A Novel Technique for Sterilization Using a Power Self-Regulated Single-Mode Microwave Cavity

sensors

Article A Novel Technique for Sterilization Using a Power Self-Regulated Single-Mode Microwave Cavity

Juan D. Reverte-Ors 1, Juan L. Pedreño-Molina 1,*, Pablo S. Fernández 2, Antonio J. Lozano-Guerrero 1, Paula M. Periago 2 and Alejandro Díaz-Morcillo 1

1 Department of Information and Communication Technologies, Universidad Politécnica de Cartagena, Plaza del Hospital, 1, 30202 Cartagena (Murcia), Spain; [email protected] (J.D.R.-O.); [email protected] (A.J.L.-G.); [email protected] (A.D.-M.) 2 Department of Food Engineering and Agricultural Equipment, Universidad Politécnica de Cartagena, Paseo Alfonso XIII, 48, 30203 Cartagena (Murcia), Spain; [email protected] (P.S.F.); [email protected] (P.M.P.) * Correspondence: [email protected]; Tel.: +34-968-32-6533

Academic Editor: Vittorio M. N. Passaro Received: 25 April 2017; Accepted: 2 June 2017; Published: 7 June 2017

Abstract: In this paper, a novel technique to achieve precise temperatures in food sterilization has been proposed. An accurate temperature profile is needed in order to reach a commitment between the total removal of pathogens inside the product and the preservation of nutritional and organoleptic characteristics. The minimal variation of the target temperature in the sample by means of a monitoring and control software platform, allowing temperature stabilization over 100 ◦C, is the main goal of this work. A cylindrical microwave oven, under pressure conditions and continuous control of the microwave supply power as function of the final temperature inside the sample, has been designed and developed with conditions of single-mode resonance. The uniform heating in the product is achieved by means of sample movement and the self-regulated power control using the measured temperature. Finally, for testing the sterilization of food with this technology, specific biological validation based on Bacillus cereus as a biosensor of heat inactivation has been incorporated as a distribution along the sample in the experimental process to measure the colony-forming units (CFUs) for different food samples (laboratory medium, soup, or fish-based animal by-products). The obtained results allow the validation of this new technology for food sterilization with precise control of the microwave system to ensure the uniform elimination of pathogens using high temperatures.

Keywords: biosensors; microbiological food safety; microwave cavity; power and temperature control; sterilization; animal by-products

1. Introduction Conventional sterilization procedures for packaged foods are based on retort treatments which are subjected to high pressure and temperatures in excess of 100 ◦C to guarantee their safety. To ensure the absence of pathogenic microorganisms, as well as toxic substances for the consumer, it is necessary to establish the food safety of the final product that will be consumed. Specifically, 0.1 mg of food contaminated with botulinum toxin can cause botulism, since ingestion of doses as low as 30–100 ng can be even lethal to the consumer [1], so the requirement for uniformity in heating to ensure microbial inactivation must be maximized [2]. Treatment of food by-products (waste) is also of upmost importance in order to efficiently reduce their environmental impact. The use of microwaves for sterilization processes can be, in many cases, very restrictive. In addition, the pH value in the food significantly influences the sterilization process.

Sensors 2017, 17, 1309; doi:10.3390/s17061309 www.mdpi.com/journal/sensors Sensors 2017, 17, 1309 2 of 13

The problems of sterilization or pasteurization of foods with microwaves are well known in the industrial field [3,4]. Although microwave-sterilized products present a lower loss of nutrients compared to those processed with conventional methods, if the process is not well adapted or suitable to the product the loss of quality can be greater with microwave treatment in some cases. The problem that exists here is the difficulty of obtaining uniform heating throughout the product using microwave technology [5,6]. In food sterilization or waste processes, this uniform control is the key for obtaining a safe product while maintaining its nutritional quality. This is the main obstacle in the use of microwaves, as well as the definition of the process criteria for microorganisms, such as Clostridium perfringens, Salmonella, and Enterobacteriaceae. The non-uniform heating of the product is determined by two fundamental reasons. First, although the exponential decay of dissipated microwave energy is only valid for the model of a plane wave hitting a semi-infinite plane product, it is a good approximation when the material in the cavity is homogeneous and lossy [7], as is the case for the products analyzed in this paper. Thus, in the case of products with high losses, such as biological ones, the wave is rapidly attenuated. As a consequence of this fact, laminar products with a thickness around 1–2 cm must be formed if a good uniformity of heating is desired. On the other hand, the electric field has a high spatial variability inside a resonant cavity, which results in a non-homogeneous heating, due to the direct relation between electric field strength and generated heat. This is true for both single-mode and multi-mode cavities. In a multi-mode cavity, the resulting electric field is a linear combination of the different resonant modes or waves excited in the cavity. Attempts to improve this spatial uniformity have traditionally been based on mechanisms of product movement (turntable, conveyor belt) or mode stirrers [3] (which produce a time variation of the electric field pattern in the cavity). With these two solutions, it is possible to obtain a dissipated energy, time-averaged, similar in all of the points of the sample, but it does not necessarily guarantee a uniformity of temperature as required in sterilization processes. In recent years, the use of microwave technology at 915 MHz [8], compared to the more developed and traditional 2.45 GHz, has been studied for these cases. Although the use of lower frequencies undoubtedly improves the uniformity of the electric field inside the applicator, allowing a greater penetration of the wave in the material, this technology is much more expensive, especially in the microwave sources, which makes it difficult to transfer to the industry. At present, current research focused on uniform electric field distributions on the surface of the treated product is based on multimode applicators, which limits the application of microwaves to a large number of industrial heating processes, including sterilization processes. Recent techniques in multimode cavity simulation platforms are based on low-temperature heating processes using multiple ports with different phase feeds [9], optimization by parameter variation with microwave CAD (computer aided design) technology [10,11], determination of the optimal point of a sample to minimize the deviation of a heating pattern [12], or the use of stirrers [7]. However, when a sterilization process with temperatures over 100 ◦C is required, microwave heating conditions to minimize the expected temperature deviation must be controlled. Recent studies have demonstrated better results for food pasteurization or sterilization in terms of retention of nutrient quality [13] and microbial elimination [14], when microwave technology is used in comparison with conventional autoclaving. Specific developments have also been simulated for sterilization processes [15]. In [16] a very high microwave power in a cylindrical cavity was employed to measure the effect of sterilization in a specific food sample. However, although in this study the sterilization process is made by using microwave systems, the uniform temperature profile throughout the sample is not confirmed. In this work, a real installation based on a single-mode pressurized cavity, online data acquisition system, control software, and a monitoring system has been designed and implemented for modelling the food sterilization process. Sensors 2017, 17, 1309 3 of 13

Another limitation to ensure real uniformity in sterilization processes is the absence of microwave-adapted biological methods to determine the degree of variability in heating achieved based on microbial inactivation at each point of the food. This implies the difficulty of using existing biosensors that can efficiently measure the effectiveness of the heating process and its variability. With reference to biological sensors, another objective of this work is to obtain and verify, by measuring CFUs as a parameter, a microwave device with high volumetric heating uniformity in temperature processes above 100 ◦C for sterilization of food and animal waste. For this, a specific geometry of the applicator has been designed, under pressure conditions, with internal movement mechanisms and with biosensors. The system is controlled online by MATLAB® software (Natick, MA, USA) that continuously determines the profile of the time-power curve to minimize the final deviation for the target temperature and ensure the pathogen inactivation. On the other hand, the development of biosensors are necessary to establish the food safety of the final whole product, with accurate information of the main points that are representative of the sample, which assures the absence of pathogenic microorganisms and toxic substances for consumption [17]. Microbiological biosensors have been used to validate conventional heat sterilization processes in which colder points are known [18]. In the case of microwave heating, uncertainty about cold spots is the main challenge to ensure the safety of food or treated products and, therefore, to characterize a suitable microorganism that can evaluate uniformity in different substrates was a key step.

2. Materials, Methods, and Experimental Set Up

2.1. Materials The electromagnetic response of different samples of soups and fish wastes have been studied and electric permittivity was characterized as a function of temperature at the frequency of around 2.45 GHz by means of a commercial dielectrometer from ITACA (Mod. Dialkitv, Valencia, Spain). This measurement is based on a resonant method, which obtains the value of the electric permittivity from the shift of the resonant frequency and the decrease of the quality factor when a dielectric material is placed in a resonant cavity [19]. The equipment allows the introduction of a standard 8 mL polypropylene vial (12 mm diameter and 75 mm length). Resonant methods are described in detail in [20]. From the permittivity measurements, the single-mode resonant cavity has been designed to perform small-sample pressure (test tube) heating tests in such a way as to guarantee uniformity of heating in the sample, and including temperature sensors that monitor the treatment of samples with a wide range of permittivity. A fiber optic Opsens (EQ013 TEMPSENS) (Quebec, QC, Canada) temperature measurement device has been introduced in the sample to obtain the temperature of the sample in real-time. The fiber optic temperature sensor has been placed in the geometrical center of the sample. Since a high uniformity is expected due to the single-mode operation, the small size of the sample, and its stir, this position becomes a meaningful indicator far away from the surface of the sample where the gradient with the outer temperatures may hinder a correct measurement. As Figure1 shows, the developed prototype is formed by the Muegge (Mod. MX1000C-112KL, Reichelsheim, Germany) power supply, offering a precise continuous control of the heating power, which has been programmed using MATLAB®; the 1 kW standard component (Panasonic Mod. 2M244-M23, Osaka, Japan) magnetron, which transforms the electrical energy into microwave electromagnetic energy; the launcher to transmit the energy of the magnetron to the heating system; the (GAE GA1118, Reichelsheim, Germany) attenuator to adapt the microwave power to the 3 mL food samples; the isolator, implemented using a circulator Muegge (Mod. MM1003A 210EC), in order to protect the magnetron by deviating the reflected microwave energy to be dissipated by means of a water load, with a connection to include an incoming power detector; and, finally, the resonant cylindrical cavity, whose height is 4.34 cm, and diameter is 13.4 cm. Sensors 2017, 17, 1309 4 of 13

Sensors 2017, 17, 1309 4 of 13 Sensors 2017, 17, 1309 4 of 13

Figure 1. Experimental microwave single-mode cylindrical cavity prototype to obtain precise temperature profiles for sterilization purposes. FigureFigure 1. Experimental1. Experimental microwave microwave single-modesingle-mode cylindr cylindricalical cavity cavity prototype prototype to obtain to obtain precise precise temperatureThetemperature excited profiles mode profiles for to for sterilization heat sterilization the sample purposes. purposes. is the transversal magnetic TM010. This mode shows invariance with the sample height and a vertical component for the electric field. TheThe designed excited cavity, mode shownto heat in the Figure sample 2, includes is the atransversal tuning system magnetic to adapt TM the010 .load This to mode be heated, shows The excited mode to heat the sample is the transversal magnetic TM . This mode shows a vibrationinvariance system with the in ordersample to height obtain and a greater a vertical heating component uniformity, for the and electric the pressure field.010 system to reach invariancehigher Thetemperatures with designed the sample cavity,over 100 height shown °C. andThein Figure atuning vertical 2, includessyst componentem has a tuning been for systemcarried the electric toout adapt using field. the stubs load toor bescrews, heated, whichThea vibration designedallow the system modification cavity, in order shown toof inobtainthe Figure frequency a greater2, includes of heating resonance a tuninguniformity, of the system cavity and to andthe adapt pressure to adapt the load systemit as tomuch beto reach heated,as a vibrationpossiblehigher system fortemperatures a wide in order range over to of obtain materials. 100 °C. a greater The The tuning vibratory heating syst uniformity,systemem has consists been and carried of the a PTFE pressure out plateusing systemwith stubs holes toor reach screws,in its higher temperaturesrotatingwhich allowsurface over the 100driven modification◦C. by The a tuning motor,of the systemfrequency moving has theof beenresonance sample carried upof the outand cavity using down and stubs with to oradapt a screws,frequency it as much which of as allow theapproximately modificationpossible for a offivewide the times range frequency per of materials. second. of resonance This The vibratorysystem of the has system cavity been consists anddesigned to of adapt aand PTFE itbuilt as plate much to withimprove as holes possible thein its for a wideuniformityrotating range of surfaceof materials. the temperature. driven The by vibratory a motor, system moving consists the sample of a PTFE up plateand withdown holes within a itsfrequency rotating surfaceof drivenapproximately by a motor, movingfive times the per sample second. up This and downsystem withhas been a frequency designed of and approximately built to improve five times the per uniformity of the temperature. second. This system has been designed and built to improve the uniformity of the temperature.

Figure 2. Designed cavity, including the tuning and vibration systems. Figure 2. Designed cavity, including the tuning and vibration systems.

The opening andFigure closing 2. Designed system cavity, has beenincluding designed the tuning to and be ablevibration to safelysystems. close the cavity when working under pressure up to 5 atm, and to be able to open it quickly to remove the sample. The system is pressurized by the combination of three designs: O-rings in screws and the cover, septum in the ﬁber area temperature sensors, and a pressure window in the connection section of the power feeding system with the resonant cavity. Sensors 2017, 17, 1309 5 of 13

The opening and closing system has been designed to be able to safely close the cavity when working under pressure up to 5 atm, and to be able to open it quickly to remove the sample. The system is pressurized by the combination of three designs: O-rings in screws and the cover, Sensors 2017, 17, 1309 5 of 13 septum in the fiber area temperature sensors, and a pressure window in the connection section of the power feeding system with the resonant cavity. 2.2. Software Implementation 2.2. Software Implementation In orderIn order to control to control the the entire entire microwave microwave heating heating system, a a platform platform including including the theinformation information fromfrom the sensors, the sensors, actuation, actuation, and and process process monitoring monitoring by means means of of adaptive adaptive control control software software has been has been developed.developed. This This control control allows allows the the supplied supplied powerpower to to be be adjusted adjusted in insuch such a way a way that that the thesample sample maintainsmaintains the desiredthe desired temperature temperature as as long long as asneeded. needed. A A flow ﬂow diagram diagram of ofthe the complete complete procedure procedure is is shownshown in Figure in Figure3. 3.

FigureFigure 3. Flow 3. Flow diagram diagram of of the the adaptive adaptive procedure procedure obtainingobtaining the the desired desired temperature temperature during during the the microwavemicrowave treatment treatment time, time, by by considering considering processing processing time ( (TimeTimeProPro), ),current current time time (Time (TimeC), criticalC), critical margin (CrMarg), sample temperature (TempS), and target temperature (TempTarg). margin (CrMarg), sample temperature (TempS), and target temperature (TempTarg).

For the sterilization process, heating over 100 °C must be carried out. For this purpose, a ◦ Forcompressed the sterilization air system is process, connected heating to the cavity over in 100 orderC to must regulate be the carried pressure, out. using For the this pressure purpose, a compressedgauge. The air critical system margin is connected is defined toas thethe cavitythreshold in orderover the to difference regulate thebetween pressure, the current using the pressuretemperature gauge. The and criticalthe final margin target for is definedthe sample. as theWhile threshold the difference over thebetween difference the target between temperature the current temperature(TempTarg and) and the the finalcurrent target temperature for the sample. for the sample While ( theTemp differences) does not between overcome the the target critical temperature margin (Temp(TargCrMarg) and) that the is currentinitially temperatureestablished, the for power the sample continues (Temp at thes) doeshighest not value, overcome in order the to critical reach the margin objective temperature with minimal time, avoiding overshoots. When this value is reached, the (CrMarg) that is initially established, the power continues at the highest value, in order to reach the objectivesystem temperature autonomously with decreases minimal the time, power avoiding to enter overshoots. the maintenance When phase this in value which is reached,the stabilization the system power is adjusted as a function of the sample temperature. This is a fine adjustment of the power in autonomously decreases the power to enter the maintenance phase in which the stabilization power order to maintain the sample temperature as constant as possible inside the defined initial threshold. is adjustedA MATLAB as a function® graphical of user the interface sample temperature.(GUI) has been Thisdeveloped is a fine to control adjustment and monitor of the the power process, in order to maintainas Figure the 4 shows. sample It allows temperature reporting as of constant the real-time as possible temperature inside and the incident defined power initial profiles. threshold. A ® MATLABAsgraphical can be observed, user interface to achieve (GUI) the desired has been beha developedvior for the to microwave control and sterilization monitor process, the process, as Figuredifferent4 shows. parameters It allows must reporting be selected of from the real-time the interface temperature software, as and shown incident in Figure power 4. The profiles. heating Asrate can allows be the observed, selection to of achievea certain thepercentage desired over behavior the maximum for the power microwave of the microwave sterilization supply. process, differentThe parametersmain objective must of including be selected the fromcritical the margin interface is to software,adaptively as establish shown the in Figuretime in4 which. The heatingthe rate allowssoftware the system selection remains of a in certain the fine percentage tuning phase, over in the order maximum to avoid large power overshoots, of the microwave as well as supply.to avoid overheating of the sample to a high temperature before being stabilized. The main objective of including the critical margin is to adaptively establish the time in which the software system remains in the fine tuning phase, in order to avoid large overshoots, as well as to avoid overheating of the sample to a high temperature before being stabilized. Sensors 2017, 17, 1309 6 of 13 Sensors 2017, 17, 1309 6 of 13

FigureFigure 4. Interface 4. Interface of of the the developed developed home-madehome-made GUI GUI to to control control the the developed developed system. system.

2.3. Microbial Determinations and Food Substrates 2.3. Microbial Determinations and Food Substrates A highly heat-resistant, spore forming microorganism, Bacillus cereus, was selected as an Aindicator, highly heat-resistant,because it is a common spore forming contaminant microorganism, and foodborneBacillus pathogen cereus of, different was selected foods as and an in indicator, its becausesporulated it is a common stage it presents contaminant high resistance and foodborne to heat. pathogen The following of different food substrates foods and (or in laboratory its sporulated stagemedia) it presents were selected high resistance for the experiments: to heat. The brain following heart infusion food substratesbroth as the (orcontrol; laboratory vegetable media) puree were selectedas food, for theand experiments: a fish-based animal brain by-product heart infusion (F-ABP), broth obtained as the control;from seabass vegetable homogenate. puree as food, and a ﬁsh-basedThe animal microorganism by-product was (F-ABP), homogenized obtained fromin the seabass different homogenate. substrates tested at a known Theconcentration, microorganism and exposed was homogenized to different in heating the different times substratesand temperatures tested at ain known the microwave concentration, and exposedinstallation to previously different heatingdescribed. times Samples and were temperatures cooled, diluted in the (ten-fold) microwave in sterile installation peptone previouslywater, and plated in Petri dishes, using brain heart infusion agar as the recovery medium. Once the described. Samples were cooled, diluted (ten-fold) in sterile peptone water, and plated in Petri dishes, medium solidified, plates were incubated at 37 °C for 48 h and the resulting colonies were counted. using brain heart infusion agar as the recovery medium. Once the medium solidiﬁed, plates were Log10 counts ◦of survivors (expressed as colony forming units, CFUs) were plotted against the incubatedexposure at 37time Cfor for each 48 temperature h and the and resulting inactivation colonies kinetics were were counted. established. Log 10 counts of survivors (expressed as colony forming units, CFUs) were plotted against the exposure time for each temperature and inactivation3. Results and kinetics Discussion were established.

3. Results3.1. Permittivity and Discussion Characterization The relative electric permittivity of the material is represented by ∗ = + ′′ and describes 3.1. Permittivity Characterization the dielectric properties affecting the reflection and attenuation of the electromagnetic field energy Thein the relative material, electric being permittivity the dielectric of theconstant material and isε′′ represented being the material by ε∗ loss= ε 0factor.+ j ε00 and describes the dielectricThe properties graphs affectingwith the results the reflection concerning and the attenuation dielectric ofconstant the electromagnetic (which defines fieldthe degree energy of in the material,polarizationε0 being of the the dielectric material to constant be subjected and εto00 beingan electric the materialfield) and loss the factor.loss factor (which indicates the capacity of dissipation of electric energy within the material, demonstrated as heat [21]) are The graphs with the results concerning the dielectric constant (which defines the degree of shown in Figures 5 and 6, respectively, depending on the temperature reached in each test. Since the polarization of the material to be subjected to an electric field) and the loss factor (which indicates Dielkitv equipment does not allow pressurized measurements, the dielectric characterization of the the capacityproducts ofwas dissipation limited under of electric100 °C. energyThe selected within materials the material, are brain demonstrated heart infusion asbroth heat (BHI), [21]) are shownfish-based in Figures animal5 and by-products6, respectively, (F-ABP), depending and vegetable on the soup, temperature which consists reached of in30% each vegetables, test. Since the Dielkitvwhere the equipment main ingredients does not are allowcarrots, pressurized potato, celery, measurements, onions, tomato, the turnip, dielectric pumpkin, characterization corn starch, of the productsand dehydrated was limited milk. under 100 ◦C. The selected materials are brain heart infusion broth (BHI), fish-based animal by-products (F-ABP), and vegetable soup, which consists of 30% vegetables, where the main ingredients are carrots, potato, celery, onions, tomato, turnip, pumpkin, corn starch, and dehydrated milk. Sensors 2017, 17, 1309 7 of 13

All of the samples presented a density close to 1 due to their high water content, although they differed in their lipid content. The mass used was in the range of 3 to 10 g, as this is a representative of the size used in food studies.

Sensors 2017, 17, 1309 7 of 13 70 All of the samples' presented a density close to 1 due to their high waterBHI content, although they Sensorsdiffered2017, 17 ,in 1309 their lipid65 content. The mass used was in the range of 3 to 10 g,Vegetal as this Soupis a representative 7 of 13 of the size used in food studies. F-ABP 60 80

55 75 20 40 60 80 Temperature (°C) 70

' BHI 65Figure 5. Dielectric constant for the samples under study near 2.45Vegetal GHz. Soup F-ABP The resonant60 frequencies of an empty cavity differ from the ones of a fully-loaded cavity proportionally to a factor 1/√ε′. The perturbational theory then indicates that a shift in the resonant frequency is directly related to a deviation in the dielectric constant of an inserted sample, but the 55 tuning system allows the modification of the cavity geometry in order to keep the resonant 20 40 60 80 frequency at 2.45 GHz, i.e., the operation frequency of the magnetron source. Thus, both the cavity Temperature (°C) dimensions and the screws were designed in CST Microwave Studio to be able to tune for a wide range of materials. Figure 5. Dielectric constant for the samples under study near 2.45 GHz. Figure 5. Dielectric constant for the samples under study near 2.45 GHz. 25 The resonant23 frequencies of an empty cavity differ from the ones of a fully-loaded cavity proportionally to a factor 1/√ε′. The perturbational theory then indicates that a shift in the resonant 21 frequency is directly related to a deviation in the dielectric constant of an inserted sample, but the 19 tuning system allows the modification of the cavity geometry in order to keep the resonant 17 frequency at 2.45 GHz, i.e., the operation frequency of the magnetron source. Thus, both the cavity dimensions and" the15 screws were designed in CST Microwave Studio to beBHI able to tune for a wide range of materials.13 Vegetal Soup 11 F-ABP 259 237 215 20 40 60 80 19 Temperature (°C) 17

" 15Figure 6. Loss factor for the samples under study near 2.45BHI GHz. 13 Figure 6. Loss factor for the samples under study near 2.45 GHz.Vegetal Soup 11 All ofAlthough the samples the behavior presented of the a densitydielectric close proper toties 1 due corresponding to their high towater theF-ABP measured content, samples although is they 9 differedalmost in theirlinear, lipid an adjustment content. The based mass on usedsecond-order was in thepolynomial range of regression 3 to 10 g, ashas this been is superposed a representative to of 7 the sizeadequately used in show food the studies. trend for and . 5 The resonant frequencies of an empty cavity differ from the ones of a fully-loaded cavity 20√ 40 60 80 proportionally to a factor 1/ ε0. The perturbational theory then indicates that a shift in the resonant Temperature (°C) frequency is directly related to a deviation in the dielectric constant of an inserted sample, but the tuning system allows the modiﬁcation of the cavity geometry in order to keep the resonant frequency at 2.45 GHz, i.e., the operationFigure frequency 6. Loss factor of for the the magnetron samples under source. studyThus, near 2.45 both GHz. the cavity dimensions and the screws were designed in CST Microwave Studio to be able to tune for a wide range of materials. Although the behavior of the dielectric properties corresponding to the measured samples is almostAlthough linear, the an behavior adjustment of based the dielectric on second-order properties polynomial corresponding regression to has the been measured superposed samples to is almostadequately linear, an show adjustment the trend for based and on second-order. polynomial regression has been superposed to 0 00 adequately show the trend for ε and ε .

3.2. Resonance

The reﬂection coefﬁcient of BHI and F-ABP are shown in Figure7. Measurements have been carried out directly by measuring the cavity depicted in Figure2 with the corresponding samples. A Rohde and Schwarz Vector Network Analyzer ZVA 67 (München, Germany) has been used for the measurements with a WR-340 calibration kit. Simulated results have been obtained using the developed model in CST Microwave Studio (Darmstadt, Germany) with the measured values of permittivity obtained from Figures5 and6 at room temperature. Slight differences may be due to Sensors 2017, 17, 1309 8 of 13

3.2. Resonance The reflection coefficient of BHI and F-ABP are shown in Figure 7. Measurements have been carried out directly by measuring the cavity depicted in Figure 2 with the corresponding samples. A Rohde and Schwarz Vector Network Analyzer ZVA 67 (München, Germany) has been used for the measurements with a WR-340 calibration kit. Simulated results have been obtained using the Sensors developed2017, 17, 1309 model in CST Microwave Studio (Darmstadt, Germany) with the measured values of8 of 13 permittivity obtained from Figures 5 and 6 at room temperature. Slight differences may be due to inaccuracies in the CAD model or in the permittivity model in the whole frequency range of 2–3 inaccuraciesGHz. The in theloaded CAD system model shows or in a theclear permittivity resonance at model frequencies in the near whole 2.45 frequency GHz and rangethis frequency of 2–3 GHz. The loadedcan be systemeasily reached shows using a clear the resonancetuning screws. at frequencies near 2.45 GHz and this frequency can be easily reached using the tuning screws.

FigureFigure 7. Measured 7. Measured and and simulated simulated reﬂection reflection coefﬁcientcoefficient for for BHI BHI and and F-ABP. F-ABP.

3.3. Temperature3.3. Temperature Profiles Profiles Table1Table shows 1 shows conditions conditions for somefor some of theof the experiments experiments carriedcarried out. Under Under these these conditions, conditions, the the temperaturetemperature profiles profiles drawn drawn in Figurein Figure8 have 8 have been been obtained. obtained. AA 6060 °C◦C target target temperature temperature has hasbeen been reached with no need of the pressure system. An error of −0.5% with a deviation of ±0.1 °C has been reached with no need of the pressure system. An error of −0.5% with a deviation of ±0.1 ◦C has been obtained. When the pressure system has been activated two examples for 100 °C◦ and 110 °C have◦ obtained.been Whenreported. the For pressure these cases, system errors has of been1.7% and activated 2.2% with two deviations examples of for ±1.0 100 °C andC and ±1.1 110 °C wereC have ◦ ◦ been reported.obtained, respectively. For these cases, errors of 1.7% and 2.2% with deviations of ±1.0 C and ±1.1 C were obtained, respectively. The peak shown in Figure8aTable corresponds 1. Conditions to thefor the behavior experiments. of the power control system when trying to follow a fast incrementProcess of temperature,Pressure the occurrence Time of small Objective overshoots being frequent. The fit of the temperatureLow temp. profile to the requiredNo one allows 100 s the validation 60 °C of the stability for the implemented microwaveHigh heating temp. system. WhenYes a slower temperature 50 s increase 100 °C is required (Figure8b), the peaks are usually veryHigh temp. small. Yes 50 s 110 °C

Table 1. Conditions for the experiments.

Process Pressure Time Objective Low temp. No 100 s 60 ◦C High temp. Yes 50 s 100 ◦C High temp. Yes 50 s 110 ◦C

SensorsSensors2017, 17 2017, 1309, 17, 1309 9 of 139 of 13

Figure 8. Temperature proﬁles of developed experiments. (a) Low-temperature process; and (b) Figure 8. Temperature profiles of developed experiments. (a) Low-temperature process; and (b) high-temperaturehigh-temperature process. process.

3.4. Heat InactivationThe peak shown of Bacillus in Figure cereus 8a corresponds to the behavior of the power control system when trying to follow a fast increment of temperature, the occurrence of small overshoots being frequent. TheThe numberfit of the oftemperature survivors (expressedprofile to the as required log10 CFU/mL) one allows at the different validation heating of the times stability was for plotted the for each temperatureimplemented tested.microwave Although heating some system. survivor When a curves slower presented temperature tailing increase and is shoulders required (Figure (especially when8b), several the peaks log cycles are usually of inactivation very small. were achieved), since correlation coefficients were higher than 0.9 it was considered that, in general, they followed a first-order kinetic, although some curves could also be3.4. fitted Heat withInactivation models of Bacillus considering cereus shoulders and tailing. A non-uniform temperature would lead to a lowerThe inactivation number of rate survivors or to a (expressed deviation as from log10 linearity CFU/mL) (tailing), at different so asheating neither times were was observed, plotted for it was concludedeach temperature that homogeneous tested. Although heating some took place.survivor The curves log-linear presented primary tailing inactivation and shoulders model (especially considers that thewhen microbial several count,log cyclesN, decreasesof inactivation exponentially were achieved), with since time, correlationt. The time coefficients required forwere one higher decimal logarithmicthan 0.9 reduction it was considered under that, constant in general, temperature, they followedT, is a defined first-order in kinetic, Equation although (1) by someD(T) curves, and it is consideredcould also a first-order be fitted kineticwith models rate: considering shoulders and tailing. A non-uniform temperature would lead to a lower inactivation rate or to a deviation from linearity (tailing), so as neither were observed, it was concluded that homogeneousd log N heating1 took place. The log-linear primary 10 = − (1) inactivation model considers that the microbialdt count,D N(,T decreases) exponentially with time, t. The time required for one decimal logarithmic reduction under constant temperature, T, is defined in TheEquation microwave (1) by D(T) power, and that it is isconsidered transformed a first-order in heat kinetic [22] can rate: be obtained from Equation (2):

log 001 2 P = 2π=−f ε0ε |Erms | (1) (2) The microwave power that is transformed in heat [22] can be obtained from Equation (2): where ε = 8.8542 × 10−12 F/m is the free space electric permittivity, f (Hz) is the operation frequency, 0 and Erms(V/m) is the root mean square of the electric ﬁeld. From this relationship, it can be deduced that the higher the electric ﬁeld strength and loss factor the higher power values obtained will be. Sensors 2017, 17, 1309 10 of 13

=2 || (2) Sensorswhere2017, 17 , 1309= 8.8542 × 10 F/m is the free space electric permittivity, (Hz) is the operation10 of 13 frequency, and (V/m) is the root mean square of the electric field. From this relationship, it can be deduced that the higher the electric field strength and loss factor the higher power values This dissipatedobtained will power be. This generates dissipated a heatpowerQ gengeneratesin the material,a heat Qgen which,in the material, following which, the heatfollowing Equation the (3), producesheat anEquation increment (3), produces of temperature an increment and, consequently, of temperature a bacteriological and, consequently, inactivation a bacteriological is observed, inactivation is observed, ∂T 2 ρcp = kT∇ T + Qgen (3) ∂=t ∇ + (3) wherewhereT is theT is temperature,the temperature,t the t the time, time,k TkTis is thethe thermal conductivity, conductivity, ρ theρ the density, density, and and cp thecp specificthe specific heat ofheat the of material.the material. Nevertheless, Nevertheless, in in biological biological products a ahigh high loss loss factor factor value value is normally is normally related related to a highto a high value value in the in the dielectric dielectric constant, constant,and and thisthis hinders the the wave wave penetration penetration inside inside the sample, the sample, decreasingdecreasing the electricthe electric field field strength. strength. FiguresFigures9–11 9–11 show show the the evolution evolution of of the the CFUs CFUs ofof BacillusBacillus cereus cereus sporesspores at different at different heating heating times. times. A log-linear interpolation is provided and clearly predicts the evolution of the number of survivors. A log-linear interpolation is provided and clearly predicts the evolution of the number of survivors. Clearly, higher temperatures provide a better sterilization in terms of CFU showing a steeper slope. Clearly, higherInactivation temperatures shows, as provide expected, a a better temperature sterilization dependency: in terms the of higher CFU the showing target temperature, a steeper slope. Inactivationthe faster inactivation shows, as occurred expected, (Figures a temperature 9–11). This dependency: indicates that the there higher is an the accurate target temperature,temperature the fastercontrol inactivation in the occurredmicrowave (Figures system.9 –At11 all). Thisof the indicates temperatures that there studied is an (95 accurate °C, 100 temperature°C and 105 °C), control ◦ ◦ ◦ in theinactivation microwave rate system. is higher At all in of BHI the and temperatures lower in F-ABP. studied There (95 C,is 100a highC andlog-linear 105 C),correlation inactivation rate iscoefficient higher in for BHI all andthe survival lower incurves F-ABP. obtained There (a islways a high higher log-linear than 0.9) correlation and counts coefficient observed forwere all the survivalnot lower curves than obtained those obtained (always by higherconventional than 0.9)heating, and which counts indicated observed that werea homogeneous not lower heating than those obtainedis obtained by conventional [23,24]. heating, which indicated that a homogeneous heating is obtained [23,24].

7 Log CFU/mL F-ABP, 95ºC 6 Vegetal Soup, 95ºC BHI, 95ºC 5 0 100 200 300 400 500 600 Time (s)

SensorsFigure 2017Figure, 17 9., 1309Measurement 9. Measurement of of CFUs CFUs in in the the substratessubstrates indicated indicated at at a target a target temperature temperature of 95 of °C. 95 ◦C.11 of 13

10 F-ABP, 100ºC

9 Vegetal Soup, 100ºC 8

7 Log CFU/mL

5 0 100 200 300 400 500 600 Time (s)

FigureFigure 10. Measurement 10. Measurement of of CFUs CFUs in in the the substrates substrates in indicateddicated at at a atarget target temperature temperature of 100 of 100°C. ◦C.

10 F-ABP, 105ºC 9 Vegetal Soup, 105ºC BHI, 105ºC 8

7 Log CFU/mL

5 0 100 200 300 400 500 600 Time (s)

Figure 11. Measurement of CFUs in the substrates indicated at a target temperature of 105 °C.

At 105 °C a fast inactivation is observed, with 3 log decimal reductions in less than 120 s in BHI, whereas to achieve the same inactivation in F-ABP more than 200 s are necessary, as Figure 11 shows. Vegetable soup required a time in between. Differences in heat inactivation can be explained by three main reasons:  Higher loss factors lead to steeper temperature increase in the first heating stage, when a constant microwave power is applied to the cavity, since a higher loss factor yields a larger heat generation. Consequently, the target temperature is reached faster in the sample and, therefore, it is treated at the target temperature for a longer time. This rationale cannot be applied to the heating control stage, since there the power is controlled to keep the target temperature and, therefore, the heating generation is not directly related to the loss factor.  Heat transfer by conduction inside the sample is higher with lower lipid content, since the thermal conductivity of the water, as a polar molecule, is higher than that of lipids, which are non-polar molecules.  The presence of lipids can also have a protective effect on the spores of Bacillus cereus [25].

Sensors 2017, 17, 1309 11 of 13

10 F-ABP, 100ºC

9 Vegetal Soup, 100ºC 8

7 Log CFU/mL

5 0 100 200 300 400 500 600 Time (s) Sensors 2017, 17, 1309 11 of 13 Figure 10. Measurement of CFUs in the substrates indicated at a target temperature of 100 °C.

10 F-ABP, 105ºC 9 Vegetal Soup, 105ºC BHI, 105ºC 8

7 Log CFU/mL

5 0 100 200 300 400 500 600 Time (s)

FigureFigure 11. Measurement11. Measurement of of CFUs CFUs in in the the substratessubstrates in indicateddicated at at a target a target temperature temperature of 105 of °C. 105 ◦C.

At 105 °C a fast inactivation is observed, with 3 log decimal reductions in less than 120 s in BHI, At 105 ◦C a fast inactivation is observed, with 3 log decimal reductions in less than 120 s in BHI, whereas to achieve the same inactivation in F-ABP more than 200 s are necessary, as Figure 11 whereasshows. to achieveVegetable the soup same required inactivation a time in in between. F-ABP moreDifferences than 200in heat s are inactivation necessary, can as be Figure explained 11 shows. Vegetableby three soup main required reasons: a time in between. Differences in heat inactivation can be explained by three main reasons:  Higher loss factors lead to steeper temperature increase in the first heating stage, when a • Higher lossconstant factors microwave lead to steeper power is temperature applied to the increase cavity, insince the a ﬁrsthigher heating loss factor stage, yields when a larger a constant heat generation. Consequently, the target temperature is reached faster in the sample and, microwave power is applied to the cavity, since a higher loss factor yields a larger heat generation. therefore, it is treated at the target temperature for a longer time. This rationale cannot be Consequently, the target temperature is reached faster in the sample and, therefore, it is treated at applied to the heating control stage, since there the power is controlled to keep the target the targettemperature temperature and, for therefore, a longer the time. heating This generation rationale is cannot not directly be applied related to the the loss heating factor. control stage, sinceHeat there transfer the powerby conduction is controlled inside tothe keep sample the is target higher temperature with lower and,lipid therefore,content, since the the heating generationthermal is not conductivity directly related of theto water, the loss as a factor.polar molecule, is higher than that of lipids, which • Heat transferare non-polar by conduction molecules. inside the sample is higher with lower lipid content, since the thermal The conductivity presence of of lipids the can water, also as have a polar a protective molecule, effect is on higher the spores than of that Bacillus of lipids, cereus [25]. which are non-polar molecules. • The presence of lipids can also have a protective effect on the spores of Bacillus cereus [25].

4. Conclusions A single-mode cylindrical cavity has been designed jointly with a self-regulated power-supply system for achieving a selected temperature in order to study the sterilization of different samples. The correct operation of the designed cavity and the self-regulated system have been successfully verified. Parameters, such as the permittivity of the samples have been provided in a wide range of temperatures from 20 ◦C to 80 ◦C. Accurately and uniformly enforcing a selected temperature in the volume of the sample with the developed microwave system has allowed a systematic study of the evolution in the bacterial counts of three different materials to be sterilized. This system can effectively ensure thorough and uniform microbial inactivation in foods. Heat inactivation by microwave heating in different substrates (laboratory media and foods) has been tested using a foodborne pathogenic bacterium, Bacillus cereus, as a sensor. It shows a log-linear decline in all the conditions tested (temperatures between 95 ◦C and 105 ◦C) and a similar inactivation rate to conventional heating, indicating a homogenous temperature profile. Inactivation rate is higher in BHI and lower in F-ABP, which is related to the loss factor of each product and, ultimately, to the content of lipids. As an alternative, a solid-state microwave generator could have been used. In that case, the power control is more accurate and quite easier; therefore, probably smaller variations around the Sensors 2017, 17, 1309 12 of 13 goal temperature would have been obtained. However, solid-state microwave generators are very limited in power (as much as a few hundreds of watts) and are very expensive, which invalidates its use for high power applications, i.e., 1–100 kW. On the contrary, the analysis presented here deals with a magnetron source, which allows a scalable system. Single-mode cavities, small sample and a vibratory system lead to uniform temperature profile along the material, and the main challenge is to keep the temperature constant with time, as shown. The next step is the design of multi-mode applicators in order to deal with larger samples at an industrial scale. In this case, future work is envisaged to reach a spatial uniformity of temperature that guarantees pasteurization or sterilization while keeping the nutritional and organoleptic properties.

Acknowledgments: This work was supported by the Ministerio de Economía y Competitividad under the project with reference DPI2014-61857-EXP. Author Contributions: J.L.P.-M. and A.D.-M. conceived and designed the microwave system, including the feeding section, the tuning system, the pressure system, and the temperature measurement, and developed the power control software; A.J.L.-G. and J.D.R.-O. carried out the CST software simulations for modeling the cylindrical cavity, the measurements of dielectric characterization of the samples, and the manufacturing of the microwave experimental system; P.S.F. and P.M.P. conceived and developed the experiments, and analyzed the data of microorganisms; and all authors contributed to write the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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