Influence of Frequency and Electric Field Strength on the Inactivation of Escherichia coli O157:H7 During the Ohmic Heating Processing of Pomelo Juice

Influence of Frequency and Electric Field Strength on the Inactivation of Escherichia coli O157:H7 During the Ohmic Heating Processing of Pomelo Juice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Influence of Frequency and Electric Field Strength on the Inactivation of Escherichia coli O157:H7 During the Ohmic Heating Processing of Pomelo Juice Nhu Khue Doan, Quoc Dat Lai, Thi Kim Phung Le, Nhat Tam Le This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3922948/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The influence of frequency, electric field strength, and non-thermal effects during ohmic heating (OH) on the inactivation of Escherichia coli O157:H7 in pomelo juice was investigated. Pomelo juice was inoculated with a specific density of E. coli O157:H7 and then treated with OH at frequencies ranging from 50 Hz to 20 kHz and electric field strengths of 20, 30, and 40 V/cm. The results showed that 60 and 500 Hz were more effective in inactivating E. coli than other frequencies. As electric field strength increased, inactivation also increased. Transmission electron microscopy analysis revealed that the cell membrane of E. coli O157:H7 treated with OH underwent more pronounced changes than cells treated with conventional heating (CH). OH could inactivate E. coli O157:H7 at lower temperatures and in a shorter time than CH. These findings demonstrated the potential of OH for pasteurizing pomelo juice. ohmic heating pomelo juice electrical field strength frequency E. coli O157:H7 inactivation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction According to the World Health Organization (WHO), contaminated food is a worldwide threat to public health and socio-economic development [ 1 ]. Escherichia coli O157:H7 is a major foodborne human pathogen, and many cases of food poisoning caused by E. coli O157:H7 have occurred in North America, Europe, South East Asia, and other areas of the world [ 2 ][ 3 ]. Although the number of E. coli O157:H7 infections worldwide is lower than those caused by other pathogenic microorganism groups, namely Salmonella spp. and Campylobacter spp. [ 1 ], hospitalization and fatality rates for those infected with E. coli O157:H7 are higher [ 2 ]. E. coli O157:H7 is a gram-negative bacterium that causes hemorrhagic colitis, bloody diarrhea, and hemolytic uremic syndrome, which can lead to kidney failure and death, especially in children and the elderly. E. coli O157:H7 outbreaks have been associated with foods such as beef, spinach, beets, fermented sausages, unpasteurized fruit juices, apple cider, and raw milk [ 2 ][ 4 ][ 5 ]. Moreover, water is the primary source of infection with this bacterium [ 4 ]. E. coli O157:H7 is the most acid-tolerant pathogenic bacterium in acidic fruit juice (pH ≤ 4.6) under treatment conditions. The FDA has imposed regulations to control this bacterium in fruit juice (pH ≤ 4.6); it considers fruit juice to be microbiologically safe if processing inactivates at least 5 log CFU/mL of E. coli O157:H7 [ 6 ]. Heat treatment such as direct hot air/steam, hot water bath, shell, plate, or tube heat exchangers is commonly used in food processing to ensure microbiological safety [ 7 ]. However, this process may lead to nutrient loss and changes in sensory properties [ 8 ]. Particularly for fruit juices, heat treatment can cause the degradation of heat-sensitive components, such as biologically active molecules like vitamins, flavonoids, and polyphenols, and alter the taste and color of the product [ 9 ]. Due to the low heat conductivity of foods, heat treatment time is prolonged, and heat is not transferred uniformly throughout the food mass, affecting quality and wasting energy [ 10 ]. The advanced techniques such as microwave heating, radio frequency heating, and ohmic heating (OH) are methods of volumetric heating, generating heat uniformly and rapidly and combines the effects of thermal and non-thermal factors to inactivate microorganisms at sub-lethal temperature by selective heating, electroporation, and cell membrane rupturing [ 5 ] thus enabling the killing of them at a “milder” temperature [ 9 ][ 11 ]. For example, Jia et al. performed conventional heating (for 16 min) and OH (at 10 V/cm, 50Hz for 6 min) from 24 to 72◦C in Bacillus cereus suspension to decrease 5 log [ 12 ]. They observed a notable reduction in processing time when microbial inactivation occurred in the OH samples. Bacterial cells treated with OH showed considerably increased leakage of metal ions, nucleic acids, and damage to membrane structure compared to those treated with CH at equivalent heating temperatures. Khue et al compared the impact of OH (60 Hz, 30 V/cm) and CH on S. Enteritidis [ 13 ]. They showed S. Enteritidis density in PBW reduced 4.3 logs with OH, whereas with CH, the log reduction was 2.5 logs at the same treated conditions (30s, 60 o C). Among these techniques, OH has been demonstrated to be the most effective method for pasteurizing liquids, especially fruit juices [ 15 ][ 16 ]. OH can rapidly and uniformly generate heat within a food mass by passing alternating current through the food’s ions [ 15 ]. These ions move towards the oppositely charged electrodes and change direction with alternating current. The movement of ions causes friction with surrounding molecules, converting electrical energy into thermal energy inside the food mass. The amount of heat generated depends on the electric field strength of the current and the electrical conductivity of the food [ 15 ]. Although OH is a promising thermal technique for pasteurizing/sterilizing food, it has some limitations. The efficiency of the OH process depends on the electrical conductivity of each food type [ 16 ][ 17 ], as well as the applied frequency and electric field strength [ 18 ][ 19 ]. Hence, it is essential to investigate and determine suitable operating conditions for each specific food product. Previous studies have shown that food quality and the erosion of the electrodes were also affected by the frequency [ 18 ][ 19 ], so it is necessary to investigate a wide range of frequencies to determine the most suitable one for pasteurizing each specific food type. Furthermore, electric field strength also affects the heating rate, the degradation of nutritional compounds, and the inactivation of bacteria [ 20 ]; [ 21 ]; [ 22 ]. Pomelo juice was chosen as the research material because it is rich in vitamins, minerals, dietary fiber, and biologically active compounds such as carotenoids, flavonoids, and limonoids, which are beneficial for human health. These compounds have antioxidant properties, promote bone, cardiovascular, and immune health, and help prevent constipation [ 23 ][ 24 ]. Our previous study investigated the impact of frequency and electric field strength during OH on specific chemical components in pomelo juice [ 18 ]. We determined the suitable frequency and electric field strength levels to limit changes in chemical compounds. However, the effect of OH on microorganisms in pomelo juice during processing has not been studied. Therefore, we conducted a study to investigate the influence of frequency and electric field strength on the inactivation of pathogenic bacteria in pomelo juice using the OH technique. This study aimed to 1) determine the influence of frequency and electric field strength on E. coli O157:H7 inactivation in pomelo juice 2) explore the non-thermal effect of OH pasteurization based on the difference in inactivation rate and E.coli cell morphology. 2. Materials and Methods 2.2 Cultivation of E. coli O157:H7 The microorganism strain Escherichia coli O157:H7 (ATCC 43888) was purchased from the ATCC: The Global Bioresource Center (USA). After pre-culturing in TSB medium, the culture suspension was spread onto TSA medium. Before each experiment, a characteristic colony of E. coli O157:H7 from the TSA medium was inoculated into 10 ml of TSB and incubated at 37°C for 24 hours. The bacterial suspension was centrifuged at 4000 rpm for 20 minutes at 4°C, the supernatant was discarded, and the pellet was washed with 0.2% peptone water (PW) three times. Then, the pellet was resuspended in 5 ml of 0.2% PW to obtain a suspension with an E. coli O157:H7 density of about 10 10 –10 11 CFU/ml. 2.1 Sample Preparation Pomelo juice was extracted from uniform, ripe fruits with smooth and greenish-yellow rinds and an average weight of about 1 kg. The pomelo fruits were peeled, and the segments were separated and pressed using a fruit juicer (Fujiyama FJ400). The extracted juice was filtered through two layers of cloth, mixed well, poured into brown bottles, and stored at -18°C. The pomelo juice had the following properties: total soluble solid (TSS): 11 ± 0.5 °Brix; pH: 4 ± 0.2; electrical conductivity: 4 ± 0.5 (mS/cm). Before conducting experiments to investigate the effects of CH and OH processing conditions on the bacterium, the juice was thawed, and sterilized at 121°C for 15 min to ensure sterility. Then, the juice was cooled to 20°C before adding the microbial suspension. The PBW (with a final pH of 7.2 ± 0.2 at 20°C) was sterilized by autoclaving at 121°C for 15 min and then cooled to 20°C before adding the microbial suspension. Peptone buffer water (PBW), tryptic soy broth (TSB), tryptic soy agar (TSA), and eosin methylene blue (EMB) agar were obtained from Sigma-Aldrich (Singapore). 2.3 Experimental Apparatus The OH system used in this experiment, shown in Fig. 1 , was described by Doan et al. (2021). The ohmic system consisted of a function generator (FG-7005C, Emin, Korea), a power amplifier (P7000S, Yamaha, Japan), a data acquisition device (TR-71WF-T&D, Semiki, Japan), and a heating chamber with two titanium electrodes. 2.4 Experimental Procedure 2.4.1 Effect of Frequency and Electric Field Strength on E. coli O157:H7 Inactivation . A quantity of 0.5 ml of the bacterium suspension was added to 50 ml of pomelo juice/PBW to achieve a microbial density of ~ 10 8 –10 9 CFU/ml. The function generator’s frequency was adjusted from 50 Hz to 20 kHz, and the electric field strength was set to 20, 30, and 40 V/cm. The sample was heated from 20°C to the complete inactivation temperature of the bacterium. After each heating time point, we took 1 ml of the treated sample to spread plates and count colonies. The procedure is shown in Fig. 2 . 2.4.2 Evaluation of the Non-Thermal Effect on E. coli O157:H7 Inactivation . Conventional heating (CH) in a thermostatic bath has a thermal effect on the bacterium. Ohmic heating (OH) has both thermal and electrical effects. The difference in the inactivation rate of bacterium between the two methods was considered the non-thermal effect. The experiment was conducted as shown in Fig. 2 . In the OH experiment, 50 mL of pomelo juice were subjected to heating, individually reaching temperatures of 75 o C. The heating process was conducted using an electric field strength of 30 V/cm and a frequency of 60 and 500 Hz. In the CH experiment, 2 mL of pomelo juice was placed in a Pyrex glass tube, and each tube was positioned at the center of a thermostatic water bath. Adjusting the heating rate between the two methods, ohmic and conventional heating, was the same as in Figure S1 , Figure S2. Post-treatment, the 2 mL samples from both OH and CH were transferred to sterile tubes and cooled in ice water (0 o C). Importantly, the CH and OH samples underwent treatment simultaneously. Then, we took 1 ml to spread plates and count colonies. Cell morphology was also observed using TEM to evaluate the non-thermal effect on the bacterium inactivation. 2.5 Analysis Methods 2.5.1 Counting of Microbial Density. We took 1 ml of the treated sample, added 9 ml of PBW, and shook it for two minutes. The homogeneous sample was diluted with PBW, and 0.1 ml of the appropriate dilution was spread on EMB agar. If the density after heat treatment was low, 1 ml of the treated sample was spread evenly on four agar plates without dilution. All petri dishes were incubated at 37°C for 24 hours before counting. 2.5.2 Electron Microscopic Observation of Cells. PBW containing E. coli O157:H7 was heated by OH and CH from 20°C to 50°C, then cooled in ice water and analyzed by TEM (JEM 1400flash, JEOL-Japan) to observe the cell morphology according to Park and Kang’s method [ 25 ]. 2.5.3 Determination of Titanium Ion Content. The titanium ion content (from the titanium electrodes) of the pomelo juice sample was determined to assess the corrosion of the electrodes. The titanium ion content in the pomelo juice samples treated with OH at frequencies of 60, 100, 300, and 500 Hz was determined using spectroscopy (Ref. AOAC 973.36). 2.6 Statistical Analyses Each experiment was repeated three times, and the values determined were expressed as mean ± standard deviation. The difference between the mean values was analyzed for variance (ANOVA) and the difference between the LSD-tested treatments with a significance level of p < 0.05. We used the statistical analysis software Statgraphics Centurion XV. 3. Results and Discussion 3.1 Effect of Frequency on the Inactivation of E. coli O157:H7 3.1.1 Effect of Frequency on the Inactivation of E. coli O157:H7 in Pomelo Juice. Figure 3 a shows the density of E. coli in pomelo juice treated with OH at frequencies ranging from 50 Hz to 20 kHz and an electric field strength of 30 V/cm. The heating rate depended on the frequency. As the frequency increased from 50 Hz to 20 kHz, the heating rate became slower, as indicated in Table S1 . The inactivating efficiency of E. coli was influenced by the frequency. E. coli O157:H7 was fully inactivated (~ 10 8 CFU/mL) after 50 s of OH at frequencies of 50 and 60 Hz (the achieved temperatures were 57.65°C and 52.83°C, respectively) or after 55 s of OH at frequencies of 100 and 500 Hz (the achieved temperatures were 54.93°C and 52.97°C, respectively). At frequencies of 1, 5, 10, and 20 kHz, the heating rate was slower, and the time for complete inactivation of the bacterium extended to 70, 90, 105, and 185 s with temperatures of 61.2°C, 61.8°C, 59.1°C, and 57.7°C, respectively. Thus, the frequency range for effective E. coli inactivation was 50–500 Hz. Among these frequencies, 60 Hz and 500 Hz could completely inactivate E. coli O157:H7 at the lowest temperature (around 53°C). Lee et al. (2013) showed that E. coli O157:H7 in salsa was reduced to below the detection limit after 54 s of treatment at 500 Hz [ 26 ]. Park and Kang conducted a study on OH of apple juice and observed that the density of E. coli O157:H7 was reduced by more than 5 logs after OH at 55°C for 20 s [ 25 ]. Frequencies within the range of 50–500 Hz and an electric field strength of 30 V/cm, which is considered a low electric field intensity (in the range of 20–160 V/cm), have the potential to alter cell membrane conductivity [ 27 ]. In this case, the cell membrane could accumulate charge until it reaches a critical threshold and disrupts the cell’s integrity, leading to cell death (the electroporation phenomenon). Another occurrence is electrode corrosion [ 26 ]. Under the action of alternating current, existing ions in the food environment or food components are dissociated, or ions released from corroded electrode materials are affected and move toward the two poles of the electric field. Due to the alternating current, these ions constantly change direction and collide with the cell membrane, causing electrochemical reactions. When the reaction reaches a certain threshold, pores form on the cell membrane. At this point, small molecules can pass through the membrane, causing the cell to swell, ultimately leading to its destruction [ 25 ] [ 27 ]. The increase in frequency within the range of 50–500 Hz means an increase in the oscillation and the number of collisions of the ions, leading to an increase in the efficiency of cell destruction. Especially at a frequency of 60 Hz, the Schumann resonance phenomenon occurs, enhancing the ion oscillation effect and leading to the most significant impact on the cell membrane [ 28 ]. However, at frequencies higher than 500 Hz, the electric charges change direction rapidly, causing the cell not to accumulate enough charge for membrane rupture, thus reducing the microorganism-killing rate. The heating rate decreased at frequencies ≥ 1 kHz because this frequency range approaches the radio frequency spectrum, and ions in food mass vibrate and disperse, leading to a decrease in conductivity. This explanation is based on the theory of radio frequency current (from 3 kHz to 30 MHz) operating differently from direct or low-frequency alternating currents. The energy carried by the radio frequency current can propagate through space as electromagnetic waves [ 29 ], resulting in a decrease in the heating rate of the food mass at higher frequencies. Consequently, the time needed to reach a bacterium’s inactivation temperature increases. 3.1.2 Effect of Frequency on the Inactivation of E. coli O157:H7 in PBW. The frequency affected the efficiency of E. coli inactivation in pomelo juice. However, pomelo juice contains many factors that can inhibit the growth of bacteria, such as substances like flavonoids, alkaloids, steroids, terpenoids, and saponins, and a low pH [ 30 ][ 31 ]. Thus, to determine the effect of frequency on E. coli , PBW buffer was used as a control sample. PBW has a neutral pH and does not contain any microorganism-inhibiting components. The effect of frequency on E. coli in PBW is presented in Fig. 3 b. The results showed that E. coli survived in PBW (pH = 7.2) better than pomelo juice. According to Lee et al, low pH reduces the survival rate of microorganisms [ 32 ]. However, the effect of frequency on E. coli inactivation efficiency had the same trend in both PBW and pomelo juice. Thus, the frequency of OH had an impact on the bacterium-killing rate. 3.1.3 Effect of Frequency on Electrode Corrosion. The effect of frequency on electrode corrosion is shown in Table 1 . Applying OH to pomelo juice at a frequency of 60 Hz led to the highest concentration of titanium ions in the sample, at 0.38 mg/L. Increasing the frequency led to lower levels of titanium ions in the sample. Titanium ions were not detected with OH at frequencies ≥ 300 Hz. This result was consistent with the study of [ 26 ], who investigated the effect of frequency on titanium electrode corrosion when heating salsa sauce in a frequency range of 60 to 20,000 Hz. Therefore, in implementing OH applications, attention should be paid to the appropriate frequency or electrode materials to limit electrode corrosion and its impact on the quality and safety of processed products. Table 1 Effect of frequency on corrosion of titanium electrodes Frequency (Hz) Titanium ion in pomelo juie (mg/L) 60 100 300 500 1000 0.38 ± 0.02 0.36 ± 0.01 ND ND ND 3.2 Effect of Electric Field Strength on the Inactivation of E. coli O157:H7 The influence of electric field strength (20, 30, and 40 V/cm) on the inactivation of E. coli O157:H7 in pomelo juice is shown in Fig. 4 . As the electric field strength increased, the rate at which E. coli was inactivated became faster. At 60 Hz, the time required to inactivate E. coli (~ 8 log reduction) completely was 125, 50, and 25 s at 20, 30, and 40 V/cm, respectively. Higher electric field strength leads to greater heat generation ( Table S2 ), thereby requiring less time to achieve lethal temperatures. Additionally, as the electric field strength increased, the non-thermal effect also enhanced the inactivation of the microorganism. Hence, the temperature required to destroy the microorganism at a high electric field strength was lower compared to that at a low electric field strength. For example, at 60 Hz, the temperature for complete inactivation of E. coli O157:H7 in pomelo juice at 20, 30, and 40 V/cm was 65.85°C, 52.83°C, and 42.5°C, respectively. Alternatively, at 500 Hz, the temperature for complete inactivation of E. coli was 62.47°C, 52.97°C, and 43.53°C at 20, 30, and 40 V/cm with treatment times of 140, 60, and 25 s, respectively. The inactivation of E. coli at different levels of electric field strengths at 500 Hz showed a similar trend to that at 60 Hz, but the required time for inactivation was longer due to slower heating rates at electric field strengths of 20 and 30 V/cm, resulting in increased processing time. This result was consistent with Lee et al study, indicating that higher electric field strength enhances the ability to inactivate microorganisms [ 33 ]. Cho et al. (2020) performed increasing electric field strength when OH of soybean curd, and showed heating rate of soybean curd increased because of increased conductivity and the inactivation of E. coli O157:H7 in soybean curd increased because of having an effect on cell membrane potential, increasing electroporation and expulsion of intracellular [ 34 ]. 3.3 Influence of Non-Thermal Factors on E. coli Inactivation in Pomelo Juice 3.3.1 Impact of Non-Thermal Factors on E. coli Density. The survival capability of E. coli O157:H7 during OH and CH is shown in Fig. 5 . In general, the density of the bacterium decreased with increased heating time. However, the survival rate of E. coli after OH (at 60 Hz/500 Hz and 30 V/cm) and CH showed significant differences (p < 0.05). At 60 Hz, OH completely inactivated E. coli O157:H7 (~ 8 log reduction) in pomelo juice when it was heated to 52°C (50 s), whereas CH showed no signs of inactivation. It took a CH heating time of 70 s (equivalent to 72.6°C) to achieve complete inactivation. For OH at 500 Hz, a temperature of 52°C was reached in 55 s, resulting in complete inactivation of E. coli (~ 8 log reduction). CH required 75 s (equivalent to 72.3°C) for complete inactivation. The non-thermal factor increases microbial inactivation rate was showed in numerous studies [ 25 ][ 13 ]. The presence of an external electric field can affect ions with net charges, leading to the translational motion of electric charges. This electric field may destabilize the cell membrane. Moreover, the diverse molecular motions associated with life processes, including metal ions (Mg 2+ and K + ), as well as biomacromolecules such as nucleic acids and proteins, are also influenced [ 11 ][ 12 ]. Thus, OH could inactivate E. coli O157:H7 at lower temperatures and in a shorter time than CH. 3.3.2 Cell Morphology Determination. This section details the morphological changes of E. coli in PBW subjected to CH and OH. Overall, OH induced more significant changes in cell morphology compared to CH. The cells treated with CH showed intact or mildly disrupted cell structures (Fig. 6 a). At 60 Hz, OH leads to cell shrinkage and more cell membrane damage, resulting in cellular leakage (Fig. 6 b). At 500 Hz, the cell membrane remained intact, but internal proteins were significantly denatured (Fig. 6 c). These results align with the findings of Park and Kang [ 25 ], where OH causes higher cell membrane disruption compared to CH, contributing to increased bacteria inactivation and reducing the needed time and temperature for the pasteurization of pomelo juice. 4. Conclusion This study investigated the impact of frequency, electric field strength, and non-thermal effects in OH on the inactivation of E. coli O157:H7 in pomelo juice. The results showed that the most effective frequencies for inactivation were 60 and 500 Hz, and high electric field strength caused an increase in bacterial inactivation. Non-thermal influence in OH reduced the time and temperature required for pathogen destruction. It can be concluded that OH is a promising method for ensuring microbiological safety, allowing the processor to obtain good products in terms of nutritional and organoleptic quality. Applying OH technique to pasteurize pomelo juice on a pilot scale will be implemented in the next study. Declarations Acknowledgments We thank Industrial University of Ho Chi Minh for facilities support, and the National Institute of Hygiene and Epidemiology for TEM (JEM 1400flash, JEOL-Japan) device support. Ethical statements Ethics approval was not required for this research Conflict of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding : No funding Author Contributions Statement Doan Nhu Khue : The conception and design of the study, Methodology, Analysis, Software, Data curation, Interpretation of data, Writing – original draft, Writing -review and editing. Lai Quoc Dat : The conception and design of the study, Validation, Final approval of the version to be submitted. Le Thi Kim Phung : The conception and design of the study. Le Nhat Tam : Methodology Data availability statement This manuscript does not report data generation. Data will be made available on request References A. Havelaar et al., World Health Organization global estimates and regional comparisons of the burden of foodborne disease in 2010. PLoS Med. 12 (12), e1001923 (2015) J.Y. Lim, J.W. Yoon, C.J. Hovde, A brief overview of Escherichia coli O157:H7 and its plasmid O157. J. Microbiol. Biotechnol. 20 (1), 1–10 (2010) S. Singha, R. Thomas, J.N. Viswakarma, V.K. Gupta, Foodborne illnesses of Escherichia coli O157origin and its control measures. J. Food Sci. Technol. 60 (4), 1274–1283 (2023) J.M. Rangel, P.H. Sparling, C. Crowe, P.M. Griffin, D.L. Swerdlow, Epidemiology of Escherichia coli O157:H7 outbreaks, United States, 1982–2002. Emerg. Infect. Dis. 11 (4), 603–609 (2005). 10.3201/eid1104.040739 C. Hoff, C. Higa, J. Patel, K. Gee, E. Wellman, A. Vidanes, J. Schwensohn, Notes from the Field: An Outbreak of Escherichia coli O157: H7 Infections Linked to Romaine Lettuce Exposure—United States, 2019. Morb Mortal. Wkly. 70 (18), 689 (2021) FDA, Guidance for Industry: Juice HACCP Hazards and Controls Guidance First Edition; Final Guidance , vol. 17, pp. 1–36, 2004 M.J. Taylor, M.H. Tsai, H.C. Rasco, B. Tang, J., Zhu, Stability of Listeria monocytogenes in wheat flour during extended storage and isothermal treatment. Food Control. 91 , 434–439 (2018) L. Bahrami, A. Baboli, Z.M. Schimmel, K. Jafari, S. M., Williams, Efficiency of novel processing technologies for the control of Listeria monocytogenes in food products. Trends Food Sci. Technol. 96 , 61–78 (2020) S.J. Hernández-Hernández, H.M. Moreno-Vilet, L., Villanueva-Rodríguez, Current status of emerging food processing technologies in Latin America: Novel non-thermal processing. Innov. Food Sci. Emerg. Technol. 58 , 102233 (2019) G. Piette et al., Ohmic cooking of processed meats and its effects on product quality. J. Food Sci. 69 (2), fep71–fep78 (2004) D. De Halleux, G. Piette, M.L. Buteau, M. Dostie, Ohmic cooking of processed meats: Energy evaluation and food safety considerations. Can. Biosyst Eng. / Le Genie des. Biosyst au Can. 47 , 41–47 (2005) B. Aaliya et al., Recent trends in bacterial decontamination of food products by hurdle technology: A synergistic approach using thermal and non-thermal processing techniques, Food Res. Int. , vol. 147, no. June, p. 110514, 2021 J.A. Kubo, M.T. Siguemoto, É.S. Funcia, E.S. Augusto, P.E. Curet, S. Boillereaux, L. Gut, Non-thermal effects of microwave and ohmic processing on microbial and enzyme inactivation: a critical review. Curr. Opin. Food Sci. 35 , 36–48 (2020) L. Jia, L. Shao, Y. Zhao, Y. Sun, X. Li, R. Dai, Inactivation effects and mechanism of ohmic heating on Bacillus cereus, Int. J. Food Microbiol. , vol. 390, no. August, p. 110125, 2023 D.N. Khue, H.T. Tiep, L.Q. Dat, L.T. Kim, Phung, L.N. Tam, Influence of frequency and temperature on the inactivation of Salmonella enterica serovar enteritidis in Ohmic heating of pomelo juice. LWT. 129 , 109528 (2020) N.S. McKenna, Advances in radio frequency and ohmic heating of meats. J. Food Eng. 77 (2), 215–229 (2006) S.M.B. Hashemi, A. Gholamhosseinpour, M. Niakousari, Application of microwave and ohmic heating for pasteurization of cantaloupe juice: microbial inactivation and chemical properties. J. Sci. Food Agric. 99 (9), 4276–4286 (2019) G. Barbosa-Cánovas, D. Bermúdez-Aguirre, Other novel milk preservation technologies: Ultrasound, irradiation, microwave, radio frequency, ohmic heating, ultraviolet light and bacteriocins. Improv. Saf. Qual. Milk. 1 , 420–450 (2010) F. Icier, Ohmic heating of fluid foods. Novel thermal and non-thermal technologies for fluid foods , 305–367, 2012. Academic S. Sarang, S.K. Sastry, L. Knipe, Electrical conductivity of fruits and meats during ohmic heating. J. Food Eng. 87 (3), 351–356 (2008) H. Darvishi, M. Koushesh Saba, N. Behroozi-Khazaei, H. Nourbakhsh, Improving quality and quantity attributes of grape juice concentrate (molasses) using ohmic heating. J. Food Sci. Technol. 57 (4), 1362–1370 (2020) N.K. Doan, Q.D. Lai, T.K.P. Le, N.T. Le, Influences of AC frequency and electric field strength on changes in bioactive compounds in Ohmic heating of pomelo juice. Innov. Food Sci. Emerg. Technol. 72 , 102754 (2021) S. Lee, S. Ryu, D.H. Kang, Effect of Frequency and Waveform on Inactivation of Escherichia coli O157:H7 and Salmonella enterica Serovar Typhimurium in Salsa by Ohmic Heating. Appl. Environ. Microbiol. 79 (1), 10–17 (2013) S. Kulshrestha, S. Sastry, Frequency and voltage effects on enhanced diffusion during moderate electric field (MEF) treatment. Innov. Food Sci. Emerg. Technol. 4 (2), 189–194 (2003) S. Murashita, S. Kawamura, S. Koseki, Effects of ohmic heating, including electric field intensity and frequency, on thermal inactivation of bacillus subtilis spores. J. Food. Prot. 80 (1), 164–168 (2017) J.H. Mok, T. Pyatkovskyy, A. Yousef, S.K. Sastry, Combined effect of shear stress and moderate electric field on the inactivation of Escherichia coli K12 in apple juice, J. Food Eng , vol. 262, no. May, pp. 121–130, 2019 G. Xu, D. Liu, J. Chen, X. Ye, Y. Ma, J. Shi, Food Chemistry Juice components and antioxidant capacity of citrus varieties cultivated in China, 106 , pp. 545–551, 2008 M. Zhang, C. Duan, Y. Zang, Z. Huang, G. Liu, The flavonoid composition of flavedo and juice from the pummelo cultivar (Citrus grandis (L.) Osbeck) and the grapefruit cultivar (Citrus paradisi) from China. Food Chem. 129 (4), 1530–1536 (2011) I. Park, D. Kang, Effect of Electropermeabilization by Ohmic Heating for Inactivation of Escherichia coli O157: H7, Salmonella enterica Serovar Typhimurium, Juice, 79 , 23, pp. 7122–7129, 2013 M.M. Shawki, A. Gaballah, the Effect of Low Ac Electric Field on Bacterial Cell Death. Rom J. Biophys. 25 (2), 163–172 (2015) R.J.L. Montiel, I. Bardasano, J.L., Biophysical Device For The Treatment Of Neurodegenerative Diseases, in Recent Advances in Multidisciplinary Applied Physics: Proceedings of the first international meeting on Applied Physics , pp. 63–70, 2003 J. Awuah, G.B. Ramaswamy, H. S., Tang, Radio-Frequency heating in food processing: Principles and applications (CRC., 2014) E. Tripoli, M. La Guardia, S. Giammanco, D. Di Majo, M. Giammanco, Citrus flavonoids: Molecular structure, biological activity and nutritional properties: A review. Food Chem. 104 (2), 466–479 (2007) E.I. Oikeh, E.S. Omoregie, F.E. Oviasogie, K. Oriakhi, Phytochemical, antimicrobial, and antioxidant activities of different citrus juice concentrates. Food Sci. Nutr. 4 (1), 103–109 (2016) J. Lee, S. Kim, D. Kang, LWT - Food Science and Technology Effect of pH for inactivation of Escherichia coli O157: H7, Salmonella Typhimurium and Listeria monocytogenes in orange juice by ohmic heating. LWT - Food Sci. Technol. 62 (1), 83–88 (2015) S.-Y. Lee, H.-G. Sagong, S. Ryu, D.-H. Kang, Effect of continuous ohmic heating to inactivate Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes in orange juice and tomato juice. J. Appl. Microbiol. 112 (4), 723–731 (2012) D.H. Cho, E.R. Kim, S. S., Kang, Inactivation kinetics and membrane potential of pathogens in soybean curd subjected to pulsed ohmic heating depending on applied voltage and duty ratio, Appl. Environ. Microbiol. , vol. 86, no. 14, pp. e00656-20, 2020 M.T. Kubo et al., Non-thermal effects of microwave and ohmic processing on microbial and enzyme inactivation: a critical review, Curr. Opin. Food Sci. , vol. 35, no. January, pp. 36–48, 2020 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3922948","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":272678341,"identity":"b0e95980-2528-442d-a377-19543a1f0175","order_by":0,"name":"Nhu Khue Doan","email":"","orcid":"","institution":"Industrial University of Ho Chi Minh City","correspondingAuthor":false,"prefix":"","firstName":"Nhu","middleName":"Khue","lastName":"Doan","suffix":""},{"id":272678342,"identity":"911fa56b-919c-4a87-9c78-87b4b9076b13","order_by":1,"name":"Quoc Dat Lai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYBACPgYGAwaGigNQLhsRWthAWg6cAWlhJkXLwTaStLAnb/z8cd4ded0Z+QcYPpQdZjC43UBAC8+zYomD254ZbruRzMA44xxQy50DBLRI5BgAtRxOMANqYeZtO8wgOSOBoBbjHwfnQLX8JVKLmcTBBqgWRqAWfglCWnielVmcOXbYcNuZxwYHe86l8xDUws+evPlGRc1hebPjiQ8f/CizlmMjpIWBAUnBASDmIaQeVcsoGAWjYBSMAqwAAJpVRfSqJL1JAAAAAElFTkSuQmCC","orcid":"","institution":"Ho Chi Minh City University of Technology (HCMUT)","correspondingAuthor":true,"prefix":"","firstName":"Quoc","middleName":"Dat","lastName":"Lai","suffix":""},{"id":272678343,"identity":"edc65401-11bb-462f-86a5-7b17c596d177","order_by":2,"name":"Thi Kim Phung Le","email":"","orcid":"","institution":"Ho Chi Minh City University of Technology (HCMUT)","correspondingAuthor":false,"prefix":"","firstName":"Thi","middleName":"Kim Phung","lastName":"Le","suffix":""},{"id":272678344,"identity":"bcfaf546-831a-41e6-aee0-eee1bed71704","order_by":3,"name":"Nhat Tam Le","email":"","orcid":"","institution":"Industrial University of Ho Chi Minh City","correspondingAuthor":false,"prefix":"","firstName":"Nhat","middleName":"Tam","lastName":"Le","suffix":""}],"badges":[],"createdAt":"2024-02-03 06:44:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3922948/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3922948/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51135159,"identity":"b79ce322-6656-4b47-98b6-5f0e9edcf490","added_by":"auto","created_at":"2024-02-14 18:28:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":50700,"visible":true,"origin":"","legend":"\u003cp\u003eThe experimental apparatus system of ohmic heating\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3922948/v1/1958bfd2ffbecb7eb473cc57.png"},{"id":51135153,"identity":"2c3c7702-0b61-4898-aa27-ec010a8d7146","added_by":"auto","created_at":"2024-02-14 18:28:15","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":632406,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental diagram\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3922948/v1/fdc770b6956a0bf5b09a371c.jpeg"},{"id":51135163,"identity":"19aa5e76-5d8b-42a3-b18c-13bcdcffed79","added_by":"auto","created_at":"2024-02-14 18:28:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":27389,"visible":true,"origin":"","legend":"\u003cp\u003ea Density of \u003cem\u003eE. coli\u003c/em\u003e O157:H7 in pomelo juice treated with ohmic heating ranges from 50 Hz to 20 kHz at 30 V/cm\u003c/p\u003e\n\u003cp\u003eb Density of \u003cem\u003eE. coli\u003c/em\u003e O157:H7 in PBW treated with ohmic heating ranges from 50 Hz to 20 kHz at 30 V/cm\u003c/p\u003e","description":"","filename":"F3.png","url":"https://assets-eu.researchsquare.com/files/rs-3922948/v1/607f42df1a461741f98d0872.png"},{"id":51135152,"identity":"f79d5d47-2cc4-4d51-8120-c2c093879feb","added_by":"auto","created_at":"2024-02-14 18:28:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":13453,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e O157:H7 density in pomelo juice treated with Ohmic heating ranges from 20, 30, 40 V/cm at 60/500 Hz\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e(E. coli O157:H7 density at 40 V/cm, 60 Hz and 40 V/cm, 500 Hz were the same)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"F4.png","url":"https://assets-eu.researchsquare.com/files/rs-3922948/v1/956bd5236016547c06dcb345.png"},{"id":51135158,"identity":"83a6fa2e-72f2-4d62-b907-3ac94096527f","added_by":"auto","created_at":"2024-02-14 18:28:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":9645,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of ohmic and conventional heating on \u003cem\u003eE. coli\u003c/em\u003e inactivation in pomelo juice\u003c/p\u003e","description":"","filename":"F5.png","url":"https://assets-eu.researchsquare.com/files/rs-3922948/v1/5741fec21a5c62ca63162cce.png"},{"id":51135160,"identity":"f0974661-0e6c-42f6-93de-aa65a1c9595c","added_by":"auto","created_at":"2024-02-14 18:28:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":332451,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological changes of \u003cem\u003eE. coli\u003c/em\u003e O157:H7 treated with a-CH, b-OH at 60Hz, and c-OH at 500 Hz (PBW was heated from 20 to 50 \u003csup\u003eo\u003c/sup\u003eC).\u0026nbsp;\u003c/p\u003e","description":"","filename":"F6.png","url":"https://assets-eu.researchsquare.com/files/rs-3922948/v1/2e24bd55aa453102035f4669.png"},{"id":52569355,"identity":"c32f5acb-336d-4dd0-bca4-98c4ff0121fe","added_by":"auto","created_at":"2024-03-13 05:24:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1023676,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3922948/v1/87f946f6-5c9a-43f1-a04c-d172ac9a4431.pdf"},{"id":51135155,"identity":"d792250f-a25b-4938-a439-74c8fe2da11e","added_by":"auto","created_at":"2024-02-14 18:28:16","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":36671,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-3922948/v1/faee44c9f9b415c24fe05b22.docx"},{"id":51135161,"identity":"aac9806e-0d13-4723-beb7-6028c1181ccb","added_by":"auto","created_at":"2024-02-14 18:28:17","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":314204,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3922948/v1/edfa5a2d68be724e5f6b9122.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Influence of Frequency and Electric Field Strength on the Inactivation of Escherichia coli O157:H7 During the Ohmic Heating Processing of Pomelo Juice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAccording to the World Health Organization (WHO), contaminated food is a worldwide threat to public health and socio-economic development [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. \u003cem\u003eEscherichia coli\u003c/em\u003e O157:H7 is a major foodborne human pathogen, and many cases of food poisoning caused by \u003cem\u003eE. coli\u003c/em\u003e O157:H7 have occurred in North America, Europe, South East Asia, and other areas of the world [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e][\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Although the number of \u003cem\u003eE. coli\u003c/em\u003e O157:H7 infections worldwide is lower than those caused by other pathogenic microorganism groups, namely \u003cem\u003eSalmonella spp. and Campylobacter spp.\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], hospitalization and fatality rates for those infected with \u003cem\u003eE. coli O157:H7\u003c/em\u003e are higher [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. \u003cem\u003eE. coli\u003c/em\u003e O157:H7 is a gram-negative bacterium that causes hemorrhagic colitis, bloody diarrhea, and hemolytic uremic syndrome, which can lead to kidney failure and death, especially in children and the elderly. \u003cem\u003eE. coli\u003c/em\u003e O157:H7 outbreaks have been associated with foods such as beef, spinach, beets, fermented sausages, unpasteurized fruit juices, apple cider, and raw milk [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e][\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e][\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Moreover, water is the primary source of infection with this bacterium [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. \u003cem\u003eE. coli\u003c/em\u003e O157:H7 is the most acid-tolerant pathogenic bacterium in acidic fruit juice (pH\u0026thinsp;\u0026le;\u0026thinsp;4.6) under treatment conditions. The FDA has imposed regulations to control this bacterium in fruit juice (pH\u0026thinsp;\u0026le;\u0026thinsp;4.6); it considers fruit juice to be microbiologically safe if processing inactivates at least 5 log CFU/mL of \u003cem\u003eE. coli\u003c/em\u003e O157:H7 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHeat treatment such as direct hot air/steam, hot water bath, shell, plate, or tube heat exchangers is commonly used in food processing to ensure microbiological safety [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, this process may lead to nutrient loss and changes in sensory properties [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Particularly for fruit juices, heat treatment can cause the degradation of heat-sensitive components, such as biologically active molecules like vitamins, flavonoids, and polyphenols, and alter the taste and color of the product [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Due to the low heat conductivity of foods, heat treatment time is prolonged, and heat is not transferred uniformly throughout the food mass, affecting quality and wasting energy [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe advanced techniques such as microwave heating, radio frequency heating, and ohmic heating (OH) are methods of volumetric heating, generating heat uniformly and rapidly and combines the effects of thermal and non-thermal factors to inactivate microorganisms at sub-lethal temperature by selective heating, electroporation, and cell membrane rupturing [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] thus enabling the killing of them at a \u0026ldquo;milder\u0026rdquo; temperature [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e][\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. For example, Jia et al. performed conventional heating (for 16 min) and OH (at 10 V/cm, 50Hz for 6 min) from 24 to 72◦C in Bacillus cereus suspension to decrease 5 log [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. They observed a notable reduction in processing time when microbial inactivation occurred in the OH samples. Bacterial cells treated with OH showed considerably increased leakage of metal ions, nucleic acids, and damage to membrane structure compared to those treated with CH at equivalent heating temperatures. Khue et al compared the impact of OH (60 Hz, 30 V/cm) and CH on \u003cem\u003eS.\u003c/em\u003e Enteritidis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. They showed \u003cem\u003eS.\u003c/em\u003e Enteritidis density in PBW reduced 4.3 logs with OH, whereas with CH, the log reduction was 2.5 logs at the same treated conditions (30s, 60 \u003csup\u003eo\u003c/sup\u003eC). Among these techniques, OH has been demonstrated to be the most effective method for pasteurizing liquids, especially fruit juices [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e][\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOH can rapidly and uniformly generate heat within a food mass by passing alternating current through the food\u0026rsquo;s ions [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These ions move towards the oppositely charged electrodes and change direction with alternating current. The movement of ions causes friction with surrounding molecules, converting electrical energy into thermal energy inside the food mass.\u003c/p\u003e \u003cp\u003eThe amount of heat generated depends on the electric field strength of the current and the electrical conductivity of the food [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Although OH is a promising thermal technique for pasteurizing/sterilizing food, it has some limitations. The efficiency of the OH process depends on the electrical conductivity of each food type [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e][\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], as well as the applied frequency and electric field strength [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e][\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Hence, it is essential to investigate and determine suitable operating conditions for each specific food product.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that food quality and the erosion of the electrodes were also affected by the frequency [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e][\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], so it is necessary to investigate a wide range of frequencies to determine the most suitable one for pasteurizing each specific food type. Furthermore, electric field strength also affects the heating rate, the degradation of nutritional compounds, and the inactivation of bacteria [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]; [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]; [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePomelo juice was chosen as the research material because it is rich in vitamins, minerals, dietary fiber, and biologically active compounds such as carotenoids, flavonoids, and limonoids, which are beneficial for human health. These compounds have antioxidant properties, promote bone, cardiovascular, and immune health, and help prevent constipation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e][\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Our previous study investigated the impact of frequency and electric field strength during OH on specific chemical components in pomelo juice [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. We determined the suitable frequency and electric field strength levels to limit changes in chemical compounds. However, the effect of OH on microorganisms in pomelo juice during processing has not been studied. Therefore, we conducted a study to investigate the influence of frequency and electric field strength on the inactivation of pathogenic bacteria in pomelo juice using the OH technique. This study aimed to 1) determine the influence of frequency and electric field strength on \u003cem\u003eE. coli\u003c/em\u003e O157:H7 inactivation in pomelo juice 2) explore the non-thermal effect of OH pasteurization based on the difference in inactivation rate and \u003cem\u003eE.coli\u003c/em\u003e cell morphology.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cem\u003e2.2 Cultivation of\u003c/em\u003e E. coli \u003cem\u003eO157:H7\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eThe microorganism strain \u003cem\u003eEscherichia coli\u003c/em\u003e O157:H7 (ATCC 43888) was purchased from the ATCC: The Global Bioresource Center (USA). After pre-culturing in TSB medium, the culture suspension was spread onto TSA medium. Before each experiment, a characteristic colony of \u003cem\u003eE. coli\u003c/em\u003e O157:H7 from the TSA medium was inoculated into 10 ml of TSB and incubated at 37\u0026deg;C for 24 hours. The bacterial suspension was centrifuged at 4000 rpm for 20 minutes at 4\u0026deg;C, the supernatant was discarded, and the pellet was washed with 0.2% peptone water (PW) three times. Then, the pellet was resuspended in 5 ml of 0.2% PW to obtain a suspension with an \u003cem\u003eE. coli\u003c/em\u003e O157:H7 density of about 10\u003csup\u003e10\u003c/sup\u003e\u0026ndash;10\u003csup\u003e11\u003c/sup\u003e CFU/ml.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Sample Preparation\u003c/h2\u003e\n \u003cp\u003ePomelo juice was extracted from uniform, ripe fruits with smooth and greenish-yellow rinds and an average weight of about 1 kg. The pomelo fruits were peeled, and the segments were separated and pressed using a fruit juicer (Fujiyama FJ400). The extracted juice was filtered through two layers of cloth, mixed well, poured into brown bottles, and stored at -18\u0026deg;C. The pomelo juice had the following properties: total soluble solid (TSS): 11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026deg;Brix; pH: 4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2; electrical conductivity: 4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 (mS/cm).\u003c/p\u003e\n \u003cp\u003eBefore conducting experiments to investigate the effects of CH and OH processing conditions on the bacterium, the juice was thawed, and sterilized at 121\u0026deg;C for 15 min to ensure sterility. Then, the juice was cooled to 20\u0026deg;C before adding the microbial suspension. The PBW (with a final pH of 7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 at 20\u0026deg;C) was sterilized by autoclaving at 121\u0026deg;C for 15 min and then cooled to 20\u0026deg;C before adding the microbial suspension.\u003c/p\u003e\n \u003cp\u003ePeptone buffer water (PBW), tryptic soy broth (TSB), tryptic soy agar (TSA), and eosin methylene blue (EMB) agar were obtained from Sigma-Aldrich (Singapore).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Experimental Apparatus\u003c/h2\u003e\n \u003cp\u003eThe OH system used in this experiment, shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, was described by Doan et al. (2021). The ohmic system consisted of a function generator (FG-7005C, Emin, Korea), a power amplifier (P7000S, Yamaha, Japan), a data acquisition device (TR-71WF-T\u0026amp;D, Semiki, Japan), and a heating chamber with two titanium electrodes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Experimental Procedure\u003c/h2\u003e\u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003e2.4.1 Effect of Frequency and Electric Field Strength on\u003c/strong\u003e \u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003eO157:H7 Inactivation\u003c/strong\u003e. A quantity of 0.5 ml of the bacterium suspension was added to 50 ml of pomelo juice/PBW to achieve a microbial density of ~\u0026thinsp;10\u003csup\u003e8\u003c/sup\u003e\u0026ndash;10\u003csup\u003e9\u003c/sup\u003e CFU/ml. The function generator\u0026rsquo;s frequency was adjusted from 50 Hz to 20 kHz, and the electric field strength was set to 20, 30, and 40 V/cm. The sample was heated from 20\u0026deg;C to the complete inactivation temperature of the bacterium. After each heating time point, we took 1 ml of the treated sample to spread plates and count colonies. The procedure is shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003e2.4.2 Evaluation of the Non-Thermal Effect on\u003c/strong\u003e \u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003eO157:H7 Inactivation\u003c/strong\u003e. Conventional heating (CH) in a thermostatic bath has a thermal effect on the bacterium. Ohmic heating (OH) has both thermal and electrical effects. The difference in the inactivation rate of bacterium between the two methods was considered the non-thermal effect. The experiment was conducted as shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003c/span\u003e\n \u003cp\u003eIn the OH experiment, 50 mL of pomelo juice were subjected to heating, individually reaching temperatures of 75 \u003csup\u003eo\u003c/sup\u003eC. The heating process was conducted using an electric field strength of 30 V/cm and a frequency of 60 and 500 Hz. In the CH experiment, 2 mL of pomelo juice was placed in a Pyrex glass tube, and each tube was positioned at the center of a thermostatic water bath. Adjusting the heating rate between the two methods, ohmic and conventional heating, was the same as in \u003cstrong\u003eFigure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e, Figure S2.\u003c/strong\u003e Post-treatment, the 2 mL samples from both OH and CH were transferred to sterile tubes and cooled in ice water (0\u003csup\u003eo\u003c/sup\u003eC). Importantly, the CH and OH samples underwent treatment simultaneously. Then, we took 1 ml to spread plates and count colonies. Cell morphology was also observed using TEM to evaluate the non-thermal effect on the bacterium inactivation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Analysis Methods\u003c/h2\u003e\u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003e2.5.1 Counting of Microbial Density.\u003c/strong\u003e We took 1 ml of the treated sample, added 9 ml of PBW, and shook it for two minutes. The homogeneous sample was diluted with PBW, and 0.1 ml of the appropriate dilution was spread on EMB agar. If the density after heat treatment was low, 1 ml of the treated sample was spread evenly on four agar plates without dilution. All petri dishes were incubated at 37\u0026deg;C for 24 hours before counting.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003e2.5.2 Electron Microscopic Observation of Cells.\u003c/strong\u003e PBW containing \u003cem\u003eE. coli\u003c/em\u003e O157:H7 was heated by OH and CH from 20\u0026deg;C to 50\u0026deg;C, then cooled in ice water and analyzed by TEM (JEM 1400flash, JEOL-Japan) to observe the cell morphology according to Park and Kang\u0026rsquo;s method [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003e2.5.3 Determination of Titanium Ion Content.\u003c/strong\u003e The titanium ion content (from the titanium electrodes) of the pomelo juice sample was determined to assess the corrosion of the electrodes. The titanium ion content in the pomelo juice samples treated with OH at frequencies of 60, 100, 300, and 500 Hz was determined using spectroscopy (Ref. AOAC 973.36).\u003c/p\u003e\n \u003c/span\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 Statistical Analyses\u003c/h2\u003e\n \u003cp\u003eEach experiment was repeated three times, and the values determined were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. The difference between the mean values was analyzed for variance (ANOVA) and the difference between the LSD-tested treatments with a significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. We used the statistical analysis software Statgraphics Centurion XV.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Effect of Frequency on the Inactivation of E. coli O157:H7\u003c/h2\u003e\n \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.1 Effect of Frequency on the Inactivation of \u003cem\u003eE. coli\u003c/em\u003e O157:H7 in Pomelo Juice.\u003c/h2\u003e\n \u003cp\u003eFigure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea shows the density of \u003cem\u003eE. coli\u003c/em\u003e in pomelo juice treated with OH at frequencies ranging from 50 Hz to 20 kHz and an electric field strength of 30 V/cm. The heating rate depended on the frequency. As the frequency increased from 50 Hz to 20 kHz, the heating rate became slower, as indicated in \u003cstrong\u003eTable \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe inactivating efficiency of \u003cem\u003eE. coli\u003c/em\u003e was influenced by the frequency. \u003cem\u003eE. coli\u003c/em\u003e O157:H7 was fully inactivated (~\u0026thinsp;10\u003csup\u003e8\u003c/sup\u003e CFU/mL) after 50 s of OH at frequencies of 50 and 60 Hz (the achieved temperatures were 57.65\u0026deg;C and 52.83\u0026deg;C, respectively) or after 55 s of OH at frequencies of 100 and 500 Hz (the achieved temperatures were 54.93\u0026deg;C and 52.97\u0026deg;C, respectively). At frequencies of 1, 5, 10, and 20 kHz, the heating rate was slower, and the time for complete inactivation of the bacterium extended to 70, 90, 105, and 185 s with temperatures of 61.2\u0026deg;C, 61.8\u0026deg;C, 59.1\u0026deg;C, and 57.7\u0026deg;C, respectively. Thus, the frequency range for effective \u003cem\u003eE. coli\u003c/em\u003e inactivation was 50\u0026ndash;500 Hz. Among these frequencies, 60 Hz and 500 Hz could completely inactivate \u003cem\u003eE. coli\u003c/em\u003e O157:H7 at the lowest temperature (around 53\u0026deg;C). Lee et al. (2013) showed that \u003cem\u003eE. coli\u003c/em\u003e O157:H7 in salsa was reduced to below the detection limit after 54 s of treatment at 500 Hz [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. Park and Kang conducted a study on OH of apple juice and observed that the density of \u003cem\u003eE. coli\u003c/em\u003e O157:H7 was reduced by more than 5 logs after OH at 55\u0026deg;C for 20 s [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eFrequencies within the range of 50\u0026ndash;500 Hz and an electric field strength of 30 V/cm, which is considered a low electric field intensity (in the range of 20\u0026ndash;160 V/cm), have the potential to alter cell membrane conductivity [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. In this case, the cell membrane could accumulate charge until it reaches a critical threshold and disrupts the cell\u0026rsquo;s integrity, leading to cell death (the electroporation phenomenon). Another occurrence is electrode corrosion [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. Under the action of alternating current, existing ions in the food environment or food components are dissociated, or ions released from corroded electrode materials are affected and move toward the two poles of the electric field. Due to the alternating current, these ions constantly change direction and collide with the cell membrane, causing electrochemical reactions. When the reaction reaches a certain threshold, pores form on the cell membrane. At this point, small molecules can pass through the membrane, causing the cell to swell, ultimately leading to its destruction [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e] [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. The increase in frequency within the range of 50\u0026ndash;500 Hz means an increase in the oscillation and the number of collisions of the ions, leading to an increase in the efficiency of cell destruction. Especially at a frequency of 60 Hz, the Schumann resonance phenomenon occurs, enhancing the ion oscillation effect and leading to the most significant impact on the cell membrane [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, at frequencies higher than 500 Hz, the electric charges change direction rapidly, causing the cell not to accumulate enough charge for membrane rupture, thus reducing the microorganism-killing rate.\u003c/p\u003e\n \u003cp\u003eThe heating rate decreased at frequencies\u0026thinsp;\u0026ge;\u0026thinsp;1 kHz because this frequency range approaches the radio frequency spectrum, and ions in food mass vibrate and disperse, leading to a decrease in conductivity. This explanation is based on the theory of radio frequency current (from 3 kHz to 30 MHz) operating differently from direct or low-frequency alternating currents. The energy carried by the radio frequency current can propagate through space as electromagnetic waves [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e], resulting in a decrease in the heating rate of the food mass at higher frequencies. Consequently, the time needed to reach a bacterium\u0026rsquo;s inactivation temperature increases.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.1.2 Effect of Frequency on the Inactivation of\u003c/strong\u003e \u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003eO157:H7 in PBW.\u003c/strong\u003e The frequency affected the efficiency of \u003cem\u003eE. coli\u003c/em\u003e inactivation in pomelo juice. However, pomelo juice contains many factors that can inhibit the growth of bacteria, such as substances like flavonoids, alkaloids, steroids, terpenoids, and saponins, and a low pH [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e][\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. Thus, to determine the effect of frequency on \u003cem\u003eE. coli\u003c/em\u003e, PBW buffer was used as a control sample. PBW has a neutral pH and does not contain any microorganism-inhibiting components.\u003c/p\u003e\n \u003cp\u003eThe effect of frequency on \u003cem\u003eE. coli\u003c/em\u003e in PBW is presented in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb. The results showed that \u003cem\u003eE. coli\u003c/em\u003e survived in PBW (pH\u0026thinsp;=\u0026thinsp;7.2) better than pomelo juice. According to Lee et al, low pH reduces the survival rate of microorganisms [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, the effect of frequency on \u003cem\u003eE. coli\u003c/em\u003e inactivation efficiency had the same trend in both PBW and pomelo juice. Thus, the frequency of OH had an impact on the bacterium-killing rate.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.1.3 Effect of Frequency on Electrode Corrosion.\u003c/strong\u003e The effect of frequency on electrode corrosion is shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Applying OH to pomelo juice at a frequency of 60 Hz led to the highest concentration of titanium ions in the sample, at 0.38 mg/L. Increasing the frequency led to lower levels of titanium ions in the sample. Titanium ions were not detected with OH at frequencies\u0026thinsp;\u0026ge;\u0026thinsp;300 Hz. This result was consistent with the study of [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e], who investigated the effect of frequency on titanium electrode corrosion when heating salsa sauce in a frequency range of 60 to 20,000 Hz. Therefore, in implementing OH applications, attention should be paid to the appropriate frequency or electrode materials to limit electrode corrosion and its impact on the quality and safety of processed products.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffect of frequency on corrosion of titanium electrodes\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"2\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFrequency (Hz)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTitanium ion in pomelo juie (mg/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003cp\u003e1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Effect of Electric Field Strength on the Inactivation of E. coli O157:H7\u003c/h2\u003e\n \u003cp\u003eThe influence of electric field strength (20, 30, and 40 V/cm) on the inactivation of \u003cem\u003eE. coli\u003c/em\u003e O157:H7 in pomelo juice is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. As the electric field strength increased, the rate at which \u003cem\u003eE. coli\u003c/em\u003e was inactivated became faster.\u003c/p\u003e\n \u003cp\u003eAt 60 Hz, the time required to inactivate \u003cem\u003eE. coli\u003c/em\u003e (~\u0026thinsp;8 log reduction) completely was 125, 50, and 25 s at 20, 30, and 40 V/cm, respectively. Higher electric field strength leads to greater heat generation (\u003cstrong\u003eTable S2\u003c/strong\u003e), thereby requiring less time to achieve lethal temperatures. Additionally, as the electric field strength increased, the non-thermal effect also enhanced the inactivation of the microorganism. Hence, the temperature required to destroy the microorganism at a high electric field strength was lower compared to that at a low electric field strength. For example, at 60 Hz, the temperature for complete inactivation of \u003cem\u003eE. coli\u003c/em\u003e O157:H7 in pomelo juice at 20, 30, and 40 V/cm was 65.85\u0026deg;C, 52.83\u0026deg;C, and 42.5\u0026deg;C, respectively. Alternatively, at 500 Hz, the temperature for complete inactivation of \u003cem\u003eE. coli\u003c/em\u003e was 62.47\u0026deg;C, 52.97\u0026deg;C, and 43.53\u0026deg;C at 20, 30, and 40 V/cm with treatment times of 140, 60, and 25 s, respectively. The inactivation of \u003cem\u003eE. coli\u003c/em\u003e at different levels of electric field strengths at 500 Hz showed a similar trend to that at 60 Hz, but the required time for inactivation was longer due to slower heating rates at electric field strengths of 20 and 30 V/cm, resulting in increased processing time. This result was consistent with Lee et al study, indicating that higher electric field strength enhances the ability to inactivate microorganisms [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. Cho et al. (2020) performed increasing electric field strength when OH of soybean curd, and showed heating rate of soybean curd increased because of increased conductivity and the inactivation of \u003cem\u003eE. coli\u003c/em\u003e O157:H7 in soybean curd increased because of having an effect on cell membrane potential, increasing electroporation and expulsion of intracellular [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cem\u003e3.3 Influence of Non-Thermal Factors on\u003c/em\u003e E. coli \u003cem\u003eInactivation in Pomelo Juice\u003c/em\u003e\u003c/h2\u003e\u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003e3.3.1 Impact of Non-Thermal Factors on\u003c/strong\u003e \u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003eDensity.\u003c/strong\u003e The survival capability of \u003cem\u003eE. coli\u003c/em\u003e O157:H7 during OH and CH is shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. In general, the density of the bacterium decreased with increased heating time. However, the survival rate of \u003cem\u003eE. coli\u003c/em\u003e after OH (at 60 Hz/500 Hz and 30 V/cm) and CH showed significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At 60 Hz, OH completely inactivated \u003cem\u003eE. coli\u003c/em\u003e O157:H7 (~\u0026thinsp;8 log reduction) in pomelo juice when it was heated to 52\u0026deg;C (50 s), whereas CH showed no signs of inactivation. It took a CH heating time of 70 s (equivalent to 72.6\u0026deg;C) to achieve complete inactivation. For OH at 500 Hz, a temperature of 52\u0026deg;C was reached in 55 s, resulting in complete inactivation of \u003cem\u003eE. coli\u003c/em\u003e (~\u0026thinsp;8 log reduction). CH required 75 s (equivalent to 72.3\u0026deg;C) for complete inactivation. The non-thermal factor increases microbial inactivation rate was showed in numerous studies [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e][\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. The presence of an external electric field can affect ions with net charges, leading to the translational motion of electric charges. This electric field may destabilize the cell membrane. Moreover, the diverse molecular motions associated with life processes, including metal ions (Mg\u003csup\u003e2+\u003c/sup\u003e and K\u003csup\u003e+\u003c/sup\u003e), as well as biomacromolecules such as nucleic acids and proteins, are also influenced [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e][\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. Thus, OH could inactivate \u003cem\u003eE. coli\u003c/em\u003e O157:H7 at lower temperatures and in a shorter time than CH.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003e3.3.2 Cell Morphology Determination.\u003c/strong\u003e This section details the morphological changes of \u003cem\u003eE. coli\u003c/em\u003e in PBW subjected to CH and OH. Overall, OH induced more significant changes in cell morphology compared to CH. The cells treated with CH showed intact or mildly disrupted cell structures (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). At 60 Hz, OH leads to cell shrinkage and more cell membrane damage, resulting in cellular leakage (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb). At 500 Hz, the cell membrane remained intact, but internal proteins were significantly denatured (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec). These results align with the findings of Park and Kang [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e], where OH causes higher cell membrane disruption compared to CH, contributing to increased bacteria inactivation and reducing the needed time and temperature for the pasteurization of pomelo juice.\u003c/p\u003e\n \u003c/span\u003e\n\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study investigated the impact of frequency, electric field strength, and non-thermal effects in OH on the inactivation of \u003cem\u003eE. coli\u003c/em\u003e O157:H7 in pomelo juice. The results showed that the most effective frequencies for inactivation were 60 and 500 Hz, and high electric field strength caused an increase in bacterial inactivation. Non-thermal influence in OH reduced the time and temperature required for pathogen destruction. It can be concluded that OH is a promising method for ensuring microbiological safety, allowing the processor to obtain good products in terms of nutritional and organoleptic quality. Applying OH technique to pasteurize pomelo juice on a pilot scale will be implemented in the next study.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Industrial University of Ho Chi Minh for facilities support, and the National Institute of Hygiene and Epidemiology for TEM (JEM 1400flash, \u0026nbsp;JEOL-Japan) device support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical \u0026nbsp;statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval was not required for this research\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that\u003cbr\u003e\u0026nbsp;could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: No funding\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDoan Nhu Khue\u003c/strong\u003e: The conception and design of the study, \u0026nbsp;Methodology, Analysis, Software, Data curation, Interpretation of data, Writing \u0026ndash; original draft, Writing -review and editing. \u003cstrong\u003eLai Quoc Dat\u003c/strong\u003e: The conception and design of the study, Validation, Final approval of the version to be submitted. \u003cstrong\u003eLe Thi Kim Phung\u003c/strong\u003e: The conception and design of the study. \u003cstrong\u003eLe Nhat Tam\u003c/strong\u003e: Methodology\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis manuscript does not report data generation. Data will be made available on request\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eA. Havelaar et al., World Health Organization global estimates and regional comparisons of the burden of foodborne disease in 2010. PLoS Med. \u003cb\u003e12\u003c/b\u003e(12), e1001923 (2015)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ.Y. Lim, J.W. Yoon, C.J. Hovde, A brief overview of Escherichia coli O157:H7 and its plasmid O157. J. Microbiol. Biotechnol. \u003cb\u003e20\u003c/b\u003e(1), 1\u0026ndash;10 (2010)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Singha, R. Thomas, J.N. Viswakarma, V.K. Gupta, Foodborne illnesses of Escherichia coli O157origin and its control measures. J. Food Sci. Technol. \u003cb\u003e60\u003c/b\u003e(4), 1274\u0026ndash;1283 (2023)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ.M. Rangel, P.H. Sparling, C. Crowe, P.M. Griffin, D.L. Swerdlow, Epidemiology of Escherichia coli O157:H7 outbreaks, United States, 1982\u0026ndash;2002. Emerg. Infect. Dis. \u003cb\u003e11\u003c/b\u003e(4), 603\u0026ndash;609 (2005). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3201/eid1104.040739\u003c/span\u003e\u003cspan address=\"10.3201/eid1104.040739\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eC. Hoff, C. Higa, J. Patel, K. Gee, E. Wellman, A. Vidanes, J. Schwensohn, Notes from the Field: An Outbreak of Escherichia coli O157: H7 Infections Linked to Romaine Lettuce Exposure\u0026mdash;United States, 2019. Morb Mortal. Wkly. \u003cb\u003e70\u003c/b\u003e(18), 689 (2021)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFDA, \u003cem\u003eGuidance for Industry: Juice HACCP Hazards and Controls Guidance First Edition; Final Guidance\u003c/em\u003e, vol. 17, pp. 1\u0026ndash;36, 2004\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM.J. Taylor, M.H. Tsai, H.C. Rasco, B. Tang, J., Zhu, Stability of Listeria monocytogenes in wheat flour during extended storage and isothermal treatment. Food Control. \u003cb\u003e91\u003c/b\u003e, 434\u0026ndash;439 (2018)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL. Bahrami, A. Baboli, Z.M. Schimmel, K. Jafari, S. M., Williams, Efficiency of novel processing technologies for the control of Listeria monocytogenes in food products. Trends Food Sci. Technol. \u003cb\u003e96\u003c/b\u003e, 61\u0026ndash;78 (2020)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS.J. Hern\u0026aacute;ndez-Hern\u0026aacute;ndez, H.M. Moreno-Vilet, L., Villanueva-Rodr\u0026iacute;guez, Current status of emerging food processing technologies in Latin America: Novel non-thermal processing. Innov. Food Sci. Emerg. Technol. \u003cb\u003e58\u003c/b\u003e, 102233 (2019)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG. Piette et al., Ohmic cooking of processed meats and its effects on product quality. J. Food Sci. \u003cb\u003e69\u003c/b\u003e(2), fep71\u0026ndash;fep78 (2004)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD. De Halleux, G. Piette, M.L. Buteau, M. Dostie, Ohmic cooking of processed meats: Energy evaluation and food safety considerations. Can. Biosyst Eng. / Le Genie des. Biosyst au Can. \u003cb\u003e47\u003c/b\u003e, 41\u0026ndash;47 (2005)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB. Aaliya et al., Recent trends in bacterial decontamination of food products by hurdle technology: A synergistic approach using thermal and non-thermal processing techniques, \u003cem\u003eFood Res. Int.\u003c/em\u003e, vol. 147, no. June, p. 110514, 2021\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ.A. Kubo, M.T. Siguemoto, \u0026Eacute;.S. Funcia, E.S. Augusto, P.E. Curet, S. Boillereaux, L. Gut, Non-thermal effects of microwave and ohmic processing on microbial and enzyme inactivation: a critical review. Curr. Opin. Food Sci. \u003cb\u003e35\u003c/b\u003e, 36\u0026ndash;48 (2020)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL. Jia, L. Shao, Y. Zhao, Y. Sun, X. Li, R. Dai, Inactivation effects and mechanism of ohmic heating on Bacillus cereus, \u003cem\u003eInt. J. Food Microbiol.\u003c/em\u003e, vol. 390, no. August, p. 110125, 2023\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD.N. Khue, H.T. Tiep, L.Q. Dat, L.T. Kim, Phung, L.N. Tam, Influence of frequency and temperature on the inactivation of Salmonella enterica serovar enteritidis in Ohmic heating of pomelo juice. LWT. \u003cb\u003e129\u003c/b\u003e, 109528 (2020)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eN.S. McKenna, Advances in radio frequency and ohmic heating of meats. J. Food Eng. \u003cb\u003e77\u003c/b\u003e(2), 215\u0026ndash;229 (2006)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS.M.B. Hashemi, A. Gholamhosseinpour, M. Niakousari, Application of microwave and ohmic heating for pasteurization of cantaloupe juice: microbial inactivation and chemical properties. J. Sci. Food Agric. \u003cb\u003e99\u003c/b\u003e(9), 4276\u0026ndash;4286 (2019)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG. Barbosa-C\u0026aacute;novas, D. Berm\u0026uacute;dez-Aguirre, Other novel milk preservation technologies: Ultrasound, irradiation, microwave, radio frequency, ohmic heating, ultraviolet light and bacteriocins. Improv. Saf. Qual. Milk. \u003cb\u003e1\u003c/b\u003e, 420\u0026ndash;450 (2010)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eF. Icier, Ohmic heating of fluid foods. \u003cem\u003eNovel thermal and non-thermal technologies for fluid foods\u003c/em\u003e, 305\u0026ndash;367, 2012. Academic\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Sarang, S.K. Sastry, L. Knipe, Electrical conductivity of fruits and meats during ohmic heating. J. Food Eng. \u003cb\u003e87\u003c/b\u003e(3), 351\u0026ndash;356 (2008)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH. Darvishi, M. Koushesh Saba, N. Behroozi-Khazaei, H. Nourbakhsh, Improving quality and quantity attributes of grape juice concentrate (molasses) using ohmic heating. J. Food Sci. Technol. \u003cb\u003e57\u003c/b\u003e(4), 1362\u0026ndash;1370 (2020)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eN.K. Doan, Q.D. Lai, T.K.P. Le, N.T. Le, Influences of AC frequency and electric field strength on changes in bioactive compounds in Ohmic heating of pomelo juice. Innov. Food Sci. Emerg. Technol. \u003cb\u003e72\u003c/b\u003e, 102754 (2021)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Lee, S. Ryu, D.H. Kang, Effect of Frequency and Waveform on Inactivation of Escherichia coli O157:H7 and Salmonella enterica Serovar Typhimurium in Salsa by Ohmic Heating. Appl. Environ. Microbiol. \u003cb\u003e79\u003c/b\u003e(1), 10\u0026ndash;17 (2013)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Kulshrestha, S. Sastry, Frequency and voltage effects on enhanced diffusion during moderate electric field (MEF) treatment. Innov. Food Sci. Emerg. Technol. \u003cb\u003e4\u003c/b\u003e(2), 189\u0026ndash;194 (2003)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Murashita, S. Kawamura, S. Koseki, Effects of ohmic heating, including electric field intensity and frequency, on thermal inactivation of bacillus subtilis spores. J. Food. Prot. \u003cb\u003e80\u003c/b\u003e(1), 164\u0026ndash;168 (2017)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ.H. Mok, T. Pyatkovskyy, A. Yousef, S.K. Sastry, Combined effect of shear stress and moderate electric field on the inactivation of Escherichia coli K12 in apple juice, \u003cem\u003eJ. Food Eng\u003c/em\u003e, vol. 262, no. May, pp. 121\u0026ndash;130, 2019\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG. Xu, D. Liu, J. Chen, X. Ye, Y. Ma, J. Shi, Food Chemistry Juice components and antioxidant capacity of citrus varieties cultivated in China, \u003cb\u003e106\u003c/b\u003e, pp. 545\u0026ndash;551, 2008\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM. Zhang, C. Duan, Y. Zang, Z. Huang, G. Liu, The flavonoid composition of flavedo and juice from the pummelo cultivar (Citrus grandis (L.) Osbeck) and the grapefruit cultivar (Citrus paradisi) from China. Food Chem. \u003cb\u003e129\u003c/b\u003e(4), 1530\u0026ndash;1536 (2011)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eI. Park, D. Kang, Effect of Electropermeabilization by Ohmic Heating for Inactivation of Escherichia coli O157: H7, Salmonella enterica Serovar Typhimurium, Juice, \u003cb\u003e79\u003c/b\u003e, 23, pp. 7122\u0026ndash;7129, 2013\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM.M. Shawki, A. Gaballah, the Effect of Low Ac Electric Field on Bacterial Cell Death. Rom J. Biophys. \u003cb\u003e25\u003c/b\u003e(2), 163\u0026ndash;172 (2015)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR.J.L. Montiel, I. Bardasano, J.L., Biophysical Device For The Treatment Of Neurodegenerative Diseases, in \u003cem\u003eRecent Advances in Multidisciplinary Applied Physics: Proceedings of the first international meeting on Applied Physics\u003c/em\u003e, pp. 63\u0026ndash;70, 2003\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ. Awuah, G.B. Ramaswamy, H. S., Tang, \u003cem\u003eRadio-Frequency heating in food processing: Principles and applications\u003c/em\u003e (CRC., 2014)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eE. Tripoli, M. La Guardia, S. Giammanco, D. Di Majo, M. Giammanco, Citrus flavonoids: Molecular structure, biological activity and nutritional properties: A review. Food Chem. \u003cb\u003e104\u003c/b\u003e(2), 466\u0026ndash;479 (2007)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eE.I. Oikeh, E.S. Omoregie, F.E. Oviasogie, K. Oriakhi, Phytochemical, antimicrobial, and antioxidant activities of different citrus juice concentrates. Food Sci. Nutr. \u003cb\u003e4\u003c/b\u003e(1), 103\u0026ndash;109 (2016)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ. Lee, S. Kim, D. Kang, LWT - Food Science and Technology Effect of pH for inactivation of Escherichia coli O157: H7, Salmonella Typhimurium and Listeria monocytogenes in orange juice by ohmic heating. LWT - Food Sci. Technol. \u003cb\u003e62\u003c/b\u003e(1), 83\u0026ndash;88 (2015)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS.-Y. Lee, H.-G. Sagong, S. Ryu, D.-H. Kang, Effect of continuous ohmic heating to inactivate Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes in orange juice and tomato juice. J. Appl. Microbiol. \u003cb\u003e112\u003c/b\u003e(4), 723\u0026ndash;731 (2012)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD.H. Cho, E.R. Kim, S. S., Kang, Inactivation kinetics and membrane potential of pathogens in soybean curd subjected to pulsed ohmic heating depending on applied voltage and duty ratio, \u003cem\u003eAppl. Environ. Microbiol.\u003c/em\u003e, vol. 86, no. 14, pp. e00656-20, 2020\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM.T. Kubo et al., Non-thermal effects of microwave and ohmic processing on microbial and enzyme inactivation: a critical review, \u003cem\u003eCurr. Opin. Food Sci.\u003c/em\u003e, vol. 35, no. January, pp. 36\u0026ndash;48, 2020\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ohmic heating, pomelo juice, electrical field strength, frequency, E. coli O157:H7, inactivation","lastPublishedDoi":"10.21203/rs.3.rs-3922948/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3922948/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe influence of frequency, electric field strength, and non-thermal effects during ohmic heating (OH) on the inactivation of \u003cem\u003eEscherichia coli\u003c/em\u003e O157:H7 in pomelo juice was investigated. Pomelo juice was inoculated with a specific density of \u003cem\u003eE. coli\u003c/em\u003e O157:H7 and then treated with OH at frequencies ranging from 50 Hz to 20 kHz and electric field strengths of 20, 30, and 40 V/cm. The results showed that 60 and 500 Hz were more effective in inactivating \u003cem\u003eE. coli\u003c/em\u003e than other frequencies. As electric field strength increased, inactivation also increased. Transmission electron microscopy analysis revealed that the cell membrane of \u003cem\u003eE. coli\u003c/em\u003e O157:H7 treated with OH underwent more pronounced changes than cells treated with conventional heating (CH). OH could inactivate \u003cem\u003eE. coli\u003c/em\u003e O157:H7 at lower temperatures and in a shorter time than CH. These findings demonstrated the potential of OH for pasteurizing pomelo juice.\u003c/p\u003e","manuscriptTitle":"Influence of Frequency and Electric Field Strength on the Inactivation of Escherichia coli O157:H7 During the Ohmic Heating Processing of Pomelo Juice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-14 18:28:10","doi":"10.21203/rs.3.rs-3922948/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"059ad961-6ecf-42c5-b3a6-245846111002","owner":[],"postedDate":"February 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-13T05:16:00+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-14 18:28:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3922948","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3922948","identity":"rs-3922948","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}