Evaluation of Enterococcus faecium NRRL B-2354 as a potential surrogate of Salmonella in packaged paprika, white pepper and cumin powder during radio frequency heating

Food Control 108 (2020) 106833

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Evaluation of Enterococcus faecium NRRL B-2354 as a potential surrogate of Salmonella in packaged paprika, white pepper and cumin powder during radio frequency heating

T

⁎

Samet Ozturk, Fanbin Kong , Rakesh K. Singh Department of Food Science and Technology, University of Georgia, Athens, GA, USA

A R T I C LE I N FO

A B S T R A C T

Keywords: Salmonella Enterococcus faecium NRRL B-2354 Radio frequency (RF) heating Pasteurization Spices

Salmonella contamination of various seasonings and spices has occurred in the last decade. Radio frequency (RF) heating has been shown to be a potential alternative inactivation method for pathogens in low moisture foods (LMF), including spices and vegetable powders. This study aimed to (1) determine the thermal resistance (D and z values) of Salmonella and Enterococcus faecium (E. faecium) NRRL B-2354 as a promising surrogate for thermal validation studies using paprika, white pepper, and cumin powders, and (2) evaluate the efficacy of RF heating on the inactivation of both microorganisms in packaged samples. Samples with initial water activity levels (a w,25°C ) of 0.45 ± 0.05 were inoculated with Salmonella cocktail (S. Typhimurium, S. Agona, S. Montevideo, and S. Tennessee) and E. faecium NRRL B-2354 at approximately 8.5 log CFU/g. Inoculated samples were loaded in aluminum test cells and subjected to isothermal treatment in a water bath at 70, 75 and 80 °C. Samples (20 g) were also sealed in small polystyrene petri dishes and subjected to RF heating in a 27.12-MHz, 6-kW pilot scale RF unit with 105 mm electrode gap until the temperature at the geometric center reached 80 °C. The survival of both bacteria was enumerated and converted to log CFU per gram. The change in color of RF heated samples was also evaluated to assess the impact of RF heating on the product quality. Results showed that the thermal resistance of E. faecium at inactivation temperatures is higher than Salmonella in all three spices. Additionally, both microorganisms showed less thermal resistance in paprika than white pepper and cumin powder. The D80°C values of Salmonella and E. faecium were determined to be 1.21 and 1.82 min in paprika, 2.85 and 5.27 min in white pepper, and 4.47 and 9.53 min in cumin powder, respectively. Results also showed that E. faecium is a suitable surrogate for validation of RF pasteurization of Salmonella in spices and RF heating is an effective method to control the contamination of foodborne pathogens in spices.

1. Introduction Spices, such as paprika, white pepper, and cumin powder, are low moisture food (LMF) with water activity levels low enough (aw < 0.6) to limit the growth of microorganisms and thus have historically been considered safe ingredients. Foodborne pathogens in spices, however, have led to several outbreaks of food-borne illnesses and have recently become a threat to public health around the world (Authority, 2010; Rico et al., 2010; Waje, Kim, Kim, Todoriki, & Kwon, 2008). Over the last decade, outbreaks of Salmonella have occurred in white pepper (CDC, 2010), paprika (Lehmacher, Bockemuhl, & Aleksic, 1995), cumin powder (Moreira, Lourencao, Pinto, & Rall, 2009), and other spices in the United States (Zweifel & Stephan, 2012). This increase in pathogenic outbreaks and recalls associated with spices indicates that the current processing methods to eliminate or control foodborne ⁎

pathogens are not sufficient. Thus, it is critical to develop effective processing techniques and evaluation methods to inactivate Salmonella in spices used in the food industry. Steam treatment, fumigation, ethylene oxide treatment, and irradiation are a few popular methods of inactivating foodborne pathogens in LMFs, but they have public health concerns and can negatively affect food quality (Lee et al., 2006). Radio frequency (RF) heating in the range of 3 kHz–300 MHz has shown great potential as an alternative method to inactivate Salmonella in LMFs, as heat is generated in the dielectric material through molecular friction caused by ionic conduction and dipole rotation. The inactivation efficacy of RF heating on foodborne pathogens in various LMFs has been investigated in almonds (Gao, Tang, Johnson, & Wang, 2012; Gao, Tang, Villa-Rojas, Wang, & Wang, 2011; Gao, Tang, Wang, Powers, & Wang, 2010), flour (Tiwari, Wang, Tang, & Birla, 2011; Villa-Rojas, Zhu, Marks, & Tang, 2017) and

Corresponding author. E-mail address: [email protected] (F. Kong).

https://doi.org/10.1016/j.foodcont.2019.106833 Received 4 April 2019; Received in revised form 14 August 2019; Accepted 17 August 2019 Available online 20 August 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.

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2.1. Preparation of bacterial inoculation

pepper spices (Kim, Sagong, Choi, Ryu, & Kang, 2012). Process validation is crucial to establish a newly developed technology in the food industry, but validation studies using foodborne pathogens in food processing plants have strict safety limitations (Niebuhr, Laury, Acuff, & Dickson, 2008). Consequently, nonpathogenic surrogates must be used to establish reliable regulations and to validate processing conditions in an industrial setting (Enache et al., 2015). As a nonpathogenic bacteria with high heat resistance, Enterococcus faecium NRRL B-2354 (E. faecium) has been widely studied as a potential surrogate for Salmonella to validate thermal process for pasteurization of LMFs including almonds, wheat flour, and various spices (Bingol et al., 2011; Liu et al., 2018a, b; Rachon, Penaloza, & Gibbs, 2016; Tiwari et al., 2011). No study has been done, however, to evaluate the acceptability of using E. faecium as a surrogate for Salmonella in paprika, white pepper or cumin powder during RF heating, which is necessary as it may behave differently in various food matrices. The log-linear kinetic model is commonly used to estimate the efficacy of conventional thermal processing on inactivation of microorganisms using obtained survival curves, but recent studies have shown that the inactivation kinetics of novel treatment methods like RF, microwave, and ohmic heating on microorganisms do not always have traditional linear survival curves (Bermudez-Aguirre & Corradini, 2012). Different models such as Weibull model have shown a better fit in describing the inactivation kinetics. This study aimed to investigate the inactivation kinetics of Salmonella and its potential surrogate, E. faecium, as subjected to RF heating, and to validate the efficacy of RF heating for pasteurization. Three spices, paprika, white pepper and cumin powder were selected as representative spices to cover the severity of contamination in the wide range of spices. The specific objectives were; 1) to evaluate the suitability of E. faecium as a surrogate for Salmonella to validate thermal pasteurization through investigating inactivation kinetics and modeling; 2) to determine the heating pattern and obtain temperature profiles during the RF heating of spices; and 3) to validate efficacy of RF heating as a pasteurization method for the three spices by comparing bacteria survival kinetics and calculated F-values obtained from temperature-time histories.

A cocktail of four Salmonella strains (S. Typhimurium, S. Agona, S. Montevideo and S. Tennessee) was used in this study, and was selected based on the strains linked to reported Salmonella outbreaks . Additionally, Enterococcus faecium NRRL B-2354 (E. faecium), which is used as a potential surrogate of Salmonella, was used as a single strain of Enterococcus. All strains were obtained from Dr. Mark Harrison's laboratory (University of Georgia, Athens, GA) and cultured in tryptic soy broth (TSB) at 37 °C for 24 h. A loop from each strain tube was then spread onto 1- 50 × 15 mm Tryptic Soy Agar (TSA) plates and incubated at 37 °C for 24 h. Cells grown on TSA were harvested from each of the three plates and placed in separate sterile conical tubes by first washing with 20 ml 0.1% (w/v) peptone water and scraping colonies using a sterile plastic swap, followed by centrifugation for 30 min at 2,600 g. The suspended pellet was centrifuged 2–3 times, using 0.1% peptone water to remove extra nutrient or agar. Suspended pellets of each strain of the four-pathogen species were then combined to make the culture cocktails. Then, the supernatant was discarded, and the pellet was re-suspended in 3 ml 0.1% peptone water as described in Liu et al. (2018a, b). To obtain more uniform inoculation in the sample, 10 g of each spices were put in small sterile bags and inoculated with 1 ml of concentrated pellet. Inoculated samples were hand-mixed in a safety cabinet until the pellet was evenly mixed. After mixing the inoculated spice samples (10 g) in the sterile bags, samples were used to further inoculate 90 g of each spice separately, which were then mixed and stomached (Seward Stomacher, 400 Lab System, Norfolk, United Kingdom) at 260 rpm for 5 min. To confirm the uniformity of bacterial inoculation in the samples, ten of 1 g each spice was randomly selected from 100 g inoculated sample and enumerated on mTSA and eTSA plates. The bacteria concentration level in inoculated spices for isothermal and RF heating treatments was determined to be approximately 8.5 log CFU/g. In order to avoid the effect of water activity on thermal resistances of the Salmonella cocktail and E. faecium, the inoculated spice samples were placed in sterile trays and equalibriated in the Hotpack 435315 humidity chamber to ensure the desired water activity (a w,25°C = 0.45 ± 0.05) (Hildebrandt et al., 2016).

2. Materials and methods

2.2. Isothermal treatment

Paprika, white pepper, and cumin powder were purchased from a local store (Athens, GA USA). The initial water activity at 25 °C (a w , 25°C) of each sample was 0.71, 0.68 and 0.66, respectively, as measured with a water activity meter (AQUA PRE, Decagon Devices, Pullman, WA). In order to investigate effectiveness of RF heating on microorganisms in different samples with the same water activity (a w < 0.5), the samples were adjusted to a w,25°C =0.45 ± 0.05 using a Hotpack 435315 humidity chamber (SP Industries, Inc., Warminster, PA). Based on recently published studies (Liu et al., 2018a, b; Liu, Rojas, Gray, Zhu, & Tang, 2018b; Ozturk et al., 2019; Xu et al., 2019), the RF treatments developed for products with water activity of 0.45 at room temperature should be adequate for control of Salmonella in the three selected spices with water activities above 0.45 at room temperature. The composition and particle size of the spices are presented in Table 1.

To investigate thermal resistance (D and z values) of Salmonella and E. faecium in the three spices, inoculated samples (around 0.6–0.8 g) were filled in the aluminum thermal death time (TDT) test cells (Washington State University, Pullman, WA) with 4 mm thickness, and then isothermally treated in a water bath (Model: SWB-10L-2, Saratoga, CA USA) with temperatures of 70, 75 and 80 °C (Chung, Birla, & Tang, 2008; Villa-Rojas et al., 2013). To obtain thermal death curves of Salmonella and E. faecium in each spice sample, isothermal treatment was applied with the same time intervals after the predetermined come-up time (CUT) ended. CUT is defined as the time required for the sample to reach the set temperature. The CUTs for each spice were separately determined using an TDT test cell with a T-type thermocouple inserted in the center of a cell filled with non-inoculated spice samples. The CUT was used as time zero and reductions of bacterial population in each

Table 1 Proximate composition of spices (average values ± SD).

Moisture (% w/w) Ash (% w/w) Fat (% w/w) Protein (% w/w) Carbohydrate (by difference – w/w) Particle Size (mm)

Paprika

White Pepper

Cumin Powder

11.24 ± 0.47 7.74 ± 0.17 12.89 ± 0.30 14.14 ± 0.33 53.99 ± 0.31 0.34 ± 0.05

11.42 ± 0.17 6.89 ± 0.02 2.48 ± 0.15 10.40 ± 0.12 68.81 ± 1.98 0.27 ± 0.03

8.06 ± 0.23 7.62 ± 0.21 22.27 ± 2.60 17.81 ± 0.26 44.24 ± 0.51 0.39 ± 0.07

2

Food Control 108 (2020) 106833

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∑i = 1 [log N

No data, i

RMSE =

− log N

No model, i

]2

n−p N No data, i

where log

is measured log CFU/g reduction, and

(3)

log N No model, i

is

model predicted log CFU/g reduction, n is the total number of observations, and p is the number of model parameters. Integrated pathogen modelling program (IPMP) was used to estimate fitness of model and provide RSME directly (Huang, 2014). To evaluate differences between D-values among samples, the ANOVA test in Minitab 14 (Minitab Inc., State College, PA) was also performed. The z values (°C) indicate the change in temperature required to alter the thermal-deathtime by one log-cycle (90%), which were obtained from the thermal death time curves where the log of D-values was plotted against temperature, and the slope refers to the −1/z (Gaillard, Leguerinel, & Mafart, 1998), i.e.

Fig. 1. Samples packed in sealed small petri dishesfor RF heating.

sample were used for thermal resistance value calculations of both microorganisms. Isothermally treated cells were immediately removed from the water bath and placed in an ice-water bath for 90 s to stop thermal inactivation of bacteria. Each trial was carried out in triplicate.

z=

T2 − T1 D

log ( D1 ) 2

(4)

2.5. Radio frequency heating of spices 2.3. Enumeration

To investigate temperature time profiles and heating patterns during the RF heating of the spices, uninoculated samples of each spice (20 g) were loaded in small polystyrene petri dishes with diameter 50 mm and height 15 mm, and then sealed with polystyrene plastic (1 mm thickness) (Fig. 1). To determine the come-up time (CUT) to reach 80 °C from the initial temperature (23 ± 2 °C) of each sample, the sealed petri dishes were placed between two parallel electrodes with adjusted gap of 105 mm in a 27.12-MHz, 6-kW RF system (COMBI 6-S, Strayfield International, Wokingham, UK) (Fig. 2), and then subjected to RF heating. The change in temperature of samples during the RF heating was recorded using a fiber optic temperature sensor with an accuracy of ± 1 °C (Fiso Tech. Inc., Quebec, Canada) connected to a data logger to determine the CUTs at the geometric center of the cylindrical petri dishes. The obtained CUTs were applied to evaluate the efficacy of RF heating on pasteurization of spice samples inoculated with Salmonella and E. faecium. The heating uniformity of RF heated samples were also evaluated using an infrared camera (FLIR T440, FLIR Systems, Inc., North Billerica, MA, USA) with an accuracy of ± 2 °C. Only top surface thermal images were used to determine the heating uniformity index and the average temperature of RF heated samples due to the limited thickness of the sample (15 mm). The heating uniformity of RF heated sample was evaluated using the uniformity index (UI), which has been applied to evaluate heating uniformity in various RF heated LMFs (Hou, Ling, & Wang, 2014; Jiao, Johnson, Tang, & Wang, 2012; Pan, Jiao, Gautz, Tu, & Wang, 2012; Wang, Luechapattanaporn, & Tang, 2008; Wang, Tiwari, Jiao, Johnson, & Tang, 2010; Wang, Yue, Tang, & Chen, 2005). The UI value of RF heated spices was calculated using the following equation (Wang et al., 2005):

Isothermally treated samples in the test cells at the selected time intervals were transferred into sterile stomacher bags containing 0.1% peptone water, diluted at 1:10 (v/v), and homogenized for 2 min at 260 rpm with a Seward Stomacher (Seward, London, UK) (Harris, Uesugi, Abd, & McCarthy, 2012). After homogenization, 1 ml of each diluent was serially diluted in 9 ml of 0.1% peptone water to obtain the final diluent. To enumerate both Salmonella and E. faecium survivals in the sample, 0.1 ml of the final diluents was spread-plated in duplicate onto modified tryptic soy agars (mTSA and eTSA) plates, respectively. The mTSA consists of TSA agar, Yeast Extract, 0.05% Ammonium Iron (Ш) Citrate, and 0.03% Sodium Thiosulfate Pentahydrate (5H2O), and eTSA consists of TSA agar, Yeast Extract, 0.05% Ammonium Iron (Ш) Citrate and Esculin Hydrate. All plates were incubated at 37 °C for 24–48 h and counted for microbial populations which were converted to log CFU per gram. Log reductions were calculated by subtracting the survivor counts from the initial population.

2.4. Inactivation kinetics To analyze the inactivation kinetics of isothermal treatment of Salmonella and E. faecium in paprika, white pepper and cumin powder, the first order kinetics Eq. (1), and the Weibull model Eq. (2) were used (Peleg, 2006);

log

N t = − N0 D

(1)

log

N t = − ( )α N0 δ

(2)

λ=

where N0 was the initial load of microorganism (CFU/g), and N was the population of survivals (CFU/g) at time t, the isothermal treatment time (min) after CUT. D is the time needed to reduce microbial population by 10-fold at the inactivation temperature (°C). δ indicates the overall steepness of survival curve, and α is the survival curve factor which indicates whether it is linear (α = 1) or non-linear (α≠1) with a decreasing (α < 1) or increasing (α > 1) inactivation rate with time. Obtained survival data of both microorganisms after isothermal treatment was applied to fit the two models and estimate the model parameters. Root mean square error (RMSE) (log CFU/g) were applied to interpret the performance of the models (Motulsky & Cristopoulos, 2004)

Δσ Δμ

(5)

where Δσ is the increase in the standard deviation (SD) of RF heated sample (°C), and Δμ is the increase in the average temperature (°C) during the RF heating. The lower UI values indicate better heating uniformity in the heated sample. To determine the efficacy of RF heating on inactivation of both microorganisms, sealed cylindrical petri dishes filled with inoculated samples (20 g) were treated by the RF system for predetermined CUTs to reach 80 °C with different time intervals. Then the RF heated sample packages were immediately removed from container and inserted in ice-water bath for 90 s to stop further thermal inactivation of microorganism in the sample. For each treated sample, 20 g of the sample were transferred to a sterile stomacher bags containing 180 ml of 0.1% 3

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Fig. 2. Experimental set up of RF heating system. Adapted from Ozturk et al. (2018).

Fig. 3. The inactivation kinetics of Salmonella (open circles) and E. faecium (solid circles) in. paprika with a w,25°C =0.45 ± 0.05 during isothermal treatment at 70, 75 and 80 °C.

reduction of microorganism obtained from a heating process can be calculated as the ratio of lethality and the D value at reference temperature, i.e:

peptone water and homogenized for 3 min at 260 rpm with a Seward Stomacher (Seward, London, UK). The microbial population in the samples were determined following the methods described in the enumeration section.

Number of log reduction = 2.6. Lethality evaluation

t

∫ 10(T−T 0

ref )

(7)

The calculated number of log reduction were compared with measured values based on the decrease of microbial population. All graphs and tables were drawn with Excel 2018.

Obtained D- and z-values were applied to calculate the lethality of microorganisms at given temperature as described by Gaillard et al. (1998);

F=

Fref Dref

2.7. Color measurement

z dt

(6)

To evaluate the effect of RF heating on food quality, the change in color (L*, a*, and b* values) of RF heated samples was measured using a Minolta colorimeter (model CR300, Minolta Co., Osaka, Japan). L*, a*, and b* values indicate the lightness, whiteness, and yellowness of the

where F (min) is described as the total equivalent heating time at the reference temperature; T is the temperature of sample at time t, Tref is the reference temperature, and z is the z-value. Therefore, the log 4

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Fig. 4. The inactivation kinetics of Salmonella (open circles) and E. faecium (solid circles) in white pepper with a w,25°C =0.45 ± 0.05 during isothermal treatment at 70, 75 and 80 °C.

Fig. 5. The inactivation kinetics of Salmonella (open circles) and E. faecium (solid circles) in cumin powder with a w,25°C =0.45 ± 0.05 during isothermal treatment at 70, 75 and 80 °C.

Obtained survivals after isothermal treatment in the water bath were used to determine the inactivation kinetics of both microorganisms in the three spices. The inactivation kinetics of Salmonella and E. faecium in paprika, white pepper and cumin powder with a w,25°C = 0.45 ± 0.05 are presented in Figs. 3–5, respectively. Table 2 shows that the survival data of both Salmonella and E. faecium in each sample fit well to the primary models (First Order Kinetics and Weibull Model) with similar RMSE values. As seen in Table 2, survival curves of E. faecium shows a better fit for the Weilbull model while Salmonella corresponded better to the first order kinetic (log-linear) model. In the literature, the log-linear model is commonly used to evaluate thermal resistances of microorganisms. Therefore, the first order kinetic (loglinear) model was applied to obtain D-values and z-values of both microorganisms at the inactivation temperatures. The D-values at 70, 75 and 80 °C of Salmonella in white pepper were 13.8 ± 1.4, 5.3 ± 1.1 and 2.9 ± 0.8, respectively, with a z-value of 14.2 °C (Table 2). E. faecium showed significantly (p < 0.05) higher

sample, respectively. The colorimeter was calibrated before each measurement by using black and white calibration standards. Samples were spread into a plastic tray, and the top surface was flattened for color measurement at randomly selected five points. The average of five measurements was used to evaluate the effect of RF on food quality. Color data was analyzed using the ANOVA procedure with Duncan's multiple range test of SAS (SAS Institute, Cary, NC, USA). Value of P < 0.05 was used to indicate significant difference between treated and non-treated samples.

3. Result and discussion 3.1. D- and z-values of Salmonella and E. faecium in paprika, white pepper and cumin powder The CUTs of the three spices heated in aluminum test cells were determined as 120–180 s for isothermal treatment in the water bath. 5

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Table 2 Parameter estimates for the primary models fitted to the survival data of Salmonella and E. faecium in spices during isothermal treatment at different temperatures. Sample

Paprika

White Pepper

Linear Model Bacteria

Temperature (°C)

D-Value (min)

RMSE (log CFU/g)

δ(min)

α

RMSE (log CFU/g)

Salmonella

70 75 80

6.55 ± 1.12 3.09 ± 0.82 1.21 ± 0.46

0.46 0.23 0.17

7.72 ± 1.42 4.56 ± 1.12 1.78 ± 0.39

1.16 ± 0.14 0.69 ± 0.09 0.72 ± 0.04

0.56 0.32 0.21

E. faecium

70 75 80 70 75 80

9.62 ± 0.96 4.51 ± 0.68 1.82 ± 0.26 13.79 ± 1.42 5.27 ± 1.05 2.85 ± 0.79

0.21 0.35 0.52 0.37 0.19 0.44

12.86 ± 1.26 5.16 ± 0.52 2.56 ± 0.42 16.12 ± 1.69 7.09 ± 1.45 4.21 ± 1.09

1.09 0.74 0.89 1.24 0.92 0.76

± ± ± ± ± ±

0.26 0.16 0.06 0.17 0.11 0.08

0.32 0.41 0.49 0.42 0.21 0.39

70 75 80 70 75 80

23.32 ± 1.86 12.67 ± 1.32 5.27 ± 0.86 16.61 ± 1.24 8.01 ± 1.12 4.47 ± 0.89

0.53 0.24 0.41 0.58 0.35 0.24

26.19 ± 2.42 14.52 ± 1.63 6.51 ± 1.06 16.61 ± 1.24 8.01 ± 1.12 4.47 ± 0.89

1.12 0.81 0.96 1.52 0.83 0.78

± ± ± ± ± ±

0.36 0.13 0.09 0.11 0.07 0.06

0.63 0.19 0.38 0.64 0.46 0.19

70 75 80

28.31 ± 2.24 18.64 ± 1.72 9.53 ± 1.18

0.73 0.52 0.29

33.19 ± 2.56 21.04 ± 2.14 11.42 ± 1.18

1.26 ± 0.18 0.92 ± 0.26 0.86 ± 0.07

0.82 0.61 0.42

Salmonella

E. faecium

Cumin Powder

Weibull Model

Salmonella

E. faecium

* The root mean square error (RMSE) was determined by 1stOpt software using triplicate experimental data (n = 3). * Values are mean ± standard error. Parameters were estimated separately for each data set. Smaller RMSE values indicate a better fitness of the model.

Fig. 6. A comparison among the inactivation kinetics of Salmonella (A) (open circles) and E. faecium (B) (solid circles) in paprika, white pepper and cumin powder with a w,25°C =0.45 ± 0.05 during isothermal treatment at 80 °C.

and ground black pepper (Chen, Wei, Irmak, Chaves, & Subbiah, 2019; Wei et al., 2018). Thermal resistance values of Salmonella species were reported to be from a few minutes to a few hours for wheat flour (Liu et al., 2018a, b), corn flour (Vancauwenberge, Bothast & Kwolek, 1981; Ozturk et al., 2019), and non-fat dried milk powder (Michael et al., 2014). Rachon et al. (2016) reported that the D80°C values of Salmonella and E. faecium NRRL B-2354 were 1.9 and 8.7 min for culinary powder at a w,25°C = 0.65, and 8.9 and 23.8 min for chicken meat powder at a w,25°C = 0.38, respectively. The z-values of Salmonella and E. faecium in paprika, white pepper and cumin powder were measured to be 13.6 and 13.8 °C, 14.6 and 15.5 °C, and 17.6 and 19.1 °C, respectively (Fig. 7). The trends of zvalues for both microorganism in each spice sample are in good agreement with corn flour, wheat flour and cocoa powder (Liu et al., 2018a, b; Ozturk et al., 2019; Tsai et al., 2019). It is reported that E. faecium had lower log reduction than Salmonella in whole black

thermal resistance in each spice sample at the inactivation temperatures (Table 2). For example, D75°C of Salmonella and E. faecium in cumin powder were 8.0 ± 1.1 and 18.6 ± 1.7 min, respectively; for paprika, D75°C values of both microorganisms were 3.1 ± 0.8 and 4.5 ± 0.7, respectively. Fig. 6 shows a comparison among the D80°C values of Salmonella and E. faecium in paprika, white pepper and cumin powder. Both microorganisms show less thermal resistance in paprika, which indicate that heat resistance of the same serotype varies for different food matrices. It is reported that protection effect of fat or other components to microorganisms in each food matrix are different because of their various impact in aw with temperature increase (Tadapaneni, Yang, Carter, & Tang, 2017; Xu et al., 2019). In addition, the antimicrobial effect of the spices may also play an important role on the survival population of both microorganisms (Liu et al., 2017; Nabavi et al., 2015).The obtained values for thermal resistances of Salmonella and E. faecium are similar with cumin seeds, whole black peppercorn, 6

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Fig. 7. D-values of Salmonella and E. faecium in Paprika, White Pepper and Cumin Powder at a w,25°C Salmonella and continuous line for E. faecium). Experiments were done in triplicate.

90

0.45 ± 0.05 and their power trend lines (dashed line for

Syamaladevi et al., 2016), pet food and confectionery (Rachon et al., 2016), and cocoa powder (Tsai et al., 2019). The present study confirmed that E. faecium NRRL B-2354 can be used as a Salmonella surrogate in paprika, white pepper, and cumin powder due to the significantly (P < 0.05) higher heat resistance (D-value) and similar zvalue in the temperature range of 70–80 °C.

80 70

Temperature ( C)

=

60

3.2. Radio frequency heating and uniformity

50 40

Paprika

30

White Pepper

20

Cumin

Fig. 8 shows the temperature-time heating profiles of paprika, white pepper, and cumin powder during the RF heating to 80 °C with a w,25°C = 0.45 ± 0.05. The CUTs during the RF heating in each spice sample were determined to be 100 s for paprika with a heating rate of 0.43 °C s−1, 250 s for white pepper with a heating rate of 0.22 °C s−1, and 370 s for cumin powder with a heating rate of 0.15 °C s−1. The obtained CUTs were applied to inactivate both Salmonella and E. faecium in inoculated samples. The dielectric properties (DP) of food materials are the most important factors affecting the RF heating since the dielectric values play an important role in energy absorption and conversion in food, which directly affects both the rate and uniformity of heating (Tang, 2005). The heating rates for the spices confirmed the results from our previous study (Ozturk et al., 2018), i.e. higher dielectric properties resulted in faster heating (paprika > white pepper > cumin powder) with a linear increase in temperature during RF heating. Although the samples were held in a humidity chamber to obtain the same water activity, it has been reported that different food products at the same water activity level can have different moisture contents (Tadapaneni et al., 2017). Moisture content is one of the most important parameters affecting the dielectric properties of foods and directly impacts the temperature profiles upon RF heating. In addition

10 0 0

50

100

150

200

250

300

350

400

Heating Time (s) Fig. 8. Temperature-time profiles of paprika, white pepper and cumin powder (n = 3) with a w,25°C =0.45 ± 0.05 during RF heating to reach 80 °C .

peppercorn and cumin seed when thermally treated by RF heating (Chen et al., 2019; Wei et al., 2018). Other studies also showed that E. faecium NRRL B-2354 is a promising surrogate and can be used to validate thermal studies of Salmonella contaminated almonds (Bingol et al., 2011; Kopit, Kim, Siezen, Harris, & Marco, 2014), wheat flour (Liu et al., 2018a, b; Smith, Hildebrandt, Casulli, Dolan, & Marks, 2016;

Fig. 9. Temperature distribution of top surface of paprika, white pepper and cumin powder after RF heating for come up time to reach 80 °C. 7

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Fig. 10. Measured survival of Salmonella (open triangle) and E. faecium (open square) in paprika, white pepper and cumin powder and predicted survival curves (Salmonella (solid triangle); E. faecium (solid square)) during RF heating to reach 80 °C.

to moisture, the components of food products such as fat, protein, ash, or carbohydrate also affect the dielectric properties (Pongpichaiudom, Songsermpong, Tang, & Sablani, 2017). Different food products can thus have different temperature profiles when subjected to RF heating based on their chemical composition. Previous studies also showed similar linear temperature-time profiles for different LMFs when treated by RF heating, including broccoli powder (Ozturk, Kong, Trabelsi, & Singh, 2016), coffee bean (Pan et al., 2012), egg white powder (Boreddy & Subbiah, 2016), legume flours and wheat flours (Guo, Tiwari, Tang, & Wang, 2008; Guo, Wang, Tiwari, Johnson, & Tang, 2010; Jiao, Johnson, Tang, Tiwari, & Wang, 2011; Nelson & Trabelsi, 2006), chili powder (Li et al., 2016), and black and red peppers (Jeong & Kang, 2014). Heating uniformity of each RF heated sample is presented in Fig. 9, and the average temperature of the top surface of each sample was measured to be 76.2 ± 2.3 °C for paprika with UI 0.064, 73.6 ± 1.4 °C for white pepper with UI 0.057, and 72.8 ± 2.1 °C for cumin powder with UI 0.042. Due to the heat conduction through the cylindrical Petri dish and heat loss to the air, the average temperature of the surface area was less than the temperature at the geometric center of the container. As seen in Fig. 9, in each spice, the hot spots are located around the edges and the cold spots are at the center of the top surface (Fig. 9). The faster increase in temperature in paprika during RF heating resulted in more overheating on the edge area and less uniform heating on the top surface as compared to white pepper and cumin powder. Similar observations in heating profiles and uniformity were reported in previous studies for chili powder (Li et al., 2016), and black and white pepper (Ozturk et al., 2018). Detailed information about the relationship between dielectric properties and heating rate of selected spices can be found in Ozturk et al. (2018). It should be noted that the heating uniformity was evaluated based only on the top surface temperatures due to the small amount and the thin thickness of the samples loaded in the Petri dish (15 mm). Further studies with large sample size are needed to validate these results.

thermal resistance (D-value) of E. faecium. According to predictive lines, the RF heating during the CUT to reach 80 °C in paprika and white pepper resulted in a 4.16 and 3.34 log reduction of Salmonella, and 1.92 and 1.41 of E. faecium. The RF heating for CUT time to reach 80 °C in cumin powder, however, resulted in a lower log reduction of both Salmonella (2.83) and E. faecium (0.88) than those in paprika and white pepper, which is because Salmonella and E. faecium have higher D-values, and thus a higher thermal resistance in cumin powder. The experimental results aligned closely with predicted results for both microorganisms during the RF heating (Fig. 10). E. faecium showed more survivals than Salmonella in all samples during the RF heating at any given heating time because of the higher thermal resistance of E. faecium. The prediction model used in this experiment has also been used to predict population reduction of E. faecium in wheat flour during the RF heating at 80 °C (Liu et al., 2018a, b and Xu et al., 2018). Our study shows that the obtained log reductions collected from experimental results were higher than the applied predictive model, which may be because the RF heating was not uniform and created hot spots around edges of the petri dishes. As USDA regulates the pasteurization standard of at least 4 log reduction in surrogate microorganisms (FDA, 2016), the RF heating applied in this study was not enough to reach target log reductions in cumin powder with less than 3 log reduction in both microorganisms. Additional treatments, such as holding samples in the RF system at high temperature for a certain time period, could be used to achieve 4-5-log reduction. Results of this study indicated that F- value model could provide a dependable prediction for RF heating in closed systems. For open systems such as baking or roasting, however, the significant change in moisture content of sample due to the external water evaporation may occur during the treatment. As reported by Syamaladevi et al. (2016) and Wang et al. (2013), a reduction in moisture content could lead to sharp increase in the thermal resistance of microorganisms. In that case, the lethality will be more difficult to calculate using equations (5) and (6).

3.3. Lethality values from measurement and calculation based on inactivation kinetics

3.4. Effect of radio frequency heating on color Color values of paprika, white pepper and cumin powder after the RF heating ranged from 34.7 to 71.2 for L*, 7.7 to 26.7 for a*, and 22.1 to 32.2 for b*, and No significant color change was found before and after the RF heating. As the color is an important indicator of quality, this result proved that the RF heating was able to maintain the quality of spices, a remarkable advantage over conventional heating techniques like steam treatment.

Fig. 10 shows the temperature curve and corresponding microbial log reduction including both measured and predicted values calculated using equations (5) and (6). Predictive values were relatively stable (< 1 log reduction) up to 75 s of RF heating time for both microorganisms and declined rapidly through the rest of the heating time when temperature continued to increase. As seen in Fig. 10, the predicted survival curves of E. faecium in paprika, white pepper and cumin powder declined more slowly than that of Salmonella because of higher 8

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4. Conclusion The comparison between the thermal resistances of Salmonella and E. faecium inoculated in paprika, white pepper and cumin powder at 70, 75, and 80 °C shows that the E. faecium is a suitable surrogate of Salmonella in spices due to its higher thermal resistance. The inactivation kinetics of Salmonella and E. faecium during isothermal treatment can be described by two primary models with similar RMSE values. The results also showed that the RF heating is effective in reducing foodborne pathogens in spices without negatively affecting the color. E. faecium can be used as a surrogate in sealed packages for conservative validation of RF pasteurization technology. This study confirms that the RF heating could be applied as an alternative pasteurization method for spices, and it is practical to use E. faecium and the F-value to design RF pasteurization processes. Acknowledgment This work was supported by the USDA National Institute of Food and Agriculture, AFRI project [proposal number: 2012-04394; Grant Number: 2013-67005-21295]. We would like to acknowledge Dr. Mark A. Harrison and Dr. Abhinav Mishra for their help on this project. The authors also thank the Turkish Ministry of National Education Council for providing the chance to Samet Ozturk for his Ph.D study. References Authority, E. F. S. (2010). The community summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in the European Union in 2008. EFSA Journal, 8(1), 1496 410. Bermudez-Aguirre, D., & Corradini, M. G. (2012). Inactivation kinetics of Salmonella spp. under thermal and emerging treatments: A review. Food Research International, 45(2), 700–712. https://doi.org/10.1016/j.foodres.2011.05.040. Bingol, G., Yang, J. H., Brandl, M. T., Pan, Z. L., Wang, H., & McHugh, T. H. (2011). Infrared pasteurization of raw almonds. Journal of Food Engineering, 104(3), 387–393. https://doi.org/10.1016/j.jfoodeng.2010.12.034. Boreddy, S. R., & Subbiah, J. (2016). Temperature and moisture dependent dielectric properties of egg white powder. Journal of Food Engineering, 168, 60–67. https://doi. org/10.1016/j.jfoodeng.2015.07.023. CDC. (2010). Salmonella Montevideo infections associated with salami products made with contaminated imported black and red pepper — United States, July 2009–April 2010. http://www.cdc.gov/mmwr/preview/. Chen, L., Wei, X. Y., Irmak, S., Chaves, B. D., & Subbiah, J. (2019). Inactivation of Salmonella enterica and Enterococcus faecium NRRL B-2354 in cumin seeds by radiofrequency heating. Food Control, 103, 59–69. https://doi.org/10.1016/j. foodcont.2019.04.004. Chung, H. J., Birla, S. L., & Tang, J. (2008). Performance evaluation of aluminum test cell designed for determining the heat resistance of bacterial spores in foods. Lwt-Food Science and Technology, 41(8), 1351–1359. https://doi.org/10.1016/j.lwt.2007.08. 024. Enache, E., Kataoka, A., Black, D. G., Napier, C. D., Podolak, R., & Hayman, M. M. (2015). Development of a dry inoculation method for thermal challenge studies in lowmoisture foods by using talc as a carrier for Salmonella and a surrogate (Enterococcus faecium). Journal of Food Protection, 78(6), 1106–1112. https://doi.org/10.4315/ 0362-028x.jfp-14-396. FDA (2016). Hazard analysis and risk-based preventive controls for human food: Guidance for industry draft guidance. Retrieved August 21 from https://www.fda. gov/media/99581/download . Gaillard, S., Leguerinel, I., & Mafart, P. (1998). Model for combined effects of temperature, pH and water activity on thermal inactivation of Bacillus cereus spores. Journal of Food Science, 63(5), 887–889. https://doi.org/10.1111/j.1365-2621.1998. tb17920.x. Gao, M., Tang, J., Johnson, J. A., & Wang, S. (2012). Dielectric properties of ground almond shells in the development of radio frequency and microwave pasteurization. Journal of Food Engineering, 112(4), 282–287. https://doi.org/10.1016/j.jfoodeng. 2012.05.011. Gao, M., Tang, J., Villa-Rojas, R., Wang, Y., & Wang, S. (2011). Pasteurization process development for controlling Salmonella in in-shell almonds using radio frequency energy. Journal of Food Engineering, 104(2), 299–306. https://doi.org/10.1016/j. jfoodeng.2010.12.021. Gao, M., Tang, J., Wang, Y., Powers, J., & Wang, S. (2010). Almond quality as influenced by radio frequency heat treatments for disinfestation. Postharvest Biology and Technology, 58(3), 225–231. https://doi.org/10.1016/j.postharvbio.2010.06.005. Guo, W., Tiwari, G., Tang, J., & Wang, S. (2008). Frequency, moisture and temperaturedependent dielectric properties of chickpea flour. Biosystems Engineering, 101(2), 217–224. https://doi.org/10.1016/j.biosystemseng.2008.07.002. Guo, W., Wang, S., Tiwari, G., Johnson, J. A., & Tang, J. (2010). Temperature and

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