**E**ff**ects of Moisture, Temperature, and Salt Content on the Dielectric Properties of Pecan Kernels during Microwave and Radio Frequency Drying Processes**

**Jigang Zhang 1, Maoye Li 2, Jianghua Cheng 2, Jiao Wang 1, Zhien Ding 2, Xiaolong Yuan 1, Sumei Zhou <sup>3</sup> and Xinmin Liu 1,\***


Received: 12 August 2019; Accepted: 28 August 2019; Published: 2 September 2019

**Abstract:** Dielectric properties of materials influence the interaction of electromagnetic fields with and are therefore important in designing effective dielectric heating processes. We investigated the dielectric properties (DPs) of pecan kernels between 10 and 3000 MHz using a Novocontrol broadband dielectric spectrometer in a temperature range of 5–65 ◦C and a moisture content range of 10–30% wet basis (wb) at three salt levels. The dielectric constant (ε ) and loss factor (ε") of the pecan kernels decreased significantly with increasing frequency in the radio frequency (RF) band, but gradually in the measured microwave (MW) band. The moisture content and temperature increase greatly contributed to the increase in the ε and ε" of samples, and ε" increased sharply with increasing salt strength. Quadratic polynomial models were established to simulate DPs as functions of temperature and moisture content at four frequencies (27, 40, 915, and 2450 MHz), with *R2* > 0.94. The average penetration depth of pecan kernels in the RF band was greater than that in the MW band (238.17 ± 21.78 cm vs. 15.23 ± 7.36 cm; *p* < 0.01). Based on the measured DP data, the simulated and experimental temperature-time histories of pecan kernels at five moisture contents were compared within the 5 min RF heating period.

**Keywords:** pecan; dielectric properties; radio frequency (RF) heating; simulation

#### **1. Introduction**

Pecan [*Carya illinoinensis* (Wangenh.) K. Koch] is a world-famous Juglandaceae tree nut, mainly distributed in North America, including the United States andMexico, which accounts for approximately 60% and 30%, respectively, of global pecan nut production [1]. The pecan nut has gained increasing popularity due to its abundant nutrient components (high unsaturated fatty acids, protein, minerals, vitamins, phenolics, flavonoids, phytosterols, and saponins), unique buttery flavor, and potential health-promoting benefits, such as modulating blood cholesterol levels, preventing coronary heart disease, and mitigating adiposity [2–6]. Suitable postharvest drying is an essential step in maintaining the quality and active ingredients of pecan nuts [7].

The moisture content of freshly harvested pecan nuts must be promptly reduced from 25–35% wet basis (wb) to less than 6% wb through dehydration to decrease the metabolic rate, which facilitates the subsequent transport, storage, and processing of pecan nuts [8,9]. Pecan kernels are enclosed in a thick hard shell, which leads to a slow drying cycle. Often, more than three days is required to accomplish dehydration through sun or air convection drying methods [10]. Moreover, these traditional convection drying methods require a large area to lay the pecans outdoors and are susceptible to ambient climatic

circumstances, which often cause undesirable quality degradation, such as mold, being off-flavor, or discoloration [11]. Therefore, advanced drying technologies have become an urgent issue that needs to be resolved for postharvest pecan processing.

Dielectric heating is a fourth-generation heating treatment technology that is widely used for the industrial drying of food, agricultural products, and industrial materials [12,13]. When dielectric samples are subjected to a rapid reversal electric field, the electromagnetic wave passes through the material shell and interacts directly with the permanent or induced dipoles and charges inside the sample to induce underlying dipole polarization and ionic conduction with finite displacement, resulting in heat generation inside the volume [14,15]. However, to design and control the dielectric heating process, knowledge regarding the dielectric properties (DPs) of materials is essential, which can be used to determine how much polarization of dielectrics and charge conduction can occur and dissipate when subjected to an electromagnetic field [16]. The DPs of materials are normally described using the complex relative permittivity, ε\*, which is represented as follows: ε\* = ε – jε". The real part (ε ) of the formula is named the dielectric constant, which reflects the charge storing capability of the material regardless of the sample's size. The imaginary part (ε") is named the loss factor, reflecting the energy dissipation in the material due to the conversion of the electromagnetic field into heat energy. The higher the dielectric loss factor values, the higher the electromagnetic energy that is absorbed and converted by the material, and the higher the rate of the temperature increase [17]. Therefore, DPs are the most basic parameters for characterizing the interaction, thermal efficiency, and penetration depth when designing MW and RF heating processes [18].

Several studies have reported the DPs of foodstuffs over various frequency, temperature, moisture content, and salinity ranges in drying, pasteurization, and pest control [19–21]. The DPs of macadamia kernels were determined in the frequency band of 10–1,800 MHz within a temperature range of 25–100 ◦C at moisture contents of 3–24% wb by adopting open-ended coaxial probe technology [22]. Zhang et al. [23] found that the DP value of peanut kernels decreased with increasing frequency but increased with increasing temperature and moisture content. Ling et al. [24] simulated quadratic polynomial equations for the temperature, moisture content, and frequency of non-salted pistachio nuts, according to the DP data measured from 10–4500 MHz at 25 to 85 ◦C. Jeong et al. [25] found that RF heating can potentially inactivate foodborne *Salmonella enterica* in pistachios and that the inhibitory effect is controlled by the dielectric loss factor relative to the salt content. The dielectric heating of pecans can effectively prevent the attack of weevil [26], and the dielectric heating does not make the color of the epidermis darker when stored later than steam [27]. In recent years, new technologies of broadband dielectric spectroscopy have been developed to measure the DPs of materials. These new technologies have integrated systems, are easy to operate, have a broadband frequency range, and provide more accurate results in comparison to the previous method of the open-ended coaxial-line probe. However, some problems exist in the direct application of a broadband dielectric spectrometer for determining the DPs of irregularly shaped materials because this method requires close contact between the samples and parallel electrode probes during measurements. The contact problem is overcome by creating compressed cylindrical samples with flat surfaces from a ground sample to match the kernel bulk density of the pecan kernel.

In this study, the objectives were to determine the DPs of pecan nut kernels over frequencies from 10 to 3000 MHz at moisture contents between 10 to 30% at four temperature levels. Furthermore, simulations were performed based on empirical equations describing the DPs of the pecan kernels as functions of the moisture content and temperature at certain frequencies. The effect of the salt strength (mild, medium, and heavy) on the DPs of pecan nut kernels was also assessed. The penetration depth of electromagnetic energy into the pecan kernels under these different conditions was determined. Engineering insights into the implications of these DPs for the RF process during 5 min of RF heating for pecan samples with five different moisture contents were discussed.

#### **2. Materials and Methods**

#### *2.1. Materials*

Pecan fruits of the Mahan variety were harvested from a local farm in Jiande (Jiangsu Province, China) in September 2016. Ethephon aqueous solution (4000 mL kg<sup>−</sup>1) was sprayed on the surfaces of the fruit hulls to accelerate the hull separation from the shell. The in-shell pecan nuts were immersed in sterilizing liquid (NaClO, 5 mg mL<sup>−</sup>1) for 5 min and then rinsed with deionized sterile water. The surface of disinfected nuts was air-dried for further grading treatment. High-quality samples free of any defects were selected based on full and plump size and similar sense and stored at 4 ◦C until further testing. The main nutritional ingredients of the pecan samples were measured with Association of Official Analytical Chemists (AOAC) standard methods (Table 1).

**Table 1.** Chemical compositions (g/100 g, average ± SD of three replicates) of the pecan kernels.


<sup>a</sup> Protein was calculated by considering a nitrogen conversion factor of 5.3. <sup>b</sup> Carbohydrate content = 100% − (% moisture + % protein + % fat + % ash + % dietary fiber).

#### *2.2. Preparation of Pecan Kernel Samples*

Pecan nut kernels (200 g) with an initial moisture content of 30.2% wb were divided evenly into four sublots and then dried to moisture content levels of 25, 20, 15, and 10% wb at 40 ◦C in a blast drying oven. The samples with different moisture contents were then vacuum sealed in polyethylene bags at 4 ◦C until further use. The storage time was assumed to be sufficient for the residual humidity in each kernel to completely relax and reach a uniform level throughout the kernel.

To quantitatively examine the effect of the salt content on the DPs of the pecan kernels, the salted pecan samples were prepared in accordance with the method of Ling et al. [24]. A total of 150 g of fresh pecan kernels was freeze-dried to adjust the moisture content to 5%. The dried nut kernels were then divided into three equal parts that were immersed in 1000 mL of brine (NaCl 5%, 10%, and 20% w v<sup>−</sup>1) for 60 min with constant stirring to simulate the market products named lightly-, medium-, and heavily-salted nuts. These soaked salty samples were removed and freeze-dried again to obtain an end moisture content of 15%. The dried pecan samples contained the same ingredients and had three levels of salt: Light, medium, and heavy. All the samples were stored (relaxing time was sufficient) for further use at 4 ◦C in individual polyethylene bags.

#### *2.3. Preparation of Cylindrical Samples*

To obtain close contact between the samples and gold-plated parallel electrodes during the measurement of DPs, cylindrical samples whose density closely matched the actual kernel density were prepared. The prepared kernels with different moisture and salt contents were ground into powder. The powdered samples were then compressed under vacuum in a metal cylindrical mold of a hydraulic press (YP-2, Shanghai Shanyue Science Instrument Co., Ltd., Shanghai, China) to form cylindrical discs (ø: 7 mm; H: 3 mm). To control the pressure of the hydraulic press and the weights of the added powder, the density of the compressed cylindrical sheets closely matched the density of the real kernel sample.

The kernel bulk density of the pecan kernel samples with different moisture contents was determined using the liquid displacement method [24], in which toluene (C7H8) was used as the displaced liquid due to its low surface tension and non-absorption by nut kernels. The actual density was distributed within the numerical zone from 0.8150 to 1.2715 g cm−<sup>3</sup> (Table 2) as the moisture content varied between 10% and 30%. The specific heat at different densities was determined by a dual-needle probe method [28] using a thermal properties analyzer (KD2 Pro, Decagon Devices, Pullman, WA, USA).


**Table 2.** The densities and specific heat of pecan kernel at five moisture contents.

#### *2.4. Measurement of DPs*

The DPs of the cylindrical sheet samples were measured using a Novocontrol broadband dielectric spectrometer system (Novocontrol Concept 80, Montabaur, Germany). The key components of the system comprised an Alpha-A dielectric analyzer, Quatro temperature control systems, a Novocontrol BDS 1200 sample cell, a Dewar liquid nitrogen system, a computer, WinDETA data acquisition, and evaluation software. In this research, the DPs of samples with different moisture and salt contents were determined in the frequency band of 10 to 3000 MHz at four temperatures (5, 25, 45, and 65 ◦C). The temperature was accurately regulated by the Quatro temperature control systems coupled to the dielectric analyzer. Prior to measurement, the computer was switched on, as was the dielectric analyzer to keep it in a stable state. The cylindrical sheets were then sandwiched between the parallel electrodes and installed in the sample cell that could be immersed in a nitrogen environment to condition the tested samples to the set temperatures prior to each detection. The samples were first gradient frozen (10 ◦C min<sup>−</sup>1) to 5 ◦C, the equilibrium was maintained at this temperature for 3 min, and then the DPs were measured at intervals of 20 ◦C. Approximately 20 min was required for the temperature of the tested sample to increase from one level to the next.

#### *2.5. Power Penetration Depth*

The penetration depth (dp) refers to the quantitatively determined effective acting distance (in meters) between the MW or RF power and materials, where an incoming power intensity is decreased to 1/e (e = 2.7182) of its amplitude transiting the surface. The dp can be calculated using Equation (1):

$$\text{dp} = \frac{c}{2\pi f \sqrt{2\varepsilon' \left[ \sqrt{1 + \left( \frac{\varepsilon''}{\varepsilon'} \right)^2} - 1 \right]}} \tag{1}$$

where c represents the speed of light in a vacuum (3 <sup>×</sup> <sup>10</sup><sup>8</sup> m s<sup>−</sup>1), f represents the frequency (Hz), and ε and ε" are the permittivity and loss factor, respectively. The dp of the pecan samples was calculated according to the measured dielectric data at optional frequencies, temperatures, moisture contents, and salt contents.

#### *2.6. RF Heating Process*

Further research is needed to provide engineering insights into the implications of these DPs for the RF heating process, and we are seeking to explore the relationship between DPs and the RF heating process in which the heating rate of pecan is investigated as the consequence of those dielectric properties for an RF heating process, using both experiment and simulation. A 6 kW, 27.12 MHz parallel plate RF system (SO6B, Strayfield International, Wokingham, UK) was used in this research. The RF system includes a pair of parallel electrodes, controller, and RF cavity. The RF power of the system can be changed by adjusting the electrode gap between the upper and lower plates (90–190 mm) to achieve different heating rates for the material. An electrode gap of 150 mm was selected during 5 min RF heating. The pecan samples (3 kg) with different moisture levels (10%, 15%, 20%, 25%, and 30% wb) were successively placed in the polyethylene container on top of the bottom (ground) electrode for dielectric heating, where the stacked height of the sample should be less than the dp. The temperature increase at the central position of the heated sample was monitored using a fiber optical temperature sensor system (HQ-FTS-D1F00, Heqi Technologies Inc., Xian, China).

When the sample is heated in the RF cavity, the RF electric field acts as a heat source and leads to heat transfer inside the material. The heat transfer equation during RF heating can be calculated by Equations (2) and (3):

$$\frac{\partial T}{\partial t} = \frac{2\pi \cdot f \cdot \varepsilon\_0 \cdot \varepsilon'' \cdot \left| \overrightarrow{E\_m} \right|^2}{\rho \cdot \mathbb{C}\_P} \tag{2}$$

$$\overrightarrow{E\_{\rm m}}| = \frac{V}{\sqrt{\left(\varepsilon' d\_0 + d\_p\right)^2 + \left(\varepsilon'' d\_0\right)^2}}\tag{3}$$

where T is the temperature increase in the material (◦C); t is the temperature rise time (s); f is the frequency (Hz); <sup>ε</sup><sup>0</sup> is the the dielectric constant of free space (8.854×10−<sup>12</sup> Fm<sup>−</sup>1); E is the electric field intensity (Vm<sup>−</sup>1); Cp is the specific heat of material (J·kg<sup>−</sup>1C−1); is the density of the material (kg m<sup>−</sup>3); V is the voltage between the electrodes; d0 is the air gap from the top electrode plate to the upper surface of samples; and dp is the height of the sample. Equation (2) shows that T is proportional to the material's ε" and can be calculated by the measured ε" data. This increase in temperature is theoretically attributed to dielectric heating in comparison with the experimentally measured increase in RF heating.


A computer simulation model for solving coupled electromagnetic and heat transfer equations based on the RF system was constructed using COMSOL software (V4.3a, COMSOL Multiphysics, CnTech Co., Ltd., Wuhan, China). The modeling steps include creating FEMLAB (AC/DC) modules, a geometrical model of RF systems, a heat transfer module, assigning initial and boundary conditions, mesh creation and optimization, choosing solver, setting tolerance, and time steps, and solving inbuilt convergence [29]. The measured DP value of pecan kernels was put into the heat transfer module to solve the coupled heat transfer equations, and simulated temperature rise values of samples were saved. The upper electrode voltage had a constant value of 6000 V with ±5% fluctuation. The mesh system included 132,279 domain elements (tetrahedral), 10,874 boundary elements (triangular), 843 edge elements (linear), and 26 vertex elements. The direct linear system solver (UMFPACK) was used with a relative tolerance and absolute tolerance of 0.01 and 0.001, respectively, with the initial and maximum time steps of 0.001 s and 0.1 s. These computer simulations were performed by a Dell workstation (Dell, Inc, Texas, USA) with 8 GB RAM running a Windows 10 64-bit operating system (Microsoft Corporation, Albuquerque, USA).

#### **3. Results and Discussion**

#### *3.1. Frequency-Dependent DPs*

Figure 1 displays the semilog plot of ε and ε" as functions of the frequency at different moisture contents and temperatures. At moisture contents of 10 and 30% wb, both ε and ε" displayed a nonlinear decrease with increasing frequency characterized by a rapid decrease in the frequency band (10 to 300 MHz), followed by a slow decrease in the intermediate and high frequency band (300 to 3000 MHz), and this nonlinear decrease was more pronounced for increased temperatures and moisture contents. Ionic conduction is considered to be the predominant polarization mechanism at low frequencies, and the strengths gradually diminish with increasing frequency [22]. At a constant frequency, a high temperature resulted in high DP values, whereas at a constant temperature and given frequency, a high moisture content caused high DP values (Figure 1). This behavior can be explained by the fact that a high temperature and moisture content improve the ionic mobility and dipole rotation, which result in increased DP values [30]. However, at a low moisture content of 10%, all the ε and ε" values of the pecan kernels were less than 8 at any measured temperature. Similar DP values were also observed by Wang et al. [31] for walnut kernels at moisture contents of 7.5% wb, from 1 to 1800 MHz. This result may be attributed to the high fat contents of pecan kernels, as shown in Table 1.

**Figure 1.** Frequency-dependent dielectric constant (ε') and loss factor (ε") of the pecan kernels at four temperatures and moisture contents of 10% and 30%.

#### *3.2. Moisture- and Temperature-Dependent DPs*

The 3D plots of DPs at 27, 40, 915, and 2450 MHz are presented for pecan kernels with a moisture content range of 10−30% wb and a temperature range of 5–65 ◦C in Figures 2 and 3. Overall, both the ε and ε ' values of the pecan kernel samples in the RF range were significantly larger than those in the MW frequency range. At a certain frequency, an increase in the temperature and moisture content resulted in significant increases in ε and ε", although the values increased more in the high temperature and moisture content range. For instance, at 27 MHz, when the moisture content increased from 10% to 30% wb, ε increased from 3.08 to 5.51 at 5 ◦C, from 6.67 to 18.01 at 45 ◦C, and from 8.09 to 20.39 at 65 ◦C. For the same moisture content and frequency, ε" increased from 0.37 to 1.05 at 5 ◦C, from 5.15 to 13.06 at 45 ◦C, and from 7.85 to 25.86 at 65 ◦C. Compared with the values of the DPs in the RF range, the DPs at 980 and 2450 MHz (MW frequency) had much lower values for the same moisture and temperature levels. The same tendency has been observed at a given frequency in pistachio and peanut kernels [23,24], where the DPs at a high moisture content and temperature had significantly larger values than those at low moisture and temperature.

**Figure 2.** Three-dimensional representation of the dielectric constants of the pecan kernel as functions of the moisture content and temperature at frequencies of (**a**) 27, (**b**) 40, (**c**) 915, and (**d**) 2450 MHz.

**Figure 3.** Dielectric loss factors of the pecan kernel samples as functions of the moisture content and temperature at frequencies of (**a**) 27, (**b**) 40, (**c**) 915, and (**d**) 2450 MHz over a moisture content range of 10–30% wb and a temperature range of 5–65 ◦C.

The pecan kernel samples included a mixture of constituents with different dielectric behaviors and DPs. Due to the polar nature and solvent effect of water molecules, the DP values (ε , 80.4) were much larger than those of other matter; thus, the water molecule was the main dipole that was most responsible for the dielectric heating of materials. The water in the nut samples was mostly present in the form of free and bound water, in which the dipole polarization aroused by free water was considerably larger than that caused by bound water. In low-moisture nut kernels, most water molecules exist in the state of bound water in combination with proteins or carbohydrates [23]. Therefore, ε and ε" were very low, irrespective of the temperature and frequency. As the moisture content increased from 10% to 30% wb in the sample, the amount of free water, the ionic conduction, and the bulk density increased. All of these parameters contributed to the increase in the values of the DPs [32]. As displayed in Figure 3, the ε and ε" values of the pecan kernels in all RF and MW bands increased more remarkably with an increase in the moisture content at 65 ◦C than at 25 ◦C. The increasing temperature caused an increase in the thermal motion of the molecules and a decrease in the viscosity of the heated material [17]. Thus, the ionic conductivity increased.

In a batch RF or MW drying process of pecan kernels, the increase in ε" with an increased moisture content may lead to a potentially beneficial phenomenon, which is commonly referred to as the "moisture leveling effect" [30]. The high-moisture areas in the pecan samples could absorb increased electromagnetic energy and be heated preferentially. Thus, a faster heating rate and higher temperature would result in the high-moisture areas than in the low-moisture areas. Consequently, more water vaporization occurred in the high-moisture areas than in other areas, which finally led to a relatively uniform moisture content in the dried product. Conversely, the increase in ε" with increasing temperature may lead to a situation called "thermal runaway" when drying through dielectric heating [22,33]. Areas with high temperature have a large ε" value, which results in dielectric heating, which in turn causes more hot spots. This run-away phenomenon may lead to an increased vapor pressure gradient that promotes water migration from the inner part of the kernel to the surface of the sample. However, a very high temperature would adversely affect product quality. To avoid a very high temperature in dielectric heating, effective approaches should be taken to maintain a balance between the electromagnetic field input and energy output, such as surface air flowing in association with internal dielectric heating.

#### *3.3. Regression Models for the DPs of Pecans*

The polynomial regression models simulating the relationship between DPs of the pecan nuts and the temperature and moisture contents at frequencies of 27, 40, 915, and 2450 MHz are presented in Tables 3 and 4. Quadratic polynomial regression equations are the most suitable option for associating DPs with the temperature and moisture content. An analysis of variance was performed to test whether the temperature and moisture content had significant influences on the polynomial regression models (Tables 5 and 6). The linear term and interaction terms of Moisture content (M) and Temperature (T) had significant effects on the models (*p* < 0.05). All the models exhibited a good fit with the data at a significance level of *p* < 0.0001, with a coefficient of determination (R) higher than 0.9819. These results indicate that the polynomial models could be used to precisely predict the ε and ε" values of the pecan kernels in a known moisture content range of 10–30% wb, a temperature range of 5–65 ◦C, and four specific frequencies.


**Table 3.** Regression equations for the dielectric constants of the pecan kernels as functions of the moisture content (10% ≤ M ≤ 30%) and temperature (5 ◦C ≤ T ≤ 65 ◦C) at specific frequencies.

**Table 4.** Regression equations for the dielectric loss factors of the pecan kernels as functions of the moisture content (10% ≤ M ≤ 30%) and temperature (5 ◦C ≤ T ≤ 65 ◦C) at specific frequencies.


**Table 5.** Significance of the probability of the regressed models in Equations (2)–(5) for the pecan kernel samples at four specific frequencies.


M, Moisture content; T, Temperature.

**Table 6.** Significance of the regressed models in Equations (6)–(9) for the pecan kernel samples at four specific frequencies.


#### *3.4. E*ff*ect of the Salt Levels on DPs*

The DPs of the pecan kernels with different salt levels were measured at frequencies of 27, 40, 915, and 2450 MHz in the temperature range from 5 to 65 ◦C, as listed in Table 7. For a certain temperature, ε did not exhibit an obvious change with increasing salt concentration, whereas ε" increased significantly with increasing salt level, especially in the RF band. The nonsignificant effect of salt on ε is in accordance with the observations presented by other researchers for various food materials [24,34,35]. The nonsignificant effect may be because the added salts can decrease the water activity and diminish the polarization characteristics [25]. In this research, the addition of an electrolyte

(NaCl) did not considerably affect ε ; however, the addition did have a marked effect on ε". The addition of salt to the material could trigger large-scale electrophoretic migration when placed in an electric field, which would promote an increase in ε" by ionic conduction. The dielectric response of salts is closely connected with the effective nuclear charge and depends on the volume and charge of dissolved salt ions [25]. Ionic conduction mainly occurs in the RF range, and dipole rotation is the main functional mechanism of dielectric heating in the MW range [36]. The addition of salt addition provides an increased contribution to the development of ionic conduction in the RF range; thus, the increase in ε" was higher at 27 and 40 MHz than at 915 and 2450 MHz; for instance, when the salt concentration increased from non-salted to strongly salted at 25 ◦C, ε" increased by 627% at 27 MHz and 390% at 915 MHz. Furthermore, significant increases in ε and ε" were observed over the temperature range of 5 to 65 ◦C for both the salt-enriched and non-salted samples at four frequencies. For samples with salt, the ionic loss from electrophoretic migration increased with temperature. The variation of DPs in terms of the temperature and frequency was similar for both the salted and non-salted pecan kernel samples. The addition of salt significantly increased the values of ε", which indicates the need to develop a separate drying scheme for salted and non-salted pecan nuts.


**Table 7.** Dielectric properties of pecan nuts with different salt contents (moisture content: 15%).

#### *3.5. Penetration Depth*

The power dp computed from the measured ε and ε" of the pecan kernels at four frequencies, four temperatures, five moisture contents, and three salt levels is listed in Tables 8 and 9. The dp in the RF range was considerably higher than that in the MW range, and the dp decreased with increasing temperature and moisture content. For instance, the dp at 27 MHz was distributed from 1339.26 to 31.89 cm for the pecan kernels depending on the kernel moisture content and temperature, whereas the dp at 915 MHz was distributed from 47.29 to 7.15 cm. As the moisture content of the pecan samples increased from 10% to 30% wb, the dp decreased from 707.56 to 94.3 cm at 45 ◦C and 27 MHz. As the temperature rose from 5 to 65 ◦C, the dp decreased from 382.88 to 58.94 cm at 40 MHz and a moisture content of 20%. For salt-added pecan samples with a moisture content of 15%, the dp decreased with increasing salt level. These results agree with those of previous reports on peanuts [23] and pistachio kernels [24].


**Table 8.** Electromagnetic energy penetration depth for pecan nuts with different moisture contents.

**Table 9.** Electromagnetic energy penetration depth for pecan nuts with different salt contents.


The relatively shallow penetration depth at MW frequencies indicated that MW energy only penetrates into the shallow layers of pecan kernels, which results in considerable surface heating. The

high penetration depths in the RF range led to a relatively uniform distribution of the electromagnetic field in the bulk mass of pecan kernels. Thus, the uniformity of heating was improved, which is one of the most important advantages of RF heating compared with MW drying [37]. The actual stacked thickness in a batch drying process of pecan nuts should be lower than 27 and 5 cm at 27 and 2450 MHz, respectively.

#### *3.6. Comparison of Simulated and Experimental Heating Rates of Pecan Kernels*

Figure 4 shows a comparison between the experimental and simulated temperature-time profiles of pecan kernels with five moisture contents of 10%, 15%, 20%, 25%, and 30% during 5 min RF heating with an electrode gap of 150 mm. It can be seen for all the tested samples that the simulation results using measured DP values accorded with experimental test temperature profiles at five moisture levels. Within the 5 min RF heating period, all heating curves were relatively linear, and the heating rates of pecan kernels increased significantly (*p* < 0.05) with increasing moisture content from 10% to 25%. However, the heating rate then decreased as the concentration increased from 25% to 30%. The 25% sample had the highest heating rate (11.2 ◦C /min). Similar trends were also found by Zhang et al. [23] for the heating rate of peanut kernels, in which the increase in moisture content resulted in increasing ε", which in turn caused an initial increase and then a decrease in RF heating rates. Jiao et al. [38] reported that the maximum heating rate was reached when the values of ε and ε" were close to one another.

**Figure 4.** Experimental and simulated temperature-time histories of pecan kernels with a moisture content from 10% to 30% wb when subjected to RF heating for 5 min with an electrode gap of 150 mm.

#### **4. Conclusions**

The DP values of pecan kernels decreased with increasing frequency and increased with increasing water content and temperature. The ε and ε" values of the pecan kernels decreased substantially with increasing frequency in the measured RF range, whereas the values decreased gradually in the measured MW range. The addition of salt led to marginal increases in ε but sharp increases in ε", especially in the RF range. At four specific frequencies, the relationship between the DPs and the temperature and moisture content could be quantitatively described with a battery of quadratic polynomial equations, which could be adopted to precisely predict the ε and ε" values from the temperature and moisture content. The penetration depths of electromagnetic power in the pecan kernel decreased sharply with increasing frequency, temperature, and moisture content. Consequently, RF dielectric heating could provide relatively more uniform heating. Thus, RF dielectric heating is especially suited for large-scale treatments.

**Author Contributions:** Conceptualization, J.Z. and M.L.; methodology, M.L.; software, J.C.; validation, J.Z., J.C., and J.W.; formal analysis, Z.D.; investigation, J.Z.; resources, S.Z., B.L. and C.Y.; data curation, X.Y.; writing—original draft preparation, J.Z.; writing—review and editing, J.Z.; visualization, J.Z.; supervision, J.Z.; project administration, X.L.; funding acquisition, X.L.

**Funding:** The work was supported by the Fundamental Research Funds for the Central Non-Profit Scientific Institution (1610232019006) and Natural Science Foundation of Anhui Province (1908085MC70) and Key Projects of China National Tobacco Corp Sichuan Company (SCYC201703 and SCYC201806 and SCYC2018-2).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **Development of Bacterial Spore Pouches as a Tool to Evaluate the Sterilization E**ffi**ciency—A Case Study with Microwave Sterilization Using** *Clostridium sporogenes* **and** *Geobacillus stearothermophilus*

**Aswathi Soni 1,\*, Jeremy Smith 2, Richard Archer 2, Amanda Gardner 1, Kris Tong <sup>2</sup> and Gale Brightwell 1,3**


Received: 31 August 2020; Accepted: 20 September 2020; Published: 23 September 2020

**Abstract:** In this study, novel spore pouches were developed using mashed potato as a food model inoculated with either *Geobacillus stearothermophilus* or *Clostridium sporogenes* spores. These spore pouches were used to evaluate the sterilization efficiency of Coaxially induced microwave pasteurization and sterilization (CiMPAS) as a case study. CiMPAS technology combines microwave energy (915 MHz) along with hot water immersion to sterilize food in polymeric packages. The spore pouches were placed at pre-determined specific locations, especially cold spots in each food tray before being processed using two regimes (R-121 and R-65), which consisted of 121 ◦C and 65 ◦C at 12 and 22 kW, respectively, followed by recovery and enumeration of the surviving spores. To identify cold spots or the location for inoculation, mashed potato was spiked with Maillard precursors and processed through CiMPAS, followed by measurement of lightness values (\**L*-values). Inactivation equivalent to of 1–2 Log CFU/g and >6 Log CFU/g for *Geobacillus stearothermophilus* and *Clostridium sporogenes* spores, respectively was obtained on the cold spots using R-121, which comprised of a total processing time of 64.2 min. Whereas, inactivation of <1 and 2–3 Log CFU/g for *G. stearothermophilus* and *C. sporogenes* spores, respectively on the cold spots was obtained using R-65 (total processing time of 68.3 min), whereas inactivation of 1–3 Log CFU/g of *C. sporogenes* spores was obtained on the sides of the tray. The results were reproducible across three processing replicates for each regime and inactivation at the specific locations were clearly distinguishable. The study indicated a strong potential to use spore pouches as a tool for validation studies of microwave-induced sterilization.

**Keywords:** microwave; sterilization; *Geobacillus*; *Clostridium*; spores; inactivation; thermal resistance; Maillard reaction

#### **1. Introduction**

Any novel sterilization technology brings along the necessity to develop methods to ensure that the coldest regions inside the food would receive enough treatment to achieve the inactivation of microorganisms (mesophiles and thermophiles) to ensure food safety. Coaxially induced microwave pasteurization and sterilization (CiMPAS) is an emerging thermal technology that combines hot water immersion and microwave energy (915 MHz) to achieve sterilization in a shorter time as compared to the conventional technologies [1,2]. Microwave sterilization technology, originally developed at Washington State University [3], has been validated and accepted by the Food and Drug Administration

(FDA) as a thermal sterilization for producing pre-packaged, low-acid foods [2]. CiMPAS model consists of a hot water tank, a warm water tank a process vessel (with microwave outlets) and an electric cabinet. The operation can be divided into four major steps namely, preheat, hot water treatment, holding and cooling. CiMPAS technique can be controlled using various factors including the power of the microwave, time of exposure and the cooling mechanisms. Microwave sterilization uses less processing time as compared to conventional retorting and thereby the exposure of the nutrients in food to high temperatures is reduced to allow better nutrient retention [1,2,4]. However, a challenge with microwave sterilization is the non-uniform heating that could lead to the formation of cold spots in the processed products [5,6]. The presence of cold spots or regions that have been less thermally processed might result in incomplete bacterial inactivation. Hot or cold spots may also lead to uneven cooking, consequent undesirable sensory properties and nutrient losses.

Cold spots in foods processed using microwave-induced sterilization technology have been identified and reported using chemical markers [7,8] and temperature probes in previous studies [7,9]. Chemical markers, for example, products of Maillard reaction can serve as local time-temperature integrators. Maillard reaction involves a reducing sugar (e.g., ribose) that condenses with a compound possessing a free amino group (e.g., amino acid) to give a product (N-substituted glucosamine) that gets further arranged to form an Amadori rearrangement product (ARP) followed by an array of chemical reactions leading to the formation of compounds that impart the brown color [10]. One of these products is the chemical marker M-2 (4-hydroxy-5-methyl-3(2H)-furanone), which has been reported as an effective tool to monitor heating patterns of foods after microwave sterilization [10]. However, until now, the microbial inactivation on these colder spots have not been investigated or reported. Several microbial inactivation assays are required to ensure reproducibility and reliability [11,12]. A common method to test sterilization regimes is the use of spore strips [13,14] Several microbial inactivation assays are required to ensure reproducibility and reliability [11,12]. A common method to test sterilization regimes is the use of spore strips [13,14] that can be placed inside the sterilization chamber. Though this has been used an effective way to monitor sterilization, it only indicates whether the pre-determined spore numbers were either completely inactivated or not, hence either indicates presence or absence of spores, but the surviving spores cannot be enumerated. Also, they cannot be directly used for challenge testing in food for localized inoculation and recovery. Thermal processing efficiency may also be monitored by the conventional way of inoculating whole trays of food with specific strains of bacterial spores and subsequently measuring Log reductions. However, it is not possible to recover these spores from unique locations post treatment within the bulk food and hence the inactivation efficiency cannot be related to back to the spatial distribution of the cold spots.

To address this research gap, the current study investigated the use of spore pouches that were developed using food model inoculated with bacterial spores packed in microwavable Cryovac BNB1 pouch (15 mm2) (Cryovac, Hamilton, New Zealand). These pouches could be placed at specific target locations within the packaging trays filled with homogeneous food, followed by microwave sterilization to recover and enumerate the spores that survive treatment. For the formulation of spore pouches, two different typed strains of bacterial spores were used; *Geobacillus stearothermophilus* ATCC 12980 and *Clostridium sporogenes* spores NZRM 3052. The selection of two different spores was based on the significant difference in their thermal resistance as per the previously reported *D* values, which were also further confirmed in the current study. Decimal reduction time or *D*-value is defined as the time required at any specific temperature to achieve inactivation equivalent to 1 Log CFU/mL of a specific bacterial population [15]. *D* values at 121 ◦C for *C. sporogenes* have been reported as 0.5 and 0.6 min in phosphate buffer (pH 7.0) and carrot juice, respectively [16]. *G. stearothermophilus* spores have been reported to have D values of up to 5.4 min at 121 ◦C in yeast extract media [17]. The thermal resistance of these spores would also depend on their sporulation conditions and the medium in which the inactivation takes place. Hence, spores were inoculated in mashed potato in the current study instead of a diluent or buffer while being processed in CiMPAS to keep any specific effect on the thermal resistance in consideration while interpreting the results. The main objective was to test the

possibility of using spore pouches to evaluate sterilization efficiency of CiMPAS using inactivation of spores at specific regions (cold spots) inside food packaging trays. The method used to identify cold spots for spore inoculation was by comparing the difference in browning using the Lightness (L) values, which indicate the formation of a chemical marker (M2) that is one of the products of Maillard browning. As a case study, the CiMPAS system was operated in a manner to amplify inconsistencies within and between trays.

#### **2. Materials and Methods**

#### *2.1. Preparation of Spores*

*G. stearothermophilus* ATCC 12980 spores were produced by a method previously described by Sadiq et al. [18] with a slight modification. Briefly, an overnight culture was grown in tryptic soy broth (TSB) at 60 ◦C for 24 h followed by spread plating 200 μL of the overnight culture onto the sporulation agar plates. The sporulation agar plates (final pH 7.0) comprised of nutrient agar (NA; Difco) (13 g/L), MgSO4·7H2O, (0.51 g/L), KCl, (0.97 g/L), CaCl2·2H2O, (0.2 g/L); MnSO4·H2O, (0.003 g/L), FeSO4·7H2O, (0.55 mg/L) and additional agar (1.5 g/L). The inoculated plates were incubated for 14 days at 60 ◦C followed by harvesting using cold sterile water (3 mL) by scraping the entire growth surface using sterile L-shaped disposable plastic spreaders. The spores were harvested by centrifugation (8000× *g*, 10 min, 4 ◦C) and washed three times with autoclaved pre-cooled distilled water. The purified spore stock suspended in distilled water was then stored at 4 ◦C for up to a maximum of 7 days until used.

*C. sporogenes* NZRM 3052 spores were cultured and in the same way [18] with a few modifications. An overnight culture was grown in Fluid Thioglycolate (FTG) media (Fort Richards, Auckland New Zealand) at 35 ◦C for 24 h in an anaerobic chamber followed by spread plating 200 μL on to tryptic sheep blood agar (SBA) plates (Fort Richards, New Zealand). The plates were incubated in an inverted position at 37 ◦C for 7 days in anaerobic chambers with anaerobic environment generator packs (BD GasPak™ EZ pouch systems, Fort Richards, Auckland New Zealand) and an indicator strip (BBL™ GasPak™ Anaerobic Indicator Strip, Dry, Fort Richards, New Zealand). The colonies on the surface of the agar were then scraped using the L-shaped spreader with cold sterile water (3 mL) to remove the sticky portions. The slurry was then washed three times by centrifugation (8000× *g*, 10 min, 4 ◦C) using distilled water. The spore suspension was stored at 4 ◦C in an anaerobic chamber until used.

#### *2.2. Product*/*Food Model Formulation*

Mashed potato (food model) was prepared as previously reported by Soni et al. [8]. In short, to prepare 1000 g of mashed potato (food model), agar (Fort Richards, Auckland, New Zealand) (5 g) was added to boiling water (830 g) and mixed using a cake mixer at medium speed for 2 min followed by addition of potato flakes (150 g) while mixing continuously to avoid lumps. The mix was cooled to 60 ◦C, followed by addition of D-ribose (10 g) and lysine (5 g) and mixed for another 2 min. Ribose and lysine (1 and 0.5%, respectively) have been reported to show formation of brown color with increasing time in the presence of heat, which can be measured by colorimetry [8,19]. This final composition of food model was left to cool in the room temperature for 10 min and then filled into packaging trays (174 × 103 × 35 mm) to reach a total weight of 250 g, while excluding the weight of the tray (20 g). Trays were then placed in microwavable Cryovac BNB1 pouches and sealed (23 MPa, 2 s) in a Multivac C200 vacuum sealer and used for CiMPAS processing.

#### *2.3. CiMPAS Processing*

CiMPAS system (Coaxially induced microwave-pasteurization and -sterilization) as previously described by Soni et al. [8] was manufactured by Meyer Burger Germany GmbH (Hohenstein-Ernstthal, Germany) and the industrial microwave parts were manufactured by MUEGGE GmbH (Reichelsheim, Germany). CiMPAS equipment used in the current study consists of a hot water tank, a warm water tank a process vessel (with microwave outlets) and an electric cabinet. The operation can be divided

into four major steps namely, preheat, hot water treatment, holding and cooling. The sealed trays with mashed potato were placed in the CiMPAS carrier tray made up of polyether ether ketone (660 × 560 × 45 mm) which was then placed in the processing vessel (Figure 1) (filled with warm water with a conductivity of 8.4 uS/cm) for processing. A schematic representation of mashed potato food model in packaging trays placed in the carrier tray in the processing vessel is shown in Figure 1.

**Figure 1.** Schematic representation showing the mashed potato food model packed in the packaging trays and arranged in carrier tray while kept immersed in hot/warm water inside the pressure vessel in Coaxially induced microwave pasteurization and sterilization (CiMPAS).

For CiMPAS processing, two processing regimes R-121 and R-65 were chosen to determine if spore pouches were able to indicate the difference in potential inactivation when processed through two different temperatures, microwave power and, hence processing times. CiMPAS regimes R-121 and R-65 used hot water at 121 ◦C and 65 ◦C to simulate sterilization and pasteurization, respectively. The carrier tray used here consists of 12 slots for packaging trays as shown in Figure 1. The detailed steps in the processing regime R-121 are explained in Table 1. For R121, following the preheating step, hot water (121 ◦C) was flushed into the vessel, microwave power was switched on at 12 kW, and the carrier tray was moved back and forth through the antennae as seen in Figure 1 for 250 s (Table 1) and the total processing time was 64.2 min.


**Table 1.** The processing steps for R121.

Note: The temperature of the hot and warm water vessel was set at 121 and 30 ◦C, respectively. na = not applicable.

The detailed steps in the processing regime R-65 is explained in Table 2. For R-65 with hot water at 65 ◦C was flushed into the vessel, microwave power was switched on at 22 kW and the carrier tray was moved back and forth for 500 s (Table 2) and the total processing time was 68.3 min.


**Table 2.** The processing steps for R-65.

Note: The temperature of the hot and warm water vessel was set at 65 and 30 ◦C, respectively; na = not applicable.

CiMPAS regimes namely R-121 and R-65 could each only accommodate one carrier tray consisting of 12 packaging trays at a time (Figure 1). For both the regimes, as the final step cooling water (30 ◦C) was flushed into the vessel to cool the product. Processed packaging trays were removed from the carrier tray and placed into the chiller (4 ◦C) overnight before analysis. Samples were collected from three processing runs conducted on three different days separately for colorimetry and challenge testing. Controls were exposed to similar storage conditions except CiMPAS processing. Each processing run consisted of 12 samples and one control. The composition of mashed potato was not different in control; however, controls were not processed through CiMPAS and hence were untreated but were maintained at similar storage conditions along with samples for a direct comparison. The use of 12 trays for each processing run was entirely due to the machine set up where one carrier tray (Figure 1) consists of 12 slots, and hence to understand the spatial distribution of the processing effect, all the 12 slots were utilized.

#### *2.4. Identification of Cold Spots for Inoculation by Colorimetric Analysis and High-Pressure Liquid Chromatography (HPLC) Analysis of Chemical Marker 4-hydroxy-5-methyl-3(2H)-furanone (M2)*

After processing using CiMPAS, each tray was divided into nine different locations on the surface as previously described [8]. The lightness (\**L* values) were recorded using a Minolta CR20 colorimeter (Minolta Camera Co., Osaka, Japan) and using \*Lab (CIELAB) space as previously reported [19,20]. The coldest spot on each tray was identified as the location with the significantly highest *L* values

(*p* < 0.05) as the *L*-values reduce significantly as an effect of the increase in time when subjected to thermal treatment [8]. The increase in the brown color formation and hence the decrease in \**L* values has been previously validated using a kinetic study using oil bath set up at 121 ◦C [8]. To further verify the concentration of the chemical marker M2 at the cold spot, high-pressure liquid chromatography was used as previously described [9]. Mashed potato (food model) samples (1 g) was scooped out from the apparent cold and hot spots and were carefully ground using a mortar and pestle with an extraction buffer (10 mM sulphuric acid and 5 mM citric acid). The extracts were collected and stored overnight in a freezer (−18 ◦C), then thawed at room temperature before being centrifuged for 10 min at 10,897× *g*. The supernatants were collected and centrifuged again twice more to remove any debris, followed by filtration using a PTFE syringe filter (0.2 μm pore size) before being analyzed by HPLC. An Agilent 1100 HPLC system (Agilent Technology, Santa Clara, CA, USA) with diode array detector and an acid-fast analysis column (Bio-Rad Laboratories, Hercules, CA, USA) was used with a mobile phase flow rate of 1 mL/min. Absorbance was determined at 285 nm and a calibration curve was developed using the commercially available standard of M2 (Sigma, Castle Hill, NSW, Australia) with a concentration range of 0.0–1.4 mg/mL to interpolate the unknown concentrations of M2 in mashed potato (food model) extracts.

#### *2.5. Estimation of Thermal Resistance at 121* ◦*C (D Values) of C. sporogenes and G. stearothermophilus Spores*

#### 2.5.1. Estimation of Decimal Reduction Time for *C. sporogenes* and *G. stearothermophilus* Spores in Milli-Q Water

An aliquot (50 μL) of the spore suspension in Milli-Q water was added to glass capillaries (diameter of 1.8 mm, length 70 mm), which were heat-sealed and immersed in Digital High-Temperature Oil Bath (Interlab, Wellington, New Zealand) pre-set at 121 ◦C as per the method described by Soni et al. [8]. The capillary tubes were removed from the oil bath at regular interval points (0, 2, 4, 6, 8 and 10 min) and immediately transferred to an ice slurry to stop the thermal inactivation. The tubes were washed once with a 90% ethanol solution and then twice with autoclaved distilled water before breaking the capillary tubes and transferring their contents into 0.1% peptone solution (*w*/*v*) for serial dilution. The number of spores present was determined by serial diluting the sample in 0.1% peptone and plating it onto sheep blood agar plates in triplicates. The trial was carried out in three experimental and three technical replicates. For *C. sporogenes* spores, capillary tubes were removed at a time interval of 1 min starting from 0 to 6 min whereas, for *G. stearothermophilus* spores, the time points were at an interval of 2 min ranging from 0 to 10 min based on the difference in thermal resistance. The inoculated plates were incubated and enumerated as described in Section 2.1.

#### 2.5.2. Estimation of Decimal Reduction Time for *C. sporogenes* and *G. stearothermophilus* Spores in Mashed Potato

*C. sporogenes* and *G. stearothermophilus* spores were separately inoculated in mashed potato to achieve a final inoculum of ~10<sup>7</sup> and 10<sup>6</sup> CFU/g respectively. A digital high-temperature oil bath (Interlab, Wellington, New Zealand) was set at 121 ◦C. The inoculated mashed potato (10 g) was filled in oil bath capsules that were previously reported [8] and sealed followed by immersion for incubation in the oil bath (121 ◦C) while making sure that there was no dripping or leakage from the capsules. The come-up time for mashed potato was 4 min and capsules were then removed at specific time intervals and transferred into ice-slurry for cooling down. For *C. sporogenes* spores, the capsules were removed at a time interval of 2 min starting from 0 to 10 min whereas, for *G. stearothermophilus* spores, the time points were at an interval of 4 min ranging from 0 to 16 min based on the difference in thermal resistance. Three experimental replicates were conducted under the same setup once cooled down, the surviving *G. stearothermophilus* spores in each sample were recovered by serially diluting and plating onto tryptic soy agar (TSA) plates. The plates were incubated in an inverted position at 60 ◦C for 48 h before the colonies on each plate were enumerated. *C. sporogenes* spores were serially diluted and

plated onto tryptic SBA plates for enumeration. The plates were incubated in an inverted position at 37 ◦C for 48 h in anaerobic chambers (Section 2.1) followed by enumeration.

#### *2.6. Inoculation of Bacterial Spores in the Food Model*

For the challenge testing, *G. stearothermophilus* spores were inoculated using two methods: spot inoculation (using pouches) and whole tray inoculation and *C. sporogenes* spores were tested only using spot inoculation using spore pouches.

#### 2.6.1. Spot Inoculation of *G. stearothermophilus* Spores in Pouches

The original microwavable Cryovac BNB1 pouch (70 Micron, 150 mm × 200 mm) was cut into square-shaped pieces (15 mm2) and heat-sealed (HI Impulse Handsealer, Makmar, Auckland, New Zealand) with a maximum sealing thickness of 0.15 mm and a seal width of 2 mm with a sealing time of 2 s. Three sides of the pouch were sealed followed by placing 1 g of mashed potato (food model) inoculated with *G. stearothermophilus* spores (10<sup>6</sup> CFU/g) followed by sealing the fourth side (Figure 2).

**Figure 2.** Microwavable pouch (Cryovac BNB1, Barrier Bag–70 Micron, 150 mm × 200 mm packages) containing mashed potato (1 g) inoculated with *G. stearothermophilus* spores followed by sealing on the four ends using HI Impulse Handsealer (Makmar, Auckland, New Zealand) with a maximum sealing thickness of 0.15 mm and a seal width of 2 mm and a sealing time of 2 s.

These spore pouches were then placed in the desired locations inside the packaging tray already filled with mashed potato food model for challenge testing. The target locations for the inoculation were the cold spot on each tray that were pre-determined in Section 2.4. For each tray, the cold spot was separately identified as the spot with significantly higher \**L* values (*p* < 0.05) after three CiMPAS processing runs.

#### 2.6.2. Inoculation of *G. stearothermophilus* Spores in the Whole Tray of Mashed Potato (Food Model)

For the homogeneous inoculation, 250 g of mashed potato (food model) was inoculated with 1.5 mL of *G. stearothermophilus* spores (106 CFU/g), mixed for 2 s using a stomacher machine (Seward, Inc., London, England) followed by evenly spreading on to processing trays followed by packing and sealing as described in Section 2.2. For both pouch and whole tray inoculation using *G. stearothermophilus* spores, three processing replicates, were included, and three technical replicates used while plating each dilution. The processing replicates refer to three individual CiMPAS processing runs for the same regime that was conducted on separate days. Technical replicates refer to the use of three plates for spread plating and recovery for each dilution in each sample being analysed. The same replicate scheme was employed for the three studies.

#### 2.6.3. Spot Inoculation of *C. sporogenes* Spores

For the spot inoculation, *C. sporogenes* spores were inoculated in mashed potato and packed in pouches to achieve an inoculum level of 10<sup>7</sup> CFU/g as described in Section 2.6.1. The spore pouches inoculated with *C. sporogenes* spores were used for spot inoculation in three separate trials. Firstly, R-65 was used to identify the cold spot (highest \**L* value), medium heated spot (intermediate \**L* value) and hot spot (lowest \**L* value) on each of the 12 trays. In the next trial, one spore pouch was placed each at the hot, cold and medium heated spot inside the packaging tray filled with mashed potato (food model) followed by CiMPAS processing by R-121 (Table 1), which took of a total time of 64.2 min. For the second study, an inoculated pouch was placed on the coldest location (on each of the 12 packaging trays) pre-determined by *L*-values (after R-65 in a previous run) followed by CiMPAS processing by R-65 (Table 2), which took of a total time of 68.3 min. For the third study, four pouches inoculated with *C. sporogenes* spores were placed vertically on the four sides/walls of each tray, followed by filling with mashed potato (food model) to obtain a total weight of 250 g before being sealed as described in Section 2.2 and processed via R-65. For each of these three studies, three processing replicates, were included, and three technical replicates used while plating each dilution as described in 2.6.2 and the same replicate scheme was employed for the three studies.

Whole tray inoculation trials were not conducted with *C. sporogenes* spores as ~7 Log reduction in spore pouches were achieved using the preliminary trials and with the whole tray studies, the inactivation would go below detection limit of 1 CFU/mL.

In summary, for challenge testing, *G. stearothermophilus* spores were subjected to both pouch and whole tray inoculation and were processed through R-121. *C. sporogenes* spores were inoculated only in spore pouches and subjected to microwave sterilization via R-65 and R-121.

#### *2.7. Enumeration of Surviving Spores*

To enumerate the surviving *G. stearothermophilus* spores in the pouches, each pouch was washed thrice in autoclaved distilled water to remove any residual food sticking on the surface followed by cutting on an edge using a sterile knife to empty the contents into a universal tube with peptone solution (0.1%, 9 mL) (Fort Richards, Auckland, New Zealand). To enumerate the spores surviving in mashed potato (food model) trays that were homogenously inoculated, the contents were first inverted into a sterile stomacher bag (1000 mL) and a sterile L-spreader was used to scrape off any leftover mashed potato (food model) for complete recovery. In both cases, the samples were mixed using a stomacher machine (Seward, Inc., London, England) for 2 s followed by serially diluting (10−<sup>0</sup> to 10<sup>−</sup>4) and plating onto tryptic soy agar (TSA) plates. The plates were incubated in an inverted position at 60 ◦C for 48 h before the colonies on each plate were enumerated.

*C. sporogenes* spores were recovered using the same method where any food sticking on the surface each pouch was washed thrice in autoclaved distilled water to remove any residues followed by cutting on an edge using a sterile knife to empty the contents into a universal tube with peptone solution (0.1%, 9 mL) (Fort Richards, Auckland, New Zealand) followed by serially diluting and plating onto tryptic SBA plates for enumeration. The plates were incubated in an inverted position at 37 ◦C for 48 h in anaerobic chambers (Section 2.1) followed by enumeration.

#### *2.8. Statistical Analysis*

Three processing replicates were used for the \**L*-value measurements to determine the coldest spot for inoculation in subsequent trials. Once the cold spots were determined, three processing replicates were included for the inoculation studies using both strains of spores and three technical replicates were used for plating each sample. The significant differences among the *L* values and the spore numbers were analyzed using one-way ANOVA followed by post hoc analysis using Tukey's test (Minitab, version 19). Microsoft Excel was used to compute the average and standard deviation for graphical and tabular representation.

#### **3. Results and Discussion**

#### *3.1. Determination of Cold Spots for Microbial Innoculation*

The cold spots in this study were determined using the difference in browning as a result of Maillard reaction as an indicator of time-temperature exposure [21]. Maillard reaction involves a reducing sugar (e.g., ribose) that condenses with a compound possessing a free amino group (e.g., amino acid) to give a series of reactions and products, that impart the brown color as a result of one of the products called M2. Chemical marker M2 has been reported as an effective tool to monitor heating patterns of foods in microwave sterilization [21].

The mashed potato (food model) showed a visual difference in the extent of browning as expected. Lightness (\**L*) values were found to be the most appropriate method to identify the difference heat exposure acquired by the surface of mashed potato (food model) as also previously reported [21]. The identification of cold spots was done on the surface of the mashed potato (food model) on each tray which was divided into nine spots for the measurement of \**L* values [8]. The coldest spot on each tray was determined by analysing the results of three processing runs. Cold spots detected were different for the two types of processing regimes at R-65 (Table 3a) and R-121 (Table 3b). Processing using hot water at 121 ◦C showed uniform browning across the nine regions in each tray (*p* > 0.05) (Table 3b).

These results were in agreement with a previous study where the change in chemical marker (M2) as measured indirectly by \**L* values had shown temperature sensitivity that fits the Arrhenius relationship, which is a commonly used model to simulate the impact of temperature change on the reaction rate constants [21]. At the same time, Bornhorst et al. [21] also showed that the change in color or browning reached saturation after 100 ◦C due to a rapid rate of color formation. Similar uniform browning was observed with the current study with R-121 and hence, the cold spots were determined using a regime R-65 where the temperature of hot water was 65 ◦C. There was no significant difference across the nine spots measured on the control tray. However, the difference in \**L* values (9 spots/tray) post-processing in all the other trays (1–12) was due to Maillard reaction end products whose formation and concentration is affected by thermal exposure. Though the \**L* values among the 9 spots after R-121 on each tray were not significantly different (*p* > 0.05) from each other, the regions showing the highest and lowest \**L* value on each tray were further sampled (1 g) to be analysed using HPLC for the key Maillard intermediate product M2. For example, for tray 1, spot 1 was the hot spot as it showed an \**L* value of 55.1 ± 9.9 whereas the spot 3 was taken as the cold spot as it showed an \**L* value of 50.4 ± 1.7. Similarly, from each tray the spot showing highest \**L* value was chosen as the cold spot and the spot showing lowest \*L value was taken as a hot spot. After HPLC analysis, the concentration of M2 was found to be significantly higher (*p* < 0.05) at the hot spot in each tray as compared to the cold spot (Figure 3b).



*Foods* **2020** , *9*, 1342

**Figure 3.** Concentration of M2 (μg/g of mashed potato) at the hot spot (grey bars) and the cold spots (black bars) determined on each tray (**a**) and visual representation of as an example of samples from the cold spot (**b**) and hot spot (**c**) scooped out from a mashed potato tray after CiMPAS processing at R-121. Similar letters among the bars indicate no significant difference (*p* > 0.05).

The accumulation of M2 was on the higher side on each hot spot as compared to the cold spot and hence it was concluded that verified that the change in browning (though not significant, *p* > 0.05) is a result of M2 formation and the lighter regions are still indicative of a cold spot for inoculation to check the inactivation locally. In the current study, the *L*-values were analysed on the surface of the tray and there is a possibility that the cold spots could be in the interior regions of the tray.

#### *3.2. Thermal Resistance of C. sporogenes and G. stearothermophilus Spores in Mashed Potato and Milli-Q Water*

Decimal reduction time or D value is the exposure time required to achieve the killing of 90% or 1 Log CFU/mL of the living population of microbes at a predefined and controlled temperature [22]. Graphically, the D value is the inverse of the slope of the curve fitting the plot of the log10 value of the number of living cells against time. The D121 ◦C values of *C. sporogenes* spores were found to be 3.4 and 1.0 min in mashed potato and 0.1% peptone water, respectively (Figure 4a). On the other hand, the D121 ◦C values were found to be 5.6 and 2.2 min in mashed potato and 0.1% peptone water, respectively (Figure 4b).

The thermal resistance as evaluated at 121 ◦C for *C. sporogenes*, as well as *G. stearothermophilus* spores, varied significantly when the medium of inoculation was different (*p* < 0.05). This agrees with previous work by researchers, which indicates that the resistance of bacterial spores can be different attributing to several conditions including the food composition [23–25]. The current results agree with these previous indicates the importance of conducting a challenge test in food systems for validation.

#### *3.3. E*ff*ect of CiMPAS on Inactivation of Spores*

#### 3.3.1. Inactivation of *G. stearothermophilus* Spores

*G. stearothermophilus* spores inoculated in pouches were placed in specific cold spots on each packaging tray as shown in Figure 5a for the spot inoculation (Figure 5b) and the inactivation was compared with the results from whole tray inoculation (Figure 5c).

**Figure 4.** D121 ◦C values of *C. sporogenes* (**a**) and *G. stearothermophilus* (**b**) spores in autoclaved Milli Q water (triangles) and mashed potato food model (dots).

All the trays showed inactivation within the detection limit of 2 Log CFU/g. The overall inactivation range for spores inside the spore pouches placed on the coldest spot on each tray (Figure 5a) ranged from 0.2 to 0.9 Log CFU/g (Figure 5b). On the other hand, the inactivation was equivalent to a range of 0.9 to 1.7 Log CFU/g when the whole trays with mashed potato (food model) were inoculated with *G. stearothermophilus* spores (Figure 5c). Tray 6 showed a significantly higher (1.7 Log CFU/g) inactivation of spores using the whole tray inoculation method as compared to all the other trays (Figure 5c). Using pouch inoculation, significant differences among the 12 trays could be detected (Figure 5b) whereas using whole tray inoculation, only tray 6 was different from the 11 other trays (Figure 5c). This further indicates that inoculation using pouches at specific locations might enable the detection of differences that the whole tray inoculation might not.

(**c**)

**Figure 5.** Inoculation map for spore pouches (*G. stearothermophilus*), where the set up represents a carrier tray with 12 packaging trays and the black squares shows the cold spot for pouch inoculation (**a**) *G. stearothermophilus* spores surviving post CiMPAS processing (R-121) using pouch inoculation (**b**) and whole tray inoculation (**c**) Different letters in each graph indicate a significant difference (*p* < 0.05); na = Not applicable for control.

Inactivation of *G. stearothermophilus* by microwave sterilization has not previously been reported. One of the potential reasons would be the high thermal resistance of these spores, however, in the current study, they were an effective indicator of the difference in inactivation attributing to the thermal exposure.

#### 3.3.2. Inactivation of *C. sporogenes* Spores by CiMPAS

The pouches containing mashed potato inoculated with *C. sporogenes* spores were placed on three putative locations (including cold, hot and a spot that showed intermediate \**L* value) on each of the 12 processing trays. More than 7 Log reduction of *C. sporogenes* spores with a detection limit of 10 CFU/g was achieved post CiMPAS at R-121 on each of these three spots (Table 4). Since the D121 ◦C values (or the time required for 1 Log reduction) of *C. sporogenes* spores could vary from 0.9–1.4 min, which indicates that to achieve inactivation equivalent to 7 log CFU/g, these spots would have been exposed to an equivalent temperature of 121 ◦C for 6.3–9.8 min. Enrichment analysis (data not shown) indicated the presence of low numbers of survivors (1–10 CFU/g) in each tray.

**Table 4.** Inactivation of *C. sporogenes* spores at three different locations on each tray after Coaxially induced microwave pasteurization and sterilization (CiMPAS) (R-121).


Note: nd = not detectable with the detection limit of 10 CFU/g.

Inactivation of *C. sporogenes* spores has been previously reported using microwave sterilization (915-MHz, 10-kW pilot-scale MW system developed at Washington State University) when inoculated in pre-treated sliced beef (heated in boiling water with 0.5% salt) in gravy in 7-oz trays, where about 8 Log reduction was observed with F0 of 6 and 3 Log reduction with an F0 of 3 [26]. Since *C. sporogenes* are less resistant as compared to *G. stearothermophilus* spores, they showed a much better level of inactivation (>7 Log CFU/g), which is one of the requisites of commercial sterilization. Though the inactivation was uniform (Table 4), to further see the actual difference between numbers, a different CiMPAS regime with hot water at 65 ◦C but an increased number of passes to 12 and an increased microwave power of 22 kW was used for processing mashed potato while inoculated with *C. sporogenes* spore pouches in the coldest location as shown in Figure 5a. CiMPAS using R-65 showed significantly lower inactivation of *C. sporogenes* spores as compared to R-121 as expected (Figure 6).

**Figure 6.** Loci for inoculation for spore pouches (*C. sporogenes*), where the set up represents a carrier tray with 12 packaging trays and the black squares shows the cold spot for pouch inoculation(a) *C. sporogenes* spores surviving post CiMPAS processing in pouches inoculated at the coldest spot on each tray after processing through R-65. Each bar represents the average e ± standard deviation (*n* = 9) including three technical and three processing replicates. Note: Similar letters among the bars indicate no significant difference (*p* > 0.05)

Tray 9 showed significantly higher inactivation in all three processing runs (Figure 6). The consistency of results in three different processing replicates indicated that the heat distribution was not variable enough to cause any difference in inactivation of *C. sporogenes* spores and pouches could be used as an effective method when the inactivation potential at a particular location inside the food tray needs to be evaluated. In a subsequent trial, to help understand thermal exposure at the walls/sides of each tray, a pouch was placed in a vertical position against each of the four sides and processed using the same regime (R-65) and the surviving spores were enumerated (Figure 7).

The inactivation of *C. sporogenes* spores at all four different sides of each tray in this study was an assessment of any difference in thermal exposure. The results showed that the spore pouches placed near the region coming in direct contact with water, for example, side 2 in tray 12, consistently showed better inactivation (Figure 7). In this trial, the spore pouches were able to indicate the difference in thermal exposure. Hence, the current study supports the possibility of using spore-inoculated pouches and recovery of spores/vegetative cells to test the inactivation potential at quite precise locations.

The thermal resistance of *G. stearothermophilus* spores and *C. sporogenes* spores could significantly vary in a wide range of food products. For example, D121 ◦C values of *G. stearothermophilus* can range from 0. 9 to 8.5 min (average of 2.4 min) as determined after assessing the effect of different minerals, sporulation conditions and 18 different spore strains [27]. On the other hand, *C. sporogenes* spores have been reported to show a *D*<sup>121</sup> value of 0.92 min in phosphate buffer [28]. *D*<sup>121</sup> values of *C. sporogenes* spores in food has been reported to range from 1.2–1.4 min in asparagus substrate acidified with gluconolactone (GDL) [29] and 1.28 min in liquid (unnamed) media (pH 7.0) [30]. Mashed potato (food model) is a semisolid food matrix, therefore was selected to understand the influence of the specific matrix on the inactivation using CiMPAS. In the current study, the *D*<sup>121</sup> values of *C. sporogenes* and *G. stearothermophilus* spores showed a significant increase of 2.4 and 3.8 min when in mashed potato as compared to Millli Q water, hence also supports this fact that the resistance would change based on the matrix. Hence, each time a new model of food is tested, a validation study with microbial inactivation should be separately conducted and for that purpose, use of spore pouches instead or alongside of whole food inoculation would enable testing according to spatial mapping in food trays.

**Figure 7.** C. sporogenes spores in pouches surviving the CiMPAS processing (R-65) when placed vertically on four sides on each tray (**1**–**12**). Note: Similar letters among the bars indicate no significant difference (*p* > 0.05)

Inactivation equivalent to >7 Log reduction of *C. sporogenes* spores using spore pouches as observed in the current study indicates that a minimum of 6 min of average exposure at 121 ◦C was received by each tray. In the current study, up to 2 Log reduction of *G. stearothermophilus* spores using pouch inoculation also indicated that more than 6 min of average exposure would be attained by each tray. Both these findings were consistently reproducible using the pouches that were inoculated with spores in mashed potato (food model). The spores were inoculated in mashed potato just to ensure the coverage of any food-induced masking or protective effect as it has been reported for milk, meat and food with a high-fat content [31,32]. The findings indicate that placing these pouches inoculated with the desired kind of bacterial spores might help to understand if there would be any inactivation at colder regions inside the food tray in contrast to whole tray inoculation which cannot take the worst-case scenario of the coldest spot in any tray. Pouches also have potential to be formulated with different food matrix and different spore strains according to the requirements of the thermal regime being analysed (after initial standardization trials).

For this study, the lightness values on the topmost layer were taken to indicate broadly the heating experience of the food column below. However, the coldest spot could be somewhere in the mid-layer of the tray. In future studies, lower layers of the mashed potato (food model) tray will be used as a subject to measure cold spots.

#### **4. Conclusions**

Bacterial spore pouches were developed as a method to evaluate thermal exposure on specific locations inside food trays. Two strains of bacterial spores, with a significant difference in their thermal resistance (D121 ◦C) were used in this study to evaluative the inactivation using CiMPAS as a case study. CiMPAS regime in its research stage was deliberately chosen to give conditions generating variation in thermal exposure that could generate cold and hot spots. A CiMPAS regime at 121 ◦C (6 passes, 12 kW) at 915 MHz, although not yet optimised, showed >7 Log reduction in *C. sporogenes* whereas a similar treatment at 65 ◦C showed <2 Log reduction on the cold spots which were pre-determined using the difference in color as a result of Maillard browning, where higher lightness values indicate less heat exposure. Inactivation equivalent to 1–2 Log CFU/g of *G. stearothermophilus* was obtained using the regime at 121 ◦C indicating that the spores in the pouches were inactivated based on their thermal resistance and hence the pouch itself did not act as a restriction to mask any effect. Bacterial spore pouches with food matrix inoculated with spores could be used as an effective analytical tool to understand inactivation potential at specific location to understand spatial distribution effects. As microwave sterilization is an emerging technology, this method could be effectively used as part of the validation regime where non-uniform heating is an issue.

**Author Contributions:** A.S., K.T., J.S., A.G. and R.A. conceived and designed the experiments; A.S., K.T. and A.G. performed the experiments; A.S. analyzed the data; A.S. wrote the paper and A.S., K.T., A.G., G.B., J.S., R.A. and K.T. significantly edited and reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** This research was carried out as part of the Food Industry Enabling Technologies program funded by the New Zealand Ministry of Business, Innovation and Employment (contract MAUX1402). We would like to thank Raul Cruz from School of Food & Advanced Technology, Massey University, Palmerston North, New Zealand for providing his technical expertise.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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