Storage at 4 • C, compared to 10 or 25 • C, has been shown to better preserve enzymes and other antimicrobial compounds in onions [38]. It is likely that rehydration of the onions in this study at 5 • C aided in the preservation of these compounds, resulting in an environment which did not support the proliferation of either pathogen during subsequent storage at 25 • C.
Prior to dehydration, it is recommended to blanch certain vegetables to inactivate enzymes, preserve color and flavor, and reduce the microbial population [1,39]. For example, blanching is recommended for potatoes to inactivate enzymes involved in browning. However, some exceptions include onions, peppers, mushrooms, and tomatoes, because blanching results in loss of flavor and color [39]; it is also not recommended to blanch onions or bell peppers prior to freezing [40]. For consistency, this study did not utilize blanching prior to dehydration of the vegetables. Since blanching was not used, the vegetables used in this study retained their native microbiota, which may have impacted the results. Future studies could examine the fate of both L. monocytogenes and S. enterica during rehydration of blanched and dehydrated vegetables. It is possible that growth rates of both pathogens would be higher during rehydration and subsequent storage as no competition for nutrients would be required.

Conclusions
This study determined that the survival and proliferation of both L. monocytogenes and S. enterica on dehydrated vegetables during rehydration and storage was dependent on many variables, including the rehydration temperature, the temperature of storage, and the matrix characteristics. While both pathogens survived during rehydration and subsequent storage on all the dehydrated vegetables examined in this study, lower growth rates were generally observed when the vegetables were rehydrated at 5 • C and then stored at 5 • C. These results highlight the importance of holding rehydrated vegetables at refrigeration temperatures to hinder pathogen proliferation. Data for this study can be used to inform regulatory decisions surrounding time and temperature control for safety for these food products.