661 Journal of Food Protection, Vol. 78, No. 4, 2015, Pages 661-667 doi: 10.4315/0362-028X.JFP-14-508

Survival of Salm onella on Chamomile, Peppermint, and Green Tea during Storage and Subsequent Survival or Growth following Tea Brewing SUSANNE E. KELLER,1* CHRISTINA N. STAM, 1 DANA R. GRADL, 1 ZHENGZAI CHEN,2 EMILY L. LARKIN,'t SHANNON R. PICKENS,2 and STUART J. CHIRTEL3 'Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Bedford Park, Illinois 60501; institute o f Food Safety and Health, Illinois Institute of Technology, 6502 South Archer Avenue, Bedford Park, Illinois 60501; and 3Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland 20740, USA MS 14-508: Received 23 October 2014/Accepted 4 December 2014

ABSTRACT The survival o f Salmonella on dried chamomile flowers, peppermint leaves, and green tea leaves stored under different conditions was examined. Survival and growth of Salmonella was also assessed after subsequent brewing using dried inoculated teas. A Salmonella enterica serovar cocktail was inoculated onto different dried tea leaves or flowers to give starting populations of approximately 10 log CFU/g. The inoculum was allowed to dry (at ambient temperature for 24 h) onto the dried leaves or flowers prior to storage under 25 and 35°C at low (< 3 0 % relative humidity [RH]) and high (> 9 0 % RH) humidity levels. Under the four storage conditions tested, survival followed the order 25°C with low RH > 35°C with low RH > 25°C with high RH > 35 C with high RH. Salmonella losses at 25°C with low RH occurred primarily during drying, after which populations showed little decline over 6 months. In contrast, Salmonella decreased below detection after 45 days at 35°C and high RH in all teas tested. The thermal resistance of Salmonella was assessed at 55°C immediately after inoculation o f tea leaves or flowers, after drying (24 h) onto tea leaves or flowers, and after 28 days of storage at 25°C with low RH. All conditions resulted in similar Dvalues (2.78 + 0.12, 3.04 + 0.07, and 2.78 + 0.56, at 0 h, 24 h, and 28 days, respectively), indicating thermal resistance of Salmonella in brewed tea did not change after desiccation and 28 days of storage. In addition, all brewed teas tested supported the growth of Salmonella. If Salmonella survives after storage, it may also survive and grow after a home brewing process.

Salmonella is a ubiquitous foodborne pathogen that is especially hazardous for those with impaired immune systems. Despite the prevalence of Salmonella in nature, low-moisture foods, such as dried herbs and spices, are not traditionally considered as high risk with respect to salmonellosis because the low water activity (aw) is a barrier for bacterial growth. However, numerous worldwide Salmonella outbreaks have been linked to low-moisture foods, such as nuts, peanut butter, paprika-flavored potato chips, black and red pepper, and dry dog food (4-6, 8-13, 20, 26). There have been at least two documented outbreaks of salmonellosis associated with herbal teas. In 2003, a cluster of Salmonella Agona infections, predominantly among infants, occurred in Germany (25). These infections were associated with the consumption of herbal tea containing anise seed. A second outbreak caused by Salmonella Senftenberg and associated with the consump­ tion of fennel seed tea occurred in Serbia from March 2007 to September 2008 (22). These outbreaks are particularly * Author for correspondence. Tel: 708-924-0617; Fax: 708-924-0690; E-mail: [email protected]. f Present address: Center for Medical Mycology, 11100 Euclid Avenue, LKS-5028, Cleveland, OH 44106-5028, USA.

noteworthy in that most of the illnesses occurred in infants. Teas and botanical products are occasionally consumed by infants, a vulnerable population. Data from 2005 to 2007 indicate 9% of infants in the United States are fed teas containing botanical ingredients (32). Salmonella is known for the ability to persist in lowmoisture environments and may survive months or even years in some food products under optimum conditions (23, 29). This persistence in dried environments, as well as the long shelf life of dried food products may contribute to the length of some outbreaks. The actual length of survival in various dry products, such as botanical and herbal teas, is unknown but may be affected by the presence of antimicrobial compounds (24, 27, 28, 31). There is also limited information on the survival of Salmonella, if present, in botanical and herbal teas during brewing. The thermal treatments provided by a consumer during tea brewing may be insufficient to destroy the pathogen. Recommended temperatures provided by various commer­ cial sources has been reported to vary from a low of 50°C to boiling, depending on the type of tea with steeping times as low as 1 min (18, 30). Even when longer brewing times or temperatures are recommended, the use of newer types of tea-steeping devices in which tea leaves float on the surface

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of water may prevent the delivery of thermal treatments appropriate for the destruction of Salmonella. In addition, the production of low-temperature teas, or sun-tea, a method in which tea is brewed at room temperature for extended periods of time and the temperature may not exceed 50°C (7), is particularly problematic. Consequently, research is needed to conduct an appropriate risk profile for these types of products. The objective of this study was to determine the survival of Salmonella in three types of herbal tea products, chamomile, peppermint, and green tea, stored under different storage temperatures and humidity levels. In addition, Salmonella thermal resistance and its ability to grow in these teas during and after brewing were evaluated. MATERIALS AND METHODS Inoculum development. Cultures used to inoculate various teas were Salmonella enterica serovars Enteritidis, Oranienburg, Anatum, and Tennessee (L. Beuchat, University of Georgia, Griffin). Cultures were stored as stock cultures on Trypticase soy agar supplemented with 0.6% yeast extract (TSAYE; BD, Franklin Lakes, NJ) added. Initial transfers for experiments were made from isolated single colonies to 10 ml of Trypticase soy broth supplemented with 0.6% yeast extract (TSBYE; BD) and incubated 24 h at 37°C. A second transfer (100 pi) was then made to the surface of TSAYE plates (23). Cultures were harvested by adding 1 ml of sodium phosphate buffer (0.5 M, pH 7.0) to the agar surface and gently scraping the surface with a sterile L-shaped plate spreader after which the resulting cell suspension was removed and collected in a sterile 50-ml conical tube (Fisher Scientific, Fair Lawn, NJ). Equal volumes of each harvested culture serovar were mixed to create inoculum cocktails at a final concentration of approximately 10 to 11 log CFU/ml. For the assessment of thermal resistance, only one serovar, Salmonella Anatum, was used. The culture was grown and harvested as indicated previously.

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Determination of survival during storage. To determine the survival of Salmonella during storage, dried inoculated herbal products were stored under four conditions: 25°C with ambient relative humidity (RH), 25°C and high humidity, 35°C with ambient RH, and 35°C with high RH. All Whirl-Pak bags containing tea samples were stored open under each condition. Ambient humidity samples were stored in open containers in temperature-controlled incubators. Ambient humidity was £30% RH. High humidity was >97% and was maintained using desiccators with saturated potassium sulfate solution within temperature-controlled incubators. High humidity desiccators measured from 97 to 100% RH humidity. Humidity and temperature were monitored during storage on a daily basis. Dried teas were also examined visually for any sign of mold growth. No mold was observed during the duration of the storage experiments. Populations of Salmonella were enumerated prior to storage and periodically up to 6 months of storage or until populations decreased below the minimum detectable limit (1.7 log CFU/g). At each sampling point, the aw of representative samples of stored green tea leaves, peppermint leaves, and chamomile flower samples were tested as described earlier. To enumerate surviving Salmonella populations, triplicate 1-g samples of each tea type were removed from each storage condition; each was mixed with 9 ml of TSBYE and allowed to soak for 60 min before serial dilution. The mixtures were homogenized in a stomacher (250 rpm) for 30 s then diluted with BPW. Resuspended teas were diluted appropriately and spread plated in duplicate on xylose lysine desoxycholate agar (XLD; BD) and TSAYE. All plates were incubated at 37°C for 24 to 48 h and subsequently enumerated for Salmonella populations.

Background microflora in teas. Prior to use in all experiments, the background level of microflora was assessed in all teas used. Background was determined by serially diluting in buffered peptone water (BPW, 1.0%; BD) and then spreading dilutions (0.1 ml) onto TSAYE plates. Plates were incubated at 37°C for 24 h. When used for thermal resistance experiments, green tea leaves were first steam sterilized at 121°C for 20 min. Sterility was assessed by adding 1 g of tea to 9 ml of BPW, mixing thoroughly, and spread plating 0.1 ml onto TSAYE plates. Plates were incubated at 37°C for 24 h.

Analysis of thermal resistance of Salmonella Anatum on tea leaves. Thermal resistance experiments were conducted on steam-sterilized dry green tea leaves inoculated with Salmonella Anatum. Three conditions were examined: green tea leaves directly after inoculation, green tea leaves inoculated and dried 24 h, and green tea leaves, inoculated, dried 24 h, then stored for 28 days at 25°C with ambient humidity. Green tea leaves (1 g per bag) were inoculated in Whirl-Pak bags, as previously described. To determine thermal resistance, 9 ml of water preheated to 55°C was added to the tea; the bag was closed and then immediately submerged in a 55°C circulating water bath. Samples were removed in triplicate at various time intervals and placed into an ice bath to cool. Independent trials were run in triplicate starting with separately grown inoculum. To determine surviving Salmo­ nella populations, each tea sample was serially diluted with BPW and then spread plated onto TSAYE. Agar plates were incubated at 37°C for 24 to 48 h, and colonies enumerated.

Inoculation of dried tea products. Chamomile, peppermint, and gun-powder green tea (Starwest Botanicals, Sacramento CA) were weighed aseptically into small Whirl-Pak bags (Nasco, Fort Atkinson, WI) in 1-g quantities. Harvested culture (0.1 ml) was carefully pipetted onto the dried leaves and flowers. For samples used in growth experiments, the inoculum was first diluted with phosphate buffer (0.01 M, pH 7.0) to a cell concentration of approximately 5 to 6 log CFU/ml prior to inoculating the dried product as indicated. All Whirl-Pak bags were left open 24 h in a biological safety cabinet to allow the inoculated tea leaf and flowers to redry prior to subsequent experiments, except for a subset used in thermal resistance assessments. The aw of the herbal products was measured before and after inoculation and drying. The aw was determined using an AquaLab Series 4TEV Meter (Decagon Devices, Pullman, WA).

Survival of Salmonella during simulated home brewing. Survival of Salmonella in inoculated herbal tea products (green tea, peppermint leaf, and chamomile flowers) was evaluated when simulating a home tea brewing process. Previously inoculated dried teas were prepared as described earlier. Teas were inoculated and dried 24 h before brews were prepared. Three grams of dried inoculated tea sample was subsequently placed into a tea infuser basket. Water (300 ml) preheated to 90, 75, or 55°C was poured into the tea infuser over the dried inoculated tea leaves (Fig. 1). Populations of Salmonella were determined after 1, 3, 5, and 8 min of brewing by removing 1 ml and serially diluting in BPW. The temperature was recorded at each sampling time point in the middle of the tea basket by using a handheld model 5411 temperature type K probe (Fluke Corporation, Everett, WA). Appropriate dilutions were plated in duplicate on XLD and

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Statistical analysis. An analysis of covariance was performed using SAS generalized linear model and MIXED procedures (SAS version 9.3, SAS Institute Inc., Cary, NC) on the data. RH, temperature, and type combinations were created and treated as categorical variables. A common intercept was assumed and the population declines versus time for each were generated along with 95% confidence limits. The aw was treated as a continuous variable, and the aw x log time interaction was estimated. An analysis of covariance was also perfonned on the decay rates obtained under different storage conditions treating each RH x tea type x tem­ perature combination as a categorical treatment group and days as the continuous variable. Contrast statements were written to test for the difference between teas at each RH x temperature combination, between temperatures at each tea type x RH combination, and between the RH at each tea type x temperature combination. A one­ way analysis of variance comparison was used for thermal death time comparisons. RESULTS AND DISCUSSION

FIGURE 1. Glass tea infuser. The glass jar consisted o f a doublewalled cylinder (8.25 by 2.75 in.), with a stainless steel basket (2.75 by 2.0 in) to hold tea leaves and a plastic screw cap to cover the top o f the infuser. TSAYE and were subsequently incubated at 37°C for 24 h for examination and enumeration of surviving Salmonella. Survival of Salmonella was also examined during simulated brewing without an infuser. Dried inoculated tea was first added to a cup, and heated water (55 C, 250 ml) was poured over the inoculated tea leaves. Survival was determined as with the tea infuser.

Survival and growth of Salmonella in low-temperature brewed teas. Two methods were used to assess growth of Salmonella in brewed teas. For the first method, 100 ml of deionized water was first pre-equilibrated to 6, 25, and 35°C temperatures. One gram of each type of herbal tea (three types of tea, 1 g into three separate bags, and one for each temperature) was then placed into a Whirl-Pak with pre-equilibrated water and mixed thoroughly. Each bag was then inoculated with the Salmonella cocktail (prepared as previously indicated) to give an approximate initial inoculum level of 3 to 4 log CFU/ml. For the second method, water pre-equilibrated to test temperatures, was added to 1 g of previously inoculated dried teas. These dried teas were inoculated as previously indicated, except that the inoculum for the dried tea was first diluted using phosphate buffer to result in a final concentration of approximately 5 log CFU/g in tea after redrying. After the dried green tea, chamomile flowers, and peppermint leaves were thoroughly mixed with 100 ml of water, 1 ml from each bag was removed, diluted, and plated on XLD. as described previously, to determine the initial populations. The bags of teas were then reincubated at 6°C for up to 72 h and at 25 and 35°C for 24 h. At various time intervals, 1.0 ml of sample was removed, and populations of Salmonella determined, as described previously.

Survival during storage. Survival of Salmonella as indicted by populations found on XLD in inoculated dry teas subjected to different storage conditions is shown in Figure 2. Populations were also monitored on nonselective media (TSAYE, data not shown). Microbial populations on nonselective media were typically approximately 1 log higher than on XLD but followed the same patterns of survival. The humidity during storage was reflected in the aw of the stored teas. After inoculation and prior to storage, the average aw of all three types of teas was 0.77 + 0.08. Under ambient storage conditions, when the humidity never exceeded 30% RH, aw declined in the first week, then stabilized at approximately 0.25 at both 25 and 35°C. When stored at 97% RH, the aw of the dried teas increased in the first week to stabilize at 0.95, regardless of temperature of storage. The rate of decline in populations of Salmonella was affected by the temperature of storage, the humidity of storage, and the aw of the tea (P < 0.0001). Populations of Salmonella were most stable at 25°C at low humidity, where little or no decline in populations occurred throughout the 6month storage period once aw stabilized below 0.3. At 35°C and high humidity, populations declined below detection limits in all teas in less than 45 days. The relationship is similar to that observed for ground black pepper (23). With ground black pepper, Salmonella showed little population decline for up to 8 months of storage at 25 °C under low humidity but declined below detectable limits within 45 days at 35''C stored under high humidity. Despite similarities in the various teas with respect to stability under the different storage conditions, some differences based on tea type were observed. As previously noted, the populations of Salmonella on all tea types were affected by aw (P < 0.0001); consequently, rates of declines in populations were not linear until the aw became stable (Fig. 2). However, once the aw equilibrated with the humidity, population declines became linear and a rate of population decline could be calculated using the linear portion of the curve (Table 1). At 25°C and low humidity, chamomile flowers and peppermint leaves showed essen­ tially no declines in Salmonella populations once the aw became stable for the remainder of the time period

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FIGURE 2. Survival o f Salmonella on dried green tea leaves, dried peppermint leaves, and dried chamomile flowers related to temperature and humidity o f storage. (A) 25°C, ^ 30% RH; (B) 25°C, s9 0 % RH; (C) 35°C, s3 0 % RH; (D) 35 C, S!90% RH. IB Populations on green tea leaves; □ , aw o f green tea leaves; A, populations on chamomile flowers; A, a„ o f chamomile flowers; • , populations on peppermint leaves; O , aw o f peppermint leaves. Limit o f detection: 1.7 log CFUIg.

or green tea leaves. These differences may reflect the presence of antimicrobial compounds (14).

examined. A small population decline was noted for green tea (P s 0.005). When humidity was increased, chamomile flowers showed significantly greater declines than did peppermint or green tea (P =£ 0.005). An increase in temperature, while maintaining low humidity, resulted in a significant difference in rate of population declines for all three teas, with chamomile again showing the fastest rates of decline (P < 0.005). When both temperature and humidity were increased, the rate of decline was similar for both peppermint and green tea, but again significantly higher for chamomile (P < 0.005). From these results, it would appear that dried chamomile flowers represent a more hostile environment for Salmonella than either peppermint leaves

Analysis of thermal resistance related to length of storage. The D- and z-values of Salmonella have been wellexamined in previous literature (1, 16, 21); consequently, the purpose of these tests was simply to determine if differences in thermal resistance occurred with respect to previous dried storage. Salmonella in low-moisture envi­ ronments shows increased thermal resistance; however, the reasons for increased resistance are not well understood (2, 15,17,19). To determine if prior desiccation and storage on dried tea leaves changes the thermal resistance of Salmo-

TABLE 1. Rate o f decline o f Salmonella in dried chamomile flower, peppermint leaf, and green leaf teasa

25°C, 35°C, 25°C, 35°C,

RH RH RH RH

< < > >

30% 30% 90% 90%

Rate (log CFU/g/day) 0.002 -0.034 -0.124 -0.538

A* a a a

Lower

Upper

- 0 .0 0 2

0.005 -0.038

-0.378 -0.137 -0.599

95%* confidence limits

95% confidence limits

95% confidence limits Storage conditions

Green tea

Peppermint

Chamomile

Lower

Upper

Rate (log CFU/g/day)

Lower

Upper

Ab

-0 .0 0 1

0.007 -0.013 -0 .0 4 -0 .184

-0.005 B -0 .0 2 7 c -0 .0 5 4 b -0.199 b

-0 .009 -0 .0 3 -0 .0 6 -0.217

- 0 .0 0 2

Rate (log CFU/g/day)

b

-0 .0 2 1

- 0 .1 1 2

0.003 -0.017 -0 .0 4 9

b

-0.057

-0.476

- 0 .2 0 2

b

-0 .2 2

a Numbers with different letters across rows are significantly different (P £ 0.005). b Rates not significantly different from zero.

-0.013 -0.048 -0.181

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TABLE 2. Thermal resistance o f Salmonella in green tea related to length o f storage o f dried tea leavesa Time

D55-value + SD

Day 0 Day 1 Day 28

2.78 ± 0.12 3.04 + 0.07 2.78 ± 0.56

a Salmonella population: average (log CFU per milliliter); n = 3; no significant differences between D-values (P > 0.05).

nella and increases its chances of survival during brewing, a series of thermal resistance tests were conducted. For these tests, only green tea leaves were used to represent the worstcase scenario, as recommended brewing temperatures are lowest for green teas. For the first test condition, freshly harvested cells of Salmonella Anatum were applied to dried green tea leaves to represent a control for both desiccation and storage. Salmonella Anatum was utilized for this purpose, as it appeared somewhat more heat resistant than the other serotypes used (data not shown). These cells were not dried but immediately tested for thermal resistance at 55°C after application to the dried green tea leaves. Only a single temperature was tested representing the lower range of temperatures recommended for tea brewing by online commercial sources (18, 30). To determine the effect of desiccation on subsequent thermal resistance, a subset of the freshly harvested cells applied to the dry green tea leaves were allowed to dry overnight prior to testing. The aw of the inoculated green tea leaves after drying was determined to be less than 0.5. To determine the effect of desiccation and storage on subsequent thermal resistance, a second subset of the freshly harvested cells applied to the dry green tea was allowed to dry overnight, then stored at 25 °C at ambient humidity for 28 days prior to testing. There was no significant difference found in the thermal resistance between Salmonella immediately after inoculation of tea verses those after desiccation and storage CP — 0.05; Table 2). The Z)55-value for Salmonella Anatum under the conditions of this test ranged from 2.78 to 3.04 min. This value is not different from values found in the literature for the thermal resistance of Salmonella (16). D-values measured here indicate a less than 1-log reduction in population would occur during 1 min of brewing at 55°C. Although this temperature seems somewhat low for tea brewing, recommended brewing conditions for green teas ranged from 60 to 85°C for 1 to 2 min (18, 30). However, teas brewed using higher starting temperatures may never remain at high temperatures for long periods of time. Cooling may occur rapidly, depending on the surface area of the tea cup and heat loss from thermal radiation at air ambient temperatures. In addition, brewing conditions that allow tea leaves to float freely may result in nonuniform heat treatment. Attachment of the Salmonella to the surface of the tea leaf may also provide some temporary protection from extremes of heat treatment. Although the thermal resistance of Salmonella Anatum at 55°C was not different based on desiccation and storage, the measurement of

FIGURE 3. Survival o f Salmonella during home brewing at 55°C using a commercial glass jar (infuser). For populations in tea, n = 3, fo r temperature, n = 9. ■ , Populations on green tea leaves; ▲. populations on chamomile flowers; • , populations on peppermint leaves; O, temperature.

thermal resistance was more variable after storage; a larger standard deviation was found in thermal resistance using Salmonella previously dried and stored on green tea leaves (Table 2). This may be the result of stronger adhesion of the cells to the dried tea leaves after desiccation and storage. Adhesion to the surface of tea leaves could interfere with thermal transfer resulting in less precision related to the experimental method employed, and consequently increas­ ing the variability in the observed results. Simulated home brewing. In an attempt to determine the survival of Salmonella in a home brewing process, a glass jar tea infuser (Fig. 1) for home use was acquired and teas (peppermint, chamomile, and green tea) were prepared at 90, 75 and 55°C. Salmonella populations at the start of each trial ranged between 6.33 on green tea leaves to a high of 7.58 log CFU/ml on peppermint leaves. Salmonella was not detected in the brewed teas prepared using the infuser at 90 or 75°C after 1 min. The temperature, measured in the center of the tea basket, dropped from an initial of 90°C to no less than 74°C, and from 75°C to no less than 62°C during the maximum 8-min brewing time used. The glass infuser had a double body that provided insulation to prevent cooling during the brewing period, particularly when the cap was in place. Consequently, sufficient thermal treatment was delivered to obtain a minimum 5-log kill at these two temperatures. However, for teas brewed at 55°C, less than a 2-log reduction was seen for all three tea types when brewed for 8 min (Fig. 3). For most trials, the temperature of brewing did not drop below 49°C for the 8-min brewing period (Fig. 3). Results using a standard cup were similar to those found for the glass jar (data not shown). With a D55-value of 2.98 min, less than a 1-log reduction in population would have been observed after 1 min. For all teas tested, most of the reduction in Salmonella population was observed in the first minute of brewing. After 1 min, sufficient cooling of the brewed tea resulted in no further Salmonella reduction. In both green

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TABLE 3. Salmonella population in green, peppermint, and chamomile teas inoculated into liquid teas and incubated at different temperatures Salmonella population (log CFU/ml) Initial at 6°C

24 h at 6°C

72 h at 6°C

Initial at 25°C

24 h at 25°C

Initial at 35°C

24 h at 35°C

2.88 2.81 2.82

2.86 2.88 2.86

2.27 2.34 2.06

2.89 2.17 2.93

7.93 7.80 7.86

3.02 2.89 2.79

7.81 7.93 7.82

Peppermint Chamomile Green tea

tea and chamomile tea, a greater reduction occurred in the first minute of brewing than would have been predicted by the Z)55-value. These greater reductions may be due to the sampling procedure, as enumerated samples reflected both the reductions in population and the uniformity of distribution of Salmonella in the brewed tea. As the liquid in the jar was not mixed prior to sampling, some variability was likely due to nonuniform distribution of Salmonella in the brews. Nonetheless, after an initial decline at the 1-min time interval, no further decreases were observed. Previous testing with Salmonella Anatum on green tea leaves would suggest that no increased thermal resistance can be attributed to attachment to the surface of the leaves or storage up to 28 days. In addition, although the thermal resistance of Salmonella described in the literature appears to vary with serotype and heating medium, no published values would indicate extraordinary survival in hot (100 C) water. Consequently, Salmonella would not normally be expected to survive tea brewing if exposed to temperatures higher than 80°C for several minutes. However, as indicated in these home-brewing experiments, it may be unlikely that Salmonella is exposed to such temperatures for significant periods of time during such a process. A perusal of Web sites purporting to provide advice on the brewing of teas found typical recommended temperatures of 60 to 85 JC, along with brewing vessels similar to the one used here, and using loose leaf teas that would clearly allow tea leaves to float freely (18, 30). Floating on the surface of such containers could protect any Salmonella present due to both a boundary affect when attached to leaves and a surface cooling effect. The brewing temperatures to which the Salmonella is exposed may, therefore, be considerably lower than the initial value, as shown in these experiments. In addition, the amount of time to which Salmonella is actually exposed to any thermal treatment may be relatively short (< 1-min) periods of time. Thus, the actual thermal treatment received may be difficult to predict.

TABLE 4. Salmonella population in green, peppermint, and chamomile teas brewed using inoculated dried teas and incubated at 25 °C Avg Salmonella population (log CFU/ml) + SD

Green tea Peppermint tea Chamomile tea

0h

8h

24 h

3.46 ± 0.17 3.85 ± 0.33 3.70 ± 0.15

4.74 + 0.18 5.54 ± 0.21 6.10 ± 0.07

8.95 + 0.10 8.96 ± 0.07 9.19 ± 0.39

Growth of Salmonella in brewed chamomile, peppermint, and green teas. Many teas are believed to contain inhibitory compounds that would prevent growth of pathogens (3, 14, 24). If such compounds are present in significant quantities, survival during storage and subse­ quent brewing would not necessarily lead to subsequent growth in prepared teas. To measure survival or growth of Salmonella in brewed chamomile, peppermint, and green tea, brews of each tea were prepared and incubated at three different temperatures, refrigerated (6°C), room (25°C), and a temperature more optimal for growth of Salmonella (35°C). The brewed teas at each temperature were then inoculated with Salmonella at 3 to 4 log CFU/ml and populations followed for up to 72 h (Table 3). At both 25 and 35°C, populations increased to over 8 log CFU/ml within 24 h. At 6°C, populations did not increase but remained stable for over 24 h. Declines in populations at 6°C were seen at 72 h. In separate experiments, growth in brewed teas was also determined using previously inoculated chamomile flowers, green tea, and peppermint leaves that were dried for 24 h at room temperature (25°C) prior to use in experiments. Growth observed using inoculum dried overnight onto the tea leaves was similar to growth for Salmonella spiked in brewed teas (Table 4). In conclusion, Salmonella can survive for extensive periods of time (greater than 6 months) in dried chamomile flowers, peppermint leaves, and green tea leaves stored at 25°C under low humidity conditions. Increases in humidity and temperature during storage resulted in decreases in survival. These effects of storage conditions are similar to those reported by Keller et al. (23) for Salmonella in black pepper. Some differences in survival rates can be attributed to the type of matrix, as the teas had different chemical compositions. The differences with respect to the type of tea were less pronounced than the combined effects of increased temperature and humidity of storage. Higher temperature and humidity resulted in large reductions of Salmonella: however, typical storage conditions, particular­ ly in the home of a consumer, may not reduce Salmonella to levels incapable of causing infection. In addition, subse­ quent experiments indicate that Salmonella survival during brewing was independent of previous desiccation or age (up to 28 days) of cells. Consequently, survival of Salmonella during brewing can be explained by its thermal resistance in liquids. Salmonella should not survive brewing, especially when higher temperatures (>80°C) are used. However, Salmonella may survive lower brewing temperatures occasionally recommended by commercial sources. Brew-

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ing at room temperatures, methods commonly referred to as sun brewing, would be particularly dangerous, especially as Salmonella is capable of growth in the three tea types examined in this study. Although some inhibition due to naturally occurring compounds in the teas cannot be ruled out, their presence is clearly insufficient to completely prevent growth of the pathogen. As a result, should dried teas become contaminated with Salmonella during their production, the pathogen could clearly survive to create a subsequent risk to the consumer.

12.

13.

14.

15.

ACKNOWLEDGMENTS This work was supported by the U.S. Food and Drug Administration (FDA) collaborative grant 5UOIFD003801 and by appointments to the Research Participation Program at the Center for Food Safety and Applied Nutrition administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the FDA.

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