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Increased Water Activity Reduces Thermal Resistance of Salmonella enterica 1

in Peanut Butter 2

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Yingshu He,a Ye Li,a Joelle K. Salazar,a Jingyun Yang,b Mary Lou Tortorello,c and Wei 4

Zhanga 5

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Institute for Food Safety and Health, Illinois Institute of Technology, Bedford Park, Illinois, 7

USAa; Methodology Center, Pennsylvania State University, University Park, Pennsylvania, 8

USAb; U.S. Food and Drug Administration, Bedford Park, Illinois, USAc 9

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Address correspondence to Wei Zhang, zhangw@iit.edu. 11

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Running Title: Salmonella heat resistance in peanut butter 13

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Copyright © 2013, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01028-13 AEM Accepts, published online ahead of print on 31 May 2013

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ABSTRACT 19

Increased water activity in peanut butter significantly (P < 0.05) reduced the heat resistance 20

of desiccation-stressed Salmonella enterica strains when treated at 90 °C. Difference in thermal 21

resistance was less notable when treated at 126 °C. Using scanning electron microscopy, we 22

observed minor morphological changes of S. enterica cells during desiccation and rehydration 23

processes in peanut oil. 24

25

TEXT 26

Salmonellosis outbreaks linked to contaminated peanut butter products have brought 27

worldwide attention to the microbial safety of these popular food items. S. enterica serotype 28

Tennessee caused a salmonellosis outbreak in 2006-2007 linked to peanut butter, which sickened 29

425 persons and resulted in 71 (20%) hospitalizations in 44 states in the U. S. (1). This and other 30

foodborne outbreaks (2, 3) highlight the need for a re-examination of S. enterica behavior in low 31

water activity (aw) peanut butter products. 32

The water activity of peanut butter is typically 0.35 or less (2, 4-9), which precludes the 33

growth of spoilage and pathogenic microorganisms. When present in peanut butter, S. enterica 34

become heat resistant, possibly due to adaptation to the desiccation stress and the protective 35

effects of the fat content in the product (2, 4-7, 10-14). We recently demonstrated that heat 36

treatment at 72 °C for an hour resulted in less than 2-log reduction of desiccation-stressed S. 37

enterica in artificially contaminated peanut butter with an aw of 0.4 (15). In this study, we 38

evaluated the effects of desiccation and subsequent rehydration on the relative heat resistance of 39

three S. enterica serotypes: Tennessee K4643 (a human isolate from the 2006-2007 peanut butter 40

outbreak in the U. S.) (1), Enteritidis BSS-1045 (an isolate from the 2000-2001 raw almonds 41

3

outbreak in the U. S. and Canada) (16-18) and Typhimurium LT2 (19, 20). We compared two 42

commercial peanut butter formulations (regular and low-fat) to assess the influence of 43

carbohydrate and fat contents on the heat resistance of S. enterica. Most published thermal 44

challenge studies of S. enterica in peanut butter focused on heat treatments at either 72 oC or 90 45

oC (11, 21, 22), but not at the higher temperatures commonly used in commercial peanut butter 46

processing, such as dry roasting at 126 oC (22). In this study we thermally challenged S. enterica 47

strains in artificially contaminated peanut butter at both 90 oC and 126 oC. 48

Individual strains and a three-strain cocktail were grown separately as previously 49

described (15) followed by suspension in 5-mL peanut oil prior to inoculation of peanut butter 50

(aw 0.2). Bacterial cell suspensions were transferred to 500-g of peanut butter and vigorously 51

stirred for 20 min using a sampler spatula. Homogenous distribution of the cells was verified as 52

previously described (15). Inoculated samples were stored at 25 oC for 4 weeks, then serially 53

diluted and plated on BHI agar for calculating bacterial death rates. The storage simulated the 54

stress that S. enterica may typically encounter during peanut butter processing (15). The bacterial 55

population in the low-fat formulation A (33 % fat and 42% carbohydrate) decreased by an 56

average of 0.6 to 0.8 log, compared to an average of 0.9 to 1.2 log in the regular formulation E 57

(49% fat and 24% carbohydrate) (Supplemental Fig. 1). This observation is consistent with our 58

previous finding that S. enterica survived better in peanut butter with lower fat but higher 59

carbohydrate content during an extended storage period (15). 60

After the 4-wk incubation, select volumes of PBS were mixed into the spiked peanut 61

butter samples to adjust aw to 0.4, 0.6 or 0.8 for evaluating the effects of increased aw on S. 62

enterica heat resistance. The samples were incubated at 25 oC for 24 h before thermal treatment. 63

Each inoculated sample (20-g) was transferred into aluminum foil bags, sealed, compressed to a 64

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thickness of 1 mm and submerged in an oil bath for heat treatment at 90 oC or 126 oC. The 65

come-up time to reach the final treatment temperature was less than 10 s. The heat-treated 66

samples were taken at 30 s, 90 s, 5 m, 10 m and 20 m and immediately cooled on ice for 1 m. 67

Viable cell counts were enumerated as previously described (15). D-values were calculated using 68

the Bigelow model (23). Each data set was analyzed using the Weibull model (24, 25). Statistical 69

analyses were performed using SAS version 9.2 (SAS Institute, Inc., Cary, NC) and Matlab 70

7.10.0.499 (The MathWorks, Inc., Natick, MA). A P-value of <0.05 was considered statistically 71

significant. 72

Figure 1 shows the overall S. enterica population changes when treated at 90 oC and 126 73

oC over 20 min with adjusted water activities in both peanut butter formulations. More detailed 74

population dynamics are shown in supplemental figures 2 and 3. At an aw of 0.2, 90 oC 75

treatment for 20 min resulted in less than 3 log reduction of Tennessee, whereas Typhimurium 76

showed 3.4 and 7.2 log reductions in peanut butter A and E, respectively. At an aw of 0.4, 20 min 77

of heating at 90 oC resulted in 4 to 5 log reductions of both Typhimurium and Tennessee in 78

peanut butter A, compared to no detectable levels of Typhimurium and 3 to 4 log reduction of 79

Tennessee in peanut butter E. At aw of 0.8, the same thermal treatment resulted in 4.8-5.2 log 80

reduction of Typhimurium and Tennessee in peanut butter A, in contrast to no detectable levels 81

in peanut butter E. These results suggest that an increase in aw in peanut butter formulation A had 82

less of an impact on S. enterica thermal resistance than in peanut butter E, which contained a 83

higher percentage of fat but lower carbohydrate levels. At 126 oC, regardless of the adjusted 84

water activities, approximately 7 to 8 log reduction was achieved after 5 min, and at 10 min S. 85

enterica could not be detected in either peanut butter formulation. 86

The statistical difference among the D-values of the three serotypes (Table 1) was most 87

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notable in peanut butter E at an aw of 0.2, where Tennessee displayed the highest D-value (8.35 88

min) and Typhimurium the lowest (2.61 min). These observations suggest that Typhimurium was 89

considerably less heat resistant than the other two serotypes in the peanut butter formulations 90

tested. Interestingly, however, as the water activities in both formulations increased from 0.2 to 91

0.8, the difference in D-values at 90 oC among the three serotypes was not statistically 92

significant. In addition, no difference in D-values was found among the three serotypes after 93

treatment at 126 oC in either formulation. 94

To achieve a 5-log reduction in peanut butter A, significantly more time (108.08 min) was 95

required for Tennessee, compared to Typhimurium (48.14 min) and Enteritidis (66.69 min), 96

indicating that Tennessee was the most heat resistant serotype tested (Table 2). To achieve the 97

same 5-log reduction at aw of 0.8, less heating time was required for all strains; therefore, increased 98

water activity diminished the difference in thermal resistance among the different serotypes. In 99

peanut butter E at aw of 0.2, similar patterns of heat resistance were observed; however, when 100

heated at aw of 0.8, all strains decreased to below detection limits, suggesting that the higher fat 101

and lower carbohydrate contents may lead to reduced heat resistance of S. enterica. 102

Statistical comparisons of the minimum times for achieving a 5-log reduction and 103

respective D-values indicated that Typhimurium and Tennessee were the least and the most heat 104

resistant S. enterica serotypes, respectively, in both peanut butter formulations tested. The 105

serotype-specific difference in heat resistance was most significant when S. enterica was treated 106

at 90 oC in peanut butter at aw of 0.2. When subjected to a higher temperature (126 oC) or with 107

increased aw (0.8), no significant difference in heat resistance was detected among the three 108

serotypes. 109

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Cellular morphology of S. enterica during desiccation and rehydration was monitored in 110

both peanut oil (aw of 0.2) and in PBS. Peanut oil was used instead of peanut butter because the 111

separation, fixation and scanning electron microscopy (SEM) imaging of bacteria in peanut 112

butter were technically infeasible. Bacteria were prepared for SEM as previously described (26) 113

with minor modifications including primary fixation with 2.5% glutaraldehyde in 0.1M 114

cacodylate buffer at pH 7.2, and final drying with 100% hexamethyldisilazane (EMS, Hatfield, 115

Pennsylvania). Samples were examined using a JSM-6320F field emission scanning electron 116

microscope (JEOL Orion system) at an instrument magnification of ×10,000. A minimum of 30 117

bacterial cells per strain per treatment was randomly selected for cell diameter size 118

measurements. 119

Figure 2 shows the morphological alterations of Enteritidis, Typhimurium and Tennessee 120

under desiccation stress in low aw peanut oil over the 4-wk storage period and subsequent 8-h 121

rehydration in PBS. Desiccated cell diameters decreased by 21% for Enteritidis and by 8.5% for 122

Tennessee. The average cell diameters of Typhimurium did not change significantly 123

(Supplemental Table 1). Whether the reduced cell size is a response to the low water activity that 124

contributes to the increased heat resistance of S. enterica is difficult to ascertain without more 125

experimentation. Following rehydration, a slight increase in cellular size for all three serotypes 126

was observed. Decreased cell size has been reported when S. enterica express the rdar 127

morphotype at low temperature and under starvation and desiccation stresses (27-30). Because 128

low moisture is a common environmental stress which S. enterica encounters on peanut shells in 129

pre-harvest environments, in curing steps, and in finished peanut butter products, this reduced 130

cellular size may constitute an adaptation strategy to the low aw stress. Such stress adaptation 131

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may subsequently cross protect the bacteria from other environmental challenges such as heat 132

and make the desiccation-stressed S. enterica more heat resistant. 133

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ACKNOWLEDGEMENTS 135

This work was supported by the Food Research Initiative Grant no.2010-65201-20593 from 136

the USDA National Institute of Food Agriculture, Food Safety and Epidemiology: Biological 137

Approaches for Food Safety program (program code 93231). The sponsor had no role in study 138

design, data collection and analysis, decision to publish, or preparation of the manuscript. 139

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Figure 1. Box plot showing log reductions of S. enterica Typhimurium and Tennessee at 90 oC 232

and 126 oC in peanut butter A (low fat) and E (regular) with adjusted water activities. The 233

horizontal bars and stars in boxes represent median and mean values, respectively; box edges 234

represent the upper and lower hinges of the H-spread. 235

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Figure 2. Scanning electron micrographs at 10,000× magnification of fresh S. enterica cells in 237

BHI broth (A, B, and C), desiccated S. enterica cells after 1- (D, E, and F) and 4-wk (G, H, and I) 238

incubation in peanut oil (aw of 0.2) at 25 oC, desiccated S. enterica cells after 2- (J, K, and L), 4- 239

(M, N, and O) and 8-h (P, Q, and R) rehydration in PBS at 25 oC. PT30, S. Enteritidis BSS-1045; 240

LT2, S. Typhimurium LT2; TEN, S. Tennessee K4643. 241

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Table 1. D-values (min) of S. enterica in peanut butter with adjusted water activities at 90 oC 242 and 126 oC calculated based on the first-order kinetics. 243

aw

Mean ± SD (r2) a

Peanut Butter

Temp S. Enteritidis S. Typhimurium S. Tennessee Three-strain Cocktail

A 90oC 0.20 7.05±1.12 (0.93)Aa 3.71±0.74 (0.99)Ab 6.41±1.36 (0.93)Aa 6.95±1.69 (0.97)Aa A 90oC 0.40 2.64±0.36 (0.99)BCa 2.43±0.41 (0.93)Ba 2.44±0.19 (0.98)BCa 3.13±0.72 (0.95)Ba A 90oC 0.60 3.00±0.69 (0.97)Bab 2.07±0.30 (0.89)Ba 2.96±0.75 (0.97)Bab 3.14±0.94 (0.96)Bb A 90oC 0.80 1.91±0.35 (0.90)Ca 1.95±0.37 (0.87)Ba 1.89±0.37 (0.89)Ca 2.06±0.42 (0.90)Ca

A 126oC 0.20 1.20±0.52 (0.95)Aa 0.59±0.09 (0.91)Aa 1.00±0.15 (0.96)Aa 1.19±0.46 (0.94)Aa A 126oC 0.40 0.55±0.32 (0.99)Aa 0.28±0.05 (0.99)Aa 0.62±0.33 (0.92)Aa 0.44±0.19 (0.97)Aa A 126oC 0.60 0.48±0.17 (0.96)Aa 0.76±0.50 (0.96)Aa 0.70±0.29 (0.91)Aa 0.54±0.18 (0.95)Aa A 126oC 0.80 0.27±0.07 (0.98)Aa 0.30±0.06 (0.98)Aa 0.29±0.06 (0.97)Aa 0.22±0.02 (0.95)Aa

E 90oC 0.20 4.81±1.58 (0.97)Aa 2.61±0.59 (0.97)Ab 8.35±4.09 (0.88)Ac 4.84±0.95 (0.96)Aa

E 90oC 0.40 3.43±0.51 (0.98)ABa 1.35±0.20 (0.98)Bb 3.64±0.45 (0.98)Ba 3.67±0.18 (0.97)Aa

E 90oC 0.60 2.10±0.27 (0.96)BCa 1.24±0.05 (0.99)Ba 1.79±0.14 (0.98)Ca 1.94±0.36 (0.96)Ba

E 90oC 0.80 0.94±0.22 (0.89)Ca 1.15±0.44 (0.88)Ba 1.12±0.19 (0.90)Ca 1.15±0.07 (0.90)Ba

E 126oC 0.20 0.43±0.05 (0.87)Aa 0.31±0.06 (0.92)Aa 0.42±0.04 (0.91)Aa 0.54±0.08 (0.91)Aa

E 126oC 0.40 0.39±0.03 (0.93)Aa 0.29±0.05 (0.96)Aa 0.51±0.05 (0.96)Aa 0.64±0.18 (0.98)Aa

E 126oC 0.60 0.67±0.15 (0.96)Aa 0.35±0.04 (1.00)Aa 0.59±0.17 (0.89)Aa 0.43±0.06 (0.93)Aa

E 126oC 0.80 0.26±0.01 (0.89)Aa 0.95±0.55 (1.00)Aa 0.26±0.03 (0.89)Aa 0.30±0.05 (0.89)Aa

244 a D values were calculated based on 3 independent biological replicates. Different capital letters indicate 245 significant difference (P < 0.05) among D-values of the same strain under different water activities (aw) at 246 the same temperature in the same peanut butter product (columns); different lowercase letters indicate 247 significant difference (P < 0.05) among D-values of different strains under the same treatment (rows). r2, 248 coefficients of determination. 249

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Table 2. Calculated minimum time (min) for achieving 1 to 7 log reduction of S. enterica at 90 250 oC in peanut butter based on the Weibull model. 251

Peanut butter

aw Bacterial strains Calculated minimum time (m) mean ± SD a

γ2 1-log reduction 3-log reduction 5-log reduction 7-log reduction

A 0.2 S. Enteritidis 15.12±2.81A 41.12±18.18AB 66.69±37.72A 92.38±59.82A 0.95 S. Typhimurium 8.22±2.39B 27.36±7.37A 48.14±13.97A 70.12±21.92A 0.98 S. Tennessee 13.44±7.14A 55.16±43.63BC 108.08±97.44B 169.49±164.46B 0.93 Three-strain Cocktail 17.97±3.16C 68.74±11.68C 131.91±41.30B 204.91±83.37B 0.98 A 0.4 S. Enteritidis 5.27±1.34A 24.84±3.40A 51.72±9.23A 84.41±19.89A 0.97 S. Typhimurium 2.44±1.66BC 16.52±6.60A 43.09±17.69A 82.59±35.13A 0.95 S. Tennessee 6.29±1.07A 19.46±4.58A 33.03±9.18A 46.92±14.41A 0.96 Three-strain Cocktail 5.05±2.00AC 25.53±13.35A 57.01±37.43A 98.31±72.60A 0.92 A 0.6 S. Enteritidis 8.73±0.75A 25.55±4.77A 42.81±12.27A 60.53±21.03A 0.93 S. Typhimurium 3.91±0.45B 12.50±2.9A 21.80±7.22A 31.66±12.51A 0.93 S. Tennessee 7.32±1.18AC 22.32±6.96A 37.88±14.71A 53.93±23.54A 0.98 Three-strain Cocktail 6.06±1.74BC 16.70±1.10A 27.21±2.44A 37.81±6.16A 0.97 A 0.8 S. Enteritidis 3.90±0.87A 10.89±2.01A 17.55±2.96A 24.06±3.85A 0.93 S. Typhimurium 2.96±0.74A 9.41±0.62A 16.29±1.28A 23.49±2.86A 0.91 S. Tennessee 4.18±0.98A 10.37±1.69A 15.85±2.14A 21.01±2.56A 0.93 Three-strain Cocktail 3.73±0.60A 11.50±2.70A 19.93±7.12A 28.90±12.59A 0.93 E 0.2 S. Enteritidis 8.26±3.35A 31.18±1.86A 59.20±4.93AC 91.05±13.58AC 0.95 S. Typhimurium 6.32±1.84A 20.06±6.48B 34.39±11.93B 49.13±17.96B 0.98 S. Tennessee 12.08±2.63B 38.35±10.70C 67.60±29.48A 99.50±55.43A 0.96 Three-strain Cocktail 10.98±3.17B 31.34±4.53A 52.12±10.19C 73.44±18.33C 0.98 E 0.4 S. Enteritidis 6.62±2.07A 24.82±5.91A 46.14±10.38A 69.66±15.73A 0.98 S. Typhimurium 2.69±0.44B 11.80±3.31B 23.83±8.77B 38.13±16.29B 0.93 S. Tennessee 7.32±1.04A 31.92±2.76C 63.35±4.57C 99.70±7.05C 0.97 Three-strain Cocktail 8.08±0.75A 25.08±1.08A 42.59±3.46A 60.50±6.84AB 0.94 E 0.6 S. Enteritidis 5.96±1.09A 19.81±4.37A 34.92±9.65A 50.97±16.13A 0.92 S. Typhimurium 2.94±0.34B 10.90±0.49B 20.09±1.16B 30.11±2.46A 0.94 S. Tennessee 5.40±0.50A 14.83±2.88AB 24.06±7.06AB 33.28±11.96A 0.89 Three-strain Cocktail 5.94±1.63A 14.91±4.20AB 22.93±6.71AB 30.51±9.23A 0.91 E 0.8 S. Enteritidis N.A. N.A. N.A. N.A. N.A. S. Typhimurium N.A. N.A. N.A. N.A. N.A. S. Tennessee N.A. N.A. N.A. N.A. N.A. Three-strain Cocktail N.A. N.A. N.A. N.A. N.A.

252 a Values shown are based on three independent trials performed with triplicate biological replicates. 253 Different capital letters indicate significant difference (P < 0.05) among D-values of the same strain under 254 different treatments (columns); N.A., below the detection limit of the assay; r2, coefficients of 255 determination. 256