AEM Accepts, published online ahead of print on 31 May 2013 Appl. Environ. Microbiol. doi:10.1128/AEM.

01028-13 Copyright 2013, American Society for Microbiology. All Rights Reserved.

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Increased Water Activity Reduces Thermal Resistance of Salmonella enterica in Peanut Butter

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

Institute for Food Safety and Health, Illinois Institute of Technology, Bedford Park, Illinois, USAa; Methodology Center, Pennsylvania State University, University Park, Pennsylvania, USAb; U.S. Food and Drug Administration, Bedford Park, Illinois, USAc

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

Running Title: Salmonella heat resistance in peanut butter

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

ABSTRACT Increased water activity in peanut butter significantly (P < 0.05) reduced the heat resistance of desiccation-stressed Salmonella enterica strains when treated at 90 C. Difference in thermal resistance was less notable when treated at 126 C. Using scanning electron microscopy, we observed minor morphological changes of S. enterica cells during desiccation and rehydration processes in peanut oil.

TEXT Salmonellosis outbreaks linked to contaminated peanut butter products have brought worldwide attention to the microbial safety of these popular food items. S. enterica serotype Tennessee caused a salmonellosis outbreak in 2006-2007 linked to peanut butter, which sickened 425 persons and resulted in 71 (20%) hospitalizations in 44 states in the U. S. (1). This and other foodborne outbreaks (2, 3) highlight the need for a re-examination of S. enterica behavior in low water activity (aw) peanut butter products. The water activity of peanut butter is typically 0.35 or less (2, 4-9), which precludes the growth of spoilage and pathogenic microorganisms. When present in peanut butter, S. enterica become heat resistant, possibly due to adaptation to the desiccation stress and the protective effects of the fat content in the product (2, 4-7, 10-14). We recently demonstrated that heat treatment at 72 C for an hour resulted in less than 2-log reduction of desiccation-stressed S. enterica in artificially contaminated peanut butter with an aw of 0.4 (15). In this study, we evaluated the effects of desiccation and subsequent rehydration on the relative heat resistance of three S. enterica serotypes: Tennessee K4643 (a human isolate from the 2006-2007 peanut butter outbreak in the U. S.) (1), Enteritidis BSS-1045 (an isolate from the 2000-2001 raw almonds

42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64

outbreak in the U. S. and Canada) (16-18) and Typhimurium LT2 (19, 20). We compared two commercial peanut butter formulations (regular and low-fat) to assess the influence of carbohydrate and fat contents on the heat resistance of S. enterica. Most published thermal challenge studies of S. enterica in peanut butter focused on heat treatments at either 72 oC or 90
o

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

processing, such as dry roasting at 126 oC (22). In this study we thermally challenged S. enterica strains in artificially contaminated peanut butter at both 90 oC and 126 oC. Individual strains and a three-strain cocktail were grown separately as previously described (15) followed by suspension in 5-mL peanut oil prior to inoculation of peanut butter (aw 0.2). Bacterial cell suspensions were transferred to 500-g of peanut butter and vigorously stirred for 20 min using a sampler spatula. Homogenous distribution of the cells was verified as previously described (15). Inoculated samples were stored at 25 oC for 4 weeks, then serially diluted and plated on BHI agar for calculating bacterial death rates. The storage simulated the stress that S. enterica may typically encounter during peanut butter processing (15). The bacterial population in the low-fat formulation A (33 % fat and 42% carbohydrate) decreased by an average of 0.6 to 0.8 log, compared to an average of 0.9 to 1.2 log in the regular formulation E (49% fat and 24% carbohydrate) (Supplemental Fig. 1). This observation is consistent with our previous finding that S. enterica survived better in peanut butter with lower fat but higher carbohydrate content during an extended storage period (15). After the 4-wk incubation, select volumes of PBS were mixed into the spiked peanut butter samples to adjust aw to 0.4, 0.6 or 0.8 for evaluating the effects of increased aw on S. enterica heat resistance. The samples were incubated at 25 oC for 24 h before thermal treatment. Each inoculated sample (20-g) was transferred into aluminum foil bags, sealed, compressed to a

65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87

thickness of 1 mm and submerged in an oil bath for heat treatment at 90 oC or 126 oC. The come-up time to reach the final treatment temperature was less than 10 s. The heat-treated samples were taken at 30 s, 90 s, 5 m, 10 m and 20 m and immediately cooled on ice for 1 m. Viable cell counts were enumerated as previously described (15). D-values were calculated using the Bigelow model (23). Each data set was analyzed using the Weibull model (24, 25). Statistical analyses were performed using SAS version 9.2 (SAS Institute, Inc., Cary, NC) and Matlab 7.10.0.499 (The MathWorks, Inc., Natick, MA). A P-value of <0.05 was considered statistically significant. Figure 1 shows the overall S. enterica population changes when treated at 90 oC and 126
o

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

population dynamics are shown in supplemental figures 2 and 3. At an aw of 0.2, 90 oC treatment for 20 min resulted in less than 3 log reduction of Tennessee, whereas Typhimurium showed 3.4 and 7.2 log reductions in peanut butter A and E, respectively. At an aw of 0.4, 20 min of heating at 90 oC resulted in 4 to 5 log reductions of both Typhimurium and Tennessee in peanut butter A, compared to no detectable levels of Typhimurium and 3 to 4 log reduction of Tennessee in peanut butter E. At aw of 0.8, the same thermal treatment resulted in 4.8-5.2 log reduction of Typhimurium and Tennessee in peanut butter A, in contrast to no detectable levels in peanut butter E. These results suggest that an increase in aw in peanut butter formulation A had less of an impact on S. enterica thermal resistance than in peanut butter E, which contained a higher percentage of fat but lower carbohydrate levels. At 126 oC, regardless of the adjusted water activities, approximately 7 to 8 log reduction was achieved after 5 min, and at 10 min S. enterica could not be detected in either peanut butter formulation. The statistical difference among the D-values of the three serotypes (Table 1) was most

88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109

notable in peanut butter E at an aw of 0.2, where Tennessee displayed the highest D-value (8.35 min) and Typhimurium the lowest (2.61 min). These observations suggest that Typhimurium was considerably less heat resistant than the other two serotypes in the peanut butter formulations tested. Interestingly, however, as the water activities in both formulations increased from 0.2 to 0.8, the difference in D-values at 90 oC among the three serotypes was not statistically significant. In addition, no difference in D-values was found among the three serotypes after treatment at 126 oC in either formulation. To achieve a 5-log reduction in peanut butter A, significantly more time (108.08 min) was required for Tennessee, compared to Typhimurium (48.14 min) and Enteritidis (66.69 min), indicating that Tennessee was the most heat resistant serotype tested (Table 2). To achieve the same 5-log reduction at aw of 0.8, less heating time was required for all strains; therefore, increased water activity diminished the difference in thermal resistance among the different serotypes. In peanut butter E at aw of 0.2, similar patterns of heat resistance were observed; however, when heated at aw of 0.8, all strains decreased to below detection limits, suggesting that the higher fat and lower carbohydrate contents may lead to reduced heat resistance of S. enterica. Statistical comparisons of the minimum times for achieving a 5-log reduction and respective D-values indicated that Typhimurium and Tennessee were the least and the most heat resistant S. enterica serotypes, respectively, in both peanut butter formulations tested. The serotype-specific difference in heat resistance was most significant when S. enterica was treated at 90 oC in peanut butter at aw of 0.2. When subjected to a higher temperature (126 oC) or with increased aw (0.8), no significant difference in heat resistance was detected among the three serotypes.

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Cellular morphology of S. enterica during desiccation and rehydration was monitored in both peanut oil (aw of 0.2) and in PBS. Peanut oil was used instead of peanut butter because the separation, fixation and scanning electron microscopy (SEM) imaging of bacteria in peanut butter were technically infeasible. Bacteria were prepared for SEM as previously described (26) with minor modifications including primary fixation with 2.5% glutaraldehyde in 0.1M cacodylate buffer at pH 7.2, and final drying with 100% hexamethyldisilazane (EMS, Hatfield, Pennsylvania). Samples were examined using a JSM-6320F field emission scanning electron microscope (JEOL Orion system) at an instrument magnification of 10,000. A minimum of 30 bacterial cells per strain per treatment was randomly selected for cell diameter size measurements. Figure 2 shows the morphological alterations of Enteritidis, Typhimurium and Tennessee under desiccation stress in low aw peanut oil over the 4-wk storage period and subsequent 8-h rehydration in PBS. Desiccated cell diameters decreased by 21% for Enteritidis and by 8.5% for Tennessee. The average cell diameters of Typhimurium did not change significantly (Supplemental Table 1). Whether the reduced cell size is a response to the low water activity that contributes to the increased heat resistance of S. enterica is difficult to ascertain without more experimentation. Following rehydration, a slight increase in cellular size for all three serotypes was observed. Decreased cell size has been reported when S. enterica express the rdar morphotype at low temperature and under starvation and desiccation stresses (27-30). Because low moisture is a common environmental stress which S. enterica encounters on peanut shells in pre-harvest environments, in curing steps, and in finished peanut butter products, this reduced cellular size may constitute an adaptation strategy to the low aw stress. Such stress adaptation

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may subsequently cross protect the bacteria from other environmental challenges such as heat and make the desiccation-stressed S. enterica more heat resistant.

ACKNOWLEDGEMENTS This work was supported by the Food Research Initiative Grant no.2010-65201-20593 from the USDA National Institute of Food Agriculture, Food Safety and Epidemiology: Biological Approaches for Food Safety program (program code 93231). The sponsor had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Isaacs S, Aramini J, Ciebin B, Farrar JA, Ahmed R, Middleton D, Chandran AU, Harris LJ, Howes M, Chan E, Pichette AS, Campbell K, Gupta A, Lior LY, Pearce M, Clark C, Rodgers F, Jamieson F, Brophy I, Ellis A. 2005. An international outbreak of salmonellosis associated with raw almonds contaminated with a rare phage type of Salmonella Enteritidis. J Food Prot 68:191-198. Danyluk MD, Nozawa-Inoue M, Hristova KR, Scow KM, Lampinen B, Harris LJ. 2008. Survival and growth of Salmonella Enteritidis PT 30 in almond orchard soils. Journal of Applied Microbiology 104:1391-1399. Neidhardt FC. 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology. ASM, Washington DC. McClelland M, Sanderson KE, Spieth J, Clifton SW, Latreille P, Courtney L, Porwollik S, Ali J, Dante M, Du F, Hou S, Layman D, Leonard S, Nguyen C, Scott K, Holmes A, Grewal N, Mulvaney E, Ryan E, Sun H, Florea L, Miller W, Stoneking T, Nhan M, Waterston R, Wilson RK. 2001. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 413:852-856. Ma L, Zhang G, Gerner-Smidt P, Mantripragada V, Ezeoke I, Doyle MP. 2009. Thermal inactivation of Salmonella in peanut butter. J Food Prot 72:1596-1601. Woodroof J. 1983. Peanuts: Production, prcessing, products, 3rd edition, Avi Publishing Company, Westport, CT. Edwards D, Berry JJ. 1987. The efficiency of simulation-based multiple comparisons. Biometrics 43:913-928. van Boekel MA. 2002. On the use of the Weibull model to describe thermal inactivation of microbial vegetative cells. Int J Food Microbiol 74:139-159. Corradini MG, Peleg M. 2004. Demonstration of the applicability of the Weibull-log-logistic survival model to the isothermal and nonisothermal inactivation of Escherichia coli K-12 MG1655. J Food Prot 67:2617-2621. Wen J, Anantheswaran RC, Knabel SJ. 2009. Changes in barotolerance, thermotolerance, and cellular morphology throughout the life cycle of Listeria monocytogenes. Appl Environ Microbiol 75:1581-1588. White AP, Gibson DL, Kim W, Kay WW, Surette MG. 2006. Thin aggregative fimbriae and cellulose enhance long-term survival and persistence of Salmonella. J Bacteriol 188:3219-3227. Spector MP. 1998. The starvation-stress response (SSR) of Salmonella. Adv Microb Physiol 40:233-279. Rmling U, Sierralta WD, Eriksson K, Normark S. 1998. Multicellular and aggregative behaviour of Salmonella Typhimurium strains is controlled by mutations in the agfD promoter. Mol Microbiol 28:249-264. Rmling U. 2005. Characterization of the rdar morphotype, a multicellular behaviour in Enterobacteriaceae. Cell Mol Life Sci 62:1234-1246.

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Figure 1. Box plot showing log reductions of S. enterica Typhimurium and Tennessee at 90 oC and 126 oC in peanut butter A (low fat) and E (regular) with adjusted water activities. The horizontal bars and stars in boxes represent median and mean values, respectively; box edges represent the upper and lower hinges of the H-spread.

Figure 2. Scanning electron micrographs at 10,000 magnification of fresh S. enterica cells in BHI broth (A, B, and C), desiccated S. enterica cells after 1- (D, E, and F) and 4-wk (G, H, and I) incubation in peanut oil (aw of 0.2) at 25 oC, desiccated S. enterica cells after 2- (J, K, and L), 4(M, N, and O) and 8-h (P, Q, and R) rehydration in PBS at 25 oC. PT30, S. Enteritidis BSS-1045; LT2, S. Typhimurium LT2; TEN, S. Tennessee K4643.

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242 243

Table 1. D-values (min) of S. enterica in peanut butter with adjusted water activities at 90 oC and 126 oC calculated based on the first-order kinetics.
Mean SD (r2) a

Peanut Butter
A A A A A A A A

Temp 90oC 90oC 90 C 90oC 126 C 126oC 126oC 126 C 90oC 90oC 90 C 90oC 126oC 126oC 126oC 126 C
o o o o o

aw

S. Enteritidis

S. Typhimurium 3.710.74 (0.99)Ab 2.430.41 (0.93)Ba 2.070.30 (0.89) 0.590.09 (0.91) 0.280.05 (0.99) 0.760.50 (0.96) 0.300.06 (0.98)
Ba

S. Tennessee 6.411.36 (0.93)Aa 2.440.19 (0.98)BCa 2.960.75 (0.97) 1.890.37 (0.89) 1.000.15 (0.96) 0.620.33 (0.92) 0.700.29 (0.91) 0.290.06 (0.97)
Bab

Three-strain Cocktail 6.951.69 (0.97)Aa 3.130.72 (0.95)Ba 3.140.94 (0.96)Bb 2.060.42 (0.90)Ca 1.190.46 (0.94) 0.440.19 (0.97) 0.540.18 (0.95) 0.220.02 (0.95)
Aa Aa Aa Aa

0.20 7.051.12 (0.93)Aa 0.40 2.640.36 (0.99)BCa 0.60 3.000.69 (0.97) 0.20 1.200.52 (0.95)
Bab

0.80 1.910.35 (0.90)Ca

Aa

1.950.37 (0.87)Ba
Aa Aa Aa Aa

Ca Aa Aa Aa Aa

0.40 0.550.32 (0.99)Aa 0.60 0.480.17 (0.96)Aa 0.80 0.270.07 (0.98)

Aa

E E E E E E E E 244 245 246 247 248 249

a

0.20 4.811.58 (0.97)Aa 0.40 3.430.51 (0.98)ABa 0.60 2.100.27 (0.96) 0.20 0.430.05 (0.87)
BCa

2.610.59 (0.97)Ab 1.350.20 (0.98)Bb 1.240.05 (0.99) 0.310.06 (0.92) 0.290.05 (0.96) 0.350.04 (1.00) 0.950.55 (1.00)
Ba

8.354.09 (0.88)Ac 3.640.45 (0.98)Ba 1.790.14 (0.98) 1.120.19 (0.90) 0.420.04 (0.91) 0.510.05 (0.96) 0.590.17 (0.89) 0.260.03 (0.89)
Ca

4.840.95 (0.96)Aa 3.670.18 (0.97)Aa 1.940.36 (0.96)Ba 1.150.07 (0.90) 0.540.08 (0.91) 0.640.18 (0.98) 0.430.06 (0.93) 0.300.05 (0.89)
Ba Aa Aa Aa Aa

0.80 0.940.22 (0.89)Ca

Aa

1.150.44 (0.88)Ba
Aa Aa Aa Aa

Ca Aa Aa Aa Aa

0.40 0.390.03 (0.93)Aa 0.60 0.670.15 (0.96)Aa 0.80 0.260.01 (0.89)

Aa

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

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250 251
Peanut butter A

Table 2. Calculated minimum time (min) for achieving 1 to 7 log reduction of S. enterica at 90 o C in peanut butter based on the Weibull model.
aw 0.2 Bacterial strains S. Enteritidis S. Typhimurium S. Tennessee Three-strain Cocktail S. Enteritidis S. Typhimurium S. Tennessee Three-strain Cocktail S. Enteritidis S. Typhimurium S. Tennessee Three-strain Cocktail S. Enteritidis S. Typhimurium S. Tennessee Three-strain Cocktail S. Enteritidis S. Typhimurium S. Tennessee Three-strain Cocktail S. Enteritidis S. Typhimurium S. Tennessee Three-strain Cocktail S. Enteritidis S. Typhimurium S. Tennessee Three-strain Cocktail S. Enteritidis S. Typhimurium S. Tennessee Three-strain Cocktail Calculated minimum time (m) mean SD a 1-log reduction 3-log reduction 5-log reduction 7-log reduction 15.122.81A 41.1218.18AB 66.6937.72A 92.3859.82A 8.222.39B 27.367.37A 48.1413.97A 70.1221.92A A BC B 13.447.14 55.1643.63 108.0897.44 169.49164.46B C C B 17.973.16 68.7411.68 131.9141.30 204.9183.37B A A A 5.271.34 24.843.40 51.729.23 84.4119.89A BC A A 2.441.66 16.526.60 43.0917.69 82.5935.13A A A A 6.291.07 19.464.58 33.039.18 46.9214.41A AC A A 5.052.00 25.5313.35 57.0137.43 98.3172.60A A A A 8.730.75 25.554.77 42.8112.27 60.5321.03A B A A 3.910.45 12.502.9 21.807.22 31.6612.51A AC A A 7.321.18 22.326.96 37.8814.71 53.9323.54A BC A A 6.061.74 16.701.10 27.212.44 37.816.16A A A A 3.900.87 10.892.01 17.552.96 24.063.85A A A A 2.960.74 9.410.62 16.291.28 23.492.86A A A A 4.180.98 10.371.69 15.852.14 21.012.56A A A A 3.730.60 11.502.70 19.937.12 28.9012.59A A A AC 8.263.35 31.181.86 59.204.93 91.0513.58AC A B B 6.321.84 20.066.48 34.3911.93 49.1317.96B B C A 12.082.63 38.3510.70 67.6029.48 99.5055.43A B A C 10.983.17 31.344.53 52.1210.19 73.4418.33C A A A 6.622.07 24.825.91 46.1410.38 69.6615.73A B B B 2.690.44 11.803.31 23.838.77 38.1316.29B A C C 7.321.04 31.922.76 63.354.57 99.707.05C A A A 8.080.75 25.081.08 42.593.46 60.506.84AB A A A 5.961.09 19.814.37 34.929.65 50.9716.13A B B B 2.940.34 10.900.49 20.091.16 30.112.46A A AB AB 5.400.50 14.832.88 24.067.06 33.2811.96A A AB AB 5.941.63 14.914.20 22.936.71 30.519.23A N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. 2 0.95 0.98 0.93 0.98 0.97 0.95 0.96 0.92 0.93 0.93 0.98 0.97 0.93 0.91 0.93 0.93 0.95 0.98 0.96 0.98 0.98 0.93 0.97 0.94 0.92 0.94 0.89 0.91 N.A. N.A. N.A. N.A.

0.4

0.6

0.8

0.2

0.4

0.6

0.8

252 253 254 255 256

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

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