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Antimicrobial mechanism of pulsed light for the control of Escherichia coli O157:H7 and its application in carrot juice
Food Control 106 (2019) 106751
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Antimicrobial mechanism of pulsed light for the control of Escherichia coli O157:H7 and its application in carrot juice
T
Yulin Zhua, Changzhu Lib, Haiying Cuia,∗, Lin Lina,∗∗ a b
School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, China Department of Bioresource, Hunan Academy of Forestry, Changsha, 410007, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Escherichia coli O157:H7 Pulsed light Antimicrobial mechanism Enzyme activity Carrot juice
As a non-thermal sterilization method, pulsed light (PL) has been widely applied in food industries to control the microbial contamination, however little is known about the antimicrobial mechanism of PL. This study deeply investigated the antimicrobial mechanism of PL against Escherichia coli O157:H7 (E. coli O157:H7). The changes of morphology, endoenzyme, entocyte, respiratory metabolism and DNA of E. coli O157:H7 after PL treatment were emphatically studied. TEM and SEM results exhibited that cell integrity was destroyed heavily after PL treatment. Entocyte including proteins, ATP and DNA were reduced remarkably for PL-treated samples. In addition, the activity of ATPase, β-galactosidase, alkaline phosphatase, topoisomerase, as well as the metabolism were inhibited for E. coli O157:H7 with PL treatment. The electron spin resonance spectrum validated the production of hydroxyl radical by PL, thus resulting in the elevation of intracellular reactive oxygen and the acceleration of cell death. Finally, PL displayed better antibacterial activity in carrot juice without adverse effect on its quality.
1. Introduction Vegetable juices are prevailing beverages in the world due to their healthcare functions and abundant nutrition. However, they are vulnerable by different microorganism. Escherichia coli O157:H7 (E. coli O157:H7) is the representative bacteria of life-threatening foodborne pathogen in vegetable juices. E. coli O157:H7 can cause severe clinical symptoms like diarrhea, pyrexia, septicemia, further still induce hemolytic uremic syndrome to human body at all ages, especially for those with lower immunity like baby, elders and patients (Lin, Wang, He, & Cui, 2019b). In the United States, it was reported that 63153 illnesses and 2138 hospitalizations were associated with E. coli O157:H7 infections, which resulted in 20 deaths and 255 million loss in money each year (Batz, Hoffmann, & Morris, 2012). In order to minimize E. coli O157:H7 infections in vegetable juices, general physical and chemical methods are applied to reduce the microorganism load. However, undesired disadvantages such as side effect of some chemical additives, adverse impact to food quality by heat treatment restrain the application of part of the physical and chemical methods (Jeon & Ha, 2018). Pulsed light (PL) is the emerging technology gaining increased attentions as the effective, nonthermal, alternative and simple method to
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reduce the risk of microbial infection. Different pulsed light equipment contains several common components, including a flash lamp filled with Xenon gas, a high voltage power supply and a storage capacitor, which the pulsed light is produced by Xenon gas discharge flash lamp converting input electricity into light pulses of intense broad spectrum (100–1100 nm) rich in UV lights (Chen et al., 2018). PL is warranted with excellent antibacterial activity against a broad-spectrum of pathogens. Bactericidal performance of PL has been extensively verified on several food-borne pathogenic bacteria, such as salmonella, Escherichia coli, Listeria monocytogenes and Pseudomonas fluorescens, where reductions of these bacteria acquire from 1 to 3.4 log units (Hierro et al., 2011; Keklik, Demirci, & Puri, 2010; Nicorescu, Nguyen, Chevalier, & Orange, 2014). The efficiency of PL sterilization is closely associated with the input voltage power, the gap between samples and flash lamp, frequency and the number of applied pulses (Kramer, Wunderlich, & Muranyi, 2017). However, it is still not exactly known the antimicrobial mechanism of PL against a series of bacteria, especially on molecular level. Some former researches merely ascribed the germicidal effect of pulsed light to photochemical and photothermal effects, which leading to the damage of cytoderm and leakage of vacuoles (Koch, Wiacek, & Braun, 2019). Herein, the objective of this study was to deeply explore the
Corresponding author. Corresponding author. E-mail addresses: [email protected] (H. Cui), [email protected] (L. Lin).
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https://doi.org/10.1016/j.foodcont.2019.106751 Received 14 June 2019; Received in revised form 2 July 2019; Accepted 3 July 2019 Available online 09 July 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
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2.4.2. Effect of PL on entocyte of E. coli O157:H7 2.4.2.1. Detection of protein content. Briefly, the E. coli O157:H7 with or without PL treatment were centrifuged at 4000 rpm for 10 min, washed twice with sterile PBS, then collecting the precipitated bacteria. After resuspension, the bacterial suspension was crushed in ice bath by an ultrasonic sonifier (300 W, 10 min, 1.1 s of ultrasonic interval). Finally, the protein content of different samples was detected by a bicinchoninic acid (BCA) test box (Lin, Wang, & Cui, 2019a).
antibacterial mechanism of pulsed light against E. coli O157:H7 on molecular level. The effect of PL on the cytomembrane, morphology, endoenzyme, genetic material, metabolism, respiratory metabolism and proteins of E. coli O157:H7 was investigated throughout the whole research. Besides, the application of PL on the control of E. coils O157:H7 in carrot juice was investigated as well. 2. Materials and methods
2.4.2.2. Leakage of protein from E. coli O157:H7. The leakages of protein from E. coli O157:H7 were monitored by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Generally, the bacteria with or without PL treatment were rapidly centrifuged at 5000 rpm, 4 °C for 8 min, the precipitated bacteria were collected, washed with PBS and resuspended in 1 mL of sterile water. Subsequently, the different bacterial suspensions were sonicated at 300 W for 10 min to disrupt the cell. After that, the collected samples were mixed with sample buffer (250 mmol/L Tris-HCl (pH 6.8), SDS (10%, w/v), bromophenol blue (0.5%, w/v), glycerine (50%, v/v) and β-mercaptoethanol (5%, v/v)) at the ratio of 4:1 and boiled for 8 min. Finally, the mixtures were centrifuged at 8000 rpm, 4 °C for 10 min and the supernatant were collected to conduct the SDS-PAGE (Lin, Zhu, & Cui, 2018).
2.1. Materials The strains of E. coli O157:H7 CICC 21530 were purchased from China Center of Industrial Culture Collection (Beijing, China), and incubated in nutrient broth (NB) at 37 °C for 24 h to obtain the log-phase bacteria. Pulsed light equipment was assembled in Jiangsu University (Zhenjiang, Jiangsu). Other agents used in this study were provided by the local supplier without further purification. 2.2. Pulsed light treatment The prepared bacterial samples were placed in the lab-scale PL chamber, which contained two xenon lamp tubes and a sample table. PL treatment was performed at the following conditions: (A) 0.5 Hz, 10 pulse numbers, 500 J of single pulse energy. (B) 0.5 Hz, 15 pulse numbers, 500 J of single pulse energy. (C) 0.5 Hz, 20 pulse numbers, 500 J of single pulse energy. (D) 0.5 Hz, 25 pulse numbers, 500 J of single pulse energy. The bacterial samples were fixed at a 10 cm distance from xenon lamp tubes.
2.4.2.3. Measurement of ATP content. The log phase bacteria were centrifuged at 6000 rpm, 4 °C for 10 min to collect the precipitated bacteria, then washed 3 times with PBS (0.03 M, pH 7.2) and finally resuspended in 2 mL of PBS (0.03 M, pH 7.2). After above steps, the bacteria suspension was suffered from PL treatment, another sample without PL treatment was regarded as control. All samples were centrifuged again (6000 rpm, 4 °C, 10 min) to collect the precipitation. The precipitated bacteria were dissolved in 500 μL lysozyme and 500 μL TE buffer solutions, followed by treating with an ultrasonic sonifier (300 W, 10 min, 1.1 s of ultrasonic interval) to break the cell. Ultimately, the ATP contents of different samples were measured by a ATP detection kit (Cui, Zhang, Zhou, Zhao, & Lin, 2015a).
2.3. Time-killing analysis of PL against E. coli O157:H7 The bacteriocidal activity of PL against E. coli O157:H7 was tested by the plate counting method (Cui, Zhang, Li, & Lin, 2018). In brief, after different PL treatment at the time interval of 0, 1, 2, 4 and 8 h at 37 °C, each bacterial suspension (105–106 CFU/mL) in phosphate buffer (PBS, 0.03 M, pH 7.2) was taken out and serially diluted for ten times. The residual quantity of bacteria was detected by pouring 100 μL of each diluent onto nutrient agar plates and incubated at 37 °C for 24 h. The bacterial suspension without PL treatment was served as the control.
2.4.2.4. Detection of DNA content. The changes of DNA contents of E. Coil O157:H7 with or without PL treatment were analyzed by a microultraviolet spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Initially, the DNA of different samples was extracted by a DNA Kit (TIANGEN Biotechnology, Beijing, China). After extraction, all samples were put into quartzose cuvettes and measured at 260 nm to compare the changes of DNA contents. In addition, a laser scanning confocal microscope (LSCM) (TCS SP5 II, Leica, Germany) was applied to visually observe the changes of DNA of different samples (Cui, Zhao, & Lin, 2015b). In short, the bacterial suspensions with or without PL treatment were added with equal volume of 4′, 6-diamidino-2-phenylindole (DAPI, 10 μg/mL) and reacted in dark for 10 min. After reaction, a bit of the mixture was dropwise added on the microscope slide for LSCM observation.
2.4. Antibacterial mechanism of PL against E. coli O157:H7 2.4.1. Effect of PL on the morphology of E. coli O157:H7 2.4.1.1. Scanning electron microscope (SEM). The morphology of E. coli O157:H7 with different treatment was monitored by a scanning electron microscope (FEI instrument, Inc., Hillsboro, OR, USA). Different bacteriological samples were fixed overnight in 2.5% glutaraldehyde solution (in 0.1 M phosphate buffer, pH 7.0) at 4 °C. Then samples were dehydrated in the following graded series of ethanol solutions: 50, 70, 80, and 90%, and three times with 100% (Zhang, Choi, & Park, 2018). Dried samples were mounted onto brass slip using conducting resin, and then sputter-coated with gold. Subsequently, they were examined at a 10 kV electron velocity to obtain final images.
2.4.3. Effect of PL treatment on the endoenzyme of E. coli O157:H7 2.4.3.1. Adenosine triphosphatese (ATPase). As for the detection of ATPase activity, an ATPase assay kit was employed to determine the absorbance at 660 nm using an ultraviolet spectrophotometer (Agilent Cary 60, Agilent Technologies Co Ltd, USA).
2.4.1.2. Transmission electron microscopy (TEM). The changes of cell integrity of E. coli O157:H7 after PL treatment was observed by a transmission electron microscopy (TEM) (Cui, Yuan, Li, & Lin, 2017). The E. coli O157:H7 without PL treatment was regarded as the control. All bacteriological samples were centrifuged at 6000 rpm for 10 min, washed 3 times with PBS (0.03 M, pH7.2) and collected. A copper mesh was immersed into the bacterial suspension, then drying for 30 min. After that, the copper mesh loaded bacteria were stained by phosphotungstic acid for TEM observation.
2.4.3.2. β-galactosidase. The E. coli O157:H7 with or without PL treatment were centrifuged and washed to collect the precipitated bacteria. Then, 0.5 mL of each lysozyme (10 mg/mL), TE buffer and PBS were added to resuspend the bacteria. The bacteria suspensions were treated by an ultrasonic sonifier (300 W, 10 min, 1.1 s of ultrasonic interval) to break the cell. After that, 0.5 mL of 10 mg/mL onitrophenyl-β-D-galactoside (ONPG) and 1000 μL of β-galactosidase 2
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was 100 G, static field was 3460 G, frequency was 9.85 GHz and power was 19.10 Mw (Escudero et al., 2019).
reaction buffer were added into 0.5 mL of the above bacteria suspensions. The absorbances of different samples at 405 nm were monitored by an ultraviolet spectrophotometer to compare the changes of β-galactosidase activity (Cui et al., 2018).
2.4.5.2. Formation of intracellular ROS induced by PL treatment. The detection of intracellular ROS after PL treatment was performed by the aid of fluorescence probe of 2′,7′-dichlorofluorescin diacetate (DCFHDA). After centrifugation and rinse, the collected E. coli O157:H7 were resuspended in DCFH-DA solutions (10 μmol/L) to reach the final concentration of 2 × 108 CFU/mL and incubated in dark at 37 °C for 30 min to allow the penetration of DCFH-DA into bacteria. After incubation, the mixture was washed three times to remove the free DCFH-DA, then suffered from PL treatment. The sample without PL treatment was served as the control. The fluorescence intensity of different samples was recorded by a microplate reader (Infinite F50, Thermo Fisher Co., USA) at the excitation wavelength of 488 nm and emission wavelength of 525 nm (Kramer, Wunderlich, & Muranyi, 2015).
2.4.3.3. Alkaline phosphatase (AKP). The AKP activity of different samples was measured by the AKP detection kit using an ultraviolet spectrophotometer (Agilent Cary 60, Agilent Technologies Co Ltd, USA) to detect the absorbance at 520 nm. 2.4.3.4. Topoisomerase (Topo). The TopoⅠand TopoⅡwere extracted from E. coli O157:H7. The activity of TopoⅠand TopoⅡ was measured using pBR 322 DNA relaxation reaction by the following procedure: 0.5 μL pBR 322 DNA and different volume of Topo with or without PL treatment were poured into 20 μL of reaction buffer to react at 37 °C for 30 min. After that, the reaction was terminated by addition of 2 μL of sodium dodecyl sulfate (10%, w:v) and 0.5 μL of proteinase K (20 mg/ mL) for 30 min at 37 °C. Finally, the mixtures containing different samples were loaded onto agarose gel (0.8%, w:v) and performed at 115 V for 30 min (Wang, Zou, Xie, & Xie, 2014).
2.5. Application of PL treatment on the control of E. coli O157:H7 in carrot juice The carrot was cut into pieces and squeezed to obtain vegetable stock solution. Next, the stock solution was mixed with sterile water at the ratio of 1:9 to simulate carrot juice. After sterilization by an autoclave, the carrot juice was inoculated with 105–106 CFU/mL of E. coli O157:H7, followed by treating with PL at the following conditions: (A) 0.5 Hz, 20 pulse numbers, 500 J of single pulse energy. (B) 0.5 Hz, 25 pulse numbers, 500 J of single pulse energy. (C) 0.5 Hz, 30 pulse numbers, 500 J of single pulse energy. The residual amount of E. coli O157:H7 in carrot juices after PL treatment was monitored everyday for 4 days at 4 °C by plate counting method. The E. coli O157:H7 inoculated carrot juice without PL treatment was served as the control.
2.4.4. Effect of PL treatment on the vitality of E. Coil O157:H7 2.4.4.1. Oxidative respiratory metabolism. The effect of PL treatment on the oxidative respiratory metabolism of E. coli O157:H7 was measured by typical inhibitor method (Hu, Li, Dai, Cui, & Lin, 2019). Initially, the dissolved oxygen content of initial solutions (3.6 mL of 0.03 M, pH 7.2 PBS, 0.4 mL of 1% glucose, 1 mL of 106 CFU/mL bacteria) after exposure to air for 5 min was determined by a dissolved oxygen meters (INESA instrument CO., LTD., Shanghai, Chian) and marked as R0. Then the initial solutions were added with individual three kinds of inhibitors (iodoacetic acid, malonic acid and sodium phosphate) or treated with PL to determine the dissolved oxygen content and marked as R1. Next, the initial solutions were treated with respective inhibitor in combination with PL to determine the dissolved oxygen content and marked as R2. Inhibiting rate (IR) and superpose rate (DR) were calculated by the following equation.
IR = DR =
2.6. Sensory evaluation After different PL treatment (A, B and C as described in section 2.5), the carrot juices were stored at 4 °C for 2 days, and the qualities of carrot juices were evaluated in terms of their color, taste, flavor and comprehensive acceptability by 10 untrained students using a 9-point hedonic scale (1 meaned extremely dislike, 9 meaned extremely like).
R 0 − R1 × 100% R0 R1 − R2 × 100% R1
2.7. Statistical analysis
After determining the major respiratory metabolic pathway that PL played role in, the activity of some critical enzymes in this pathway was detected as well by corresponding kit.
All experiments were performed at least for three times to obtain the value of average ± standard deviation (SD). The significance analysis was tested by One-way analysis of variance (ANOVA) of SPSS software at the level of P < 0.05.
2.4.4.2. Metabolism. The metabolic level of bacteria with different treatments was measured by resazurin. In general, the bacterial suspensions with or without PL treatment were added with 100 μg/ ml of resazurin solution and placed in a shaker for 2 h in the dark. Then, the cultured bacterial suspension was centrifuged at 10,000 rpm for 4 min, and the supernatant was collected and detected under a fluorescent microplate reader at excitation wavelength of 560 nm and emission wavelength of 590 nm.
3. Results and discussion 3.1. Time-killing analysis In the time-killing analysis (Fig. 1), it could be seen that the population of bacteria in control group remained stable during 8 h incubation. In comparison with control group, the antibacterial activity of group A and group B was slight. However, in group C, the amount of E. coli O157:H7 decreased from 5.56 to 1.02 log CFU/mL within 4 h incubation, still further decreased to undetectable level after 8 h reaction. This result indicated that 0.5 Hz, 20 pulse numbers, 500 J of single pulse energy of PL treatment was enough for the sterilization of E. coli O157:H7. Thus we selected this condition for the following study of antibacterial mechanism.
2.4.5. Detection of free radicals and intracellular reactive oxygen species (ROS) 2.4.5.1. The species of free radicals generated by PL. The detection of free radicals during PL treatment was implemented by the electron spin resonance (ESR) (A300 10/12, Bruker Co., LTD., German). The bacterial suspensions (108 CFU/mL, 80 μL) after PL treatment were immediately mixed with 20 μL of 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO, Aladdin, China) and removed into a capillary, then placed in the cavity of the ESR instrument. The bacterial suspension without PL treatment was regarded as the control. The parameters of ESR instrument were set as follows: center field was 3510 G, sweep width
3.2. Morphological changes of E. coli O157:H7 The morphological changes of E. coli O157:H7 with or without PL 3
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Fig. 1. Time killing analysis of PL against E. Coil O157:H7.
Fig. 2. SEM pictures of (A) control (B) PL-treated E. Coil O157:H7. TEM pictures of (C) control (D) PL-treated E. Coil O157:H7. The white arrows indicated the malformed bacteria, and the yellow arrows indicated the sunken bacteria.
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Fig. 3. (A) SDS-PAGE picture of E. Coil O157:H7 with different treatment. C meaned control group, T indicated the bacteria treated with PL. (B) Changes of protein concentration of E. Coil O157:H7 after different treatment. (C) Changes of ATP content of E. Coil O157:H7 after different treatment. The a and b in Fig. 2B and C meaned significant difference between two samples.
Georgiou, & Habraken, 2016). As seen from Fig. 3B, the protein concentration of PL-treated bacteria was 241 μg/mL. Compared with 357 μg/mL of control group, 32.49% reduction in protein quantity after PL treatment was achieved. This conclusion was consistent with SDSPAGE result. As revealed in Fig. 3A, the protein bands of PL-treated E. coli O157:H7 were light and dim than control group, indicating the leakage of proteins after PL treatment. The reason may be ascribed to the destructive effect of PL to the cell structure of E. coli O157:H7, leading to the leakage of intracellular proteins (Qian et al., 2016).
treatment were revealed by TEM and SEM. Seen from Fig. 2A, the untreated E. coli O157:H7 exhibited bacilliform morphology and the surface looked plump and intact. Conversely, the PL-treated bacteria (Fig. 2B) were irregular and shrinking, with some hollowness exhibited on the surface. What's more, sunken and malformed bacteria were observed as well after PL treatment. TEM pictures showed the similar trend with SEM results. As seen in Fig. 2C, the untreated bacteria were intact, unbroken and smooth, with integrated cell membrane and slippy surface. In contrast, the PL-treated bacteria (Fig. 2D) were incomplete and broken. The phospholipid bilayer of bacteria after PL treatment was deformed and destroyed heavily, resulting in the leakage of entocyte in cells, which accelerated the apoptosis of E. coli O157:H7. TEM and SEM results demonstrated that PL treatment played an destructive effect on the cytomembrane and altered the cell structure of E. coli O157:H7, which can be regarded as the antimicrobial mechanism of PL against E. coli O157:H7 (Bajpai, Saha, & Basu, 2012).
3.4. Detection of ATP content The changes of ATP content were revealed in Fig. 3C. ATP is the energy factor in bacterial cells, providing power and energy to the metabolism and vitality of E. coli O157:H7. The ATP content was decreased by 59.47% after PL treatment, compared with the samples without PL treatment. Yang, Li, and Zhang (2010) found the similar tendency for the ATP contents of bacteria after pulsed electric fields treatment. In conclusion, PL treatment significantly reduced the ATP content of E. coli O157:H7, thus impacting the vitality and survival of bacteria.
3.3. Protein concentration and protein leakage Protein is the most important biomacromolecule that exists widely in various organelles and cytoplasm of bacterial cell, acting as different roles such as catalyzator and proppant to support the structure and vitality of the living cells (Zhang, Liu, Wang, Jiang, & Quek, 2016). The release of intracellular proteins and changes of protein content can be regarded as the indication for the integrality of cell structure (Omelon,
3.5. Changes of DNA DNA is the most important genetic substance that controls the
Fig. 4. (A) Changes of DNA content of E. Coil O157:H7 after different treatment. The a and b meaned significant difference between two samples. (B) CLSM pictures of control and PL-treated groups. 5
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Fig. 5. (A) Detection of different ATPase activity of E. Coil O157:H7 after different treatment. (B) The changes of β-galactosidase and AKP activity of E. Coil O157:H7 after different treatment. The a and b in Fig. 4A and B meaned significant difference between two samples. Fig. 6. Effect of PL treatment on TopoⅠand TopoⅡ in E. Coil O157:H7. The a,b, c and d indicated that the PBR 322DNA was treated with different amount (0, 2, 4 and 6 μL) of topoisomerase. The A, B, C and D indicated that the PBR 322DNA was reacted with different amount (0, 2, 4 and 6 μL) of PL-treated topoisomerase. Form Ⅰ, supercoiled DNA. Form Ⅱ, linear DNA or open circular DNA.
hydrolyze the lactose into galactose and glucose, therefore providing the energy and carbon sources for the vitality of bacteria (Watson & Chiu, 2016). ONPG is the analogue of lactose. It can be hydrolyzed to Onitrophenol under the action of β-galactosidase, and O-nitrophenol showed the absorption peak at 405 nm. Hence, the difference of β-galactosidase activity can be reflected by the changes of absorbance at 405 nm. As seen in Fig. 5B, the OD405nm value of PL-treated bacteria decreased by 36% compared with control group, indicating reduced activity of β-galactosidase after PL treatment. AKP is the enzyme exists between cytomembrane and cytoderm. As for the AKP activity (Fig. 5B), the PL-treated bacteria was 0.38 U/mg prot, compared with 0.21 U/mg prot of control group, which achieved 44.74% reduction in activity after PL treatment. This result indicated that PL treatment inhibited the activity and expression of AKP.
proliferation, growth and heredity. In normal conditions, DNA can not be detected outside the bacterial cells, unless the cell structure was destroyed by irresistible force. Fig. 4A revealed that DNA content decreased by 56% after PL treatment, when compared with control group. Phillips, Haggren, Thomas, Ishida-Jones, and Adey (1992) ascribed this phenomenon to the formation of genetically-destructive rings, hence resulting in the reduction and leakage of DNA. DAPI is the fluorochrome that can penetrate the cytomembrane and bind with DNA of bacteria (Omelon et al., 2016). The fluorescent pictures of DNA of E. coli O157:H7 with different treatment were shown in Fig. 4B. The pictures showed that the fluorescence intensity of control group was higher than PL-treated group, which demonstrated less DNA content of E. coli O157:H7 after PL treatment. The results obtained above indicated that PL treatment can accelerate the leakage of DNA from the damaged cell membrane or impede the synthesis of DNA of E. coli O157:H7, thereby DNA content of PL-treated group was much lower than E. coli O157:H7 without any treatment.
3.7. Topoisomerase DNA topoisomerase is a key enzyme in cell nucleus of bacteria, which can not only repair the topological structure of DNA, but also affect the physiological metabolism including genetic recombination, transcription and repair of nucleic acid (Chang, Nair, & Nitiss, 1995). Its main function is the catalyzing of breaking and unwinding of DNA. TopoⅠand TopoⅡ, as two main topoisomerases in bacterial cells, can unwinding the pBR 322 DNA from superhelical structure (Form Ⅰ) to linear or open circular form (Form Ⅱ) (Cantero, Campanella, Mateos, & Cortés, 2006). The effect of PL on the activity of TopoⅠand TopoⅡwas investigated by the enzyme-mediated supercoiled pBR322 relaxation. As revealed in Fig. 6, the band of superhelical form (Form Ⅰ) of pBR322 relaxation (a) became dim and light after addition of different volume of TopoⅠand TopoⅡextracting solutions (b, c, d), indicating better unwinding effect of the extracted enzyme solutions to superhelical DNA.
3.6. Effect of PL treatment on ATPase, β-galactosidase and AKP ATPase is the spherical protein which is important for energy metabolism of bacteria. Its catalytic activity depends on the native configuration and conformation of active sites and surrounding proteins. The changes of ATPase activity of E. coli O157:H7 with different treatment were shown in Fig. 5A. As the picture revealed, the activity of Na+ K+ ATPase, Ca2+ ATPase and Mg2+ ATPase of bacteria treated with PL decreased by 64.26%, 51.07% and 26.50% respectively, as compared with the E. coli O157:H7 without PL treatment. This result indicated that PL may change the structure and conformation of protein, thus resulting in the reduction of ATPase activity. β-galactosidase is a hydrolase exists in the cytoplasm. It can 6
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Table 1 The inhibiting rate (IR) of iodoacetic acid, malonic acid, sodium phosphate and PL. Inhibitors
R0/μmol O2 (g.min)−1
R1/μmol O2 (g.min)−1
IR (%)
Iodoacetic acid Malonic acid Sodium phosphate PL
0.64 0.58 0.61 0.59
0.52 0.41 0.57 0.37
18.75 29.31 6.56 37.29
Table 2 The superpose rate (DR) of PL with different inhibitors. Inhibitors
R2/μmol O2 (g.min)−1
DR (%)
Iodoacetic acid + PL Malonic acid + PL Sodium phosphate + PL
0.41 0.39 0.45
21.15 4.88 26.67
However, After addition of PL-treated TopoⅠand TopoⅡ(B,C,D), the unwinding effect to superhelical DNA (Form Ⅰ) was not apparent compared with TopoⅠand TopoⅡ without PL treatment. The results indicated that PL affected the activity of TopoⅠand TopoⅡ, thereby impacting the metabolism of nucleic acid and bacterial growth. 3.8. Oxidative respiration metabolism and metabolism Fig. 7. The tricarboxylic acid cycle pathway.
The metabolism of glucose mainly rely on three pathways in bacterial cells, including embden-meyerhof-parnas (EMP) pathway, tricarboxylic acid cycle (TCA) and hexose monophosphate pathway (HMP) pathway. Iodoacetic acid, malonic acid and sodium phosphate are the typical inhibitors of EMP, TCA and HMP pathways respectively. The inhibiting rate (IR) and superpose rate (DR) were shown in Table 1 and Table 2. As the table presented, the PL possessed the highest IR than iodoacetic acid, malonic acid and sodium phosphate, indicating PL possessed of better inhibiting ability to the metabolic pathways of E. coli O157:H7. Superpose rate (DR) was used to determine the pathway that PL mainly imposed effort on. Smaller superpose rate (DR) meaned weaker cooperative effect between typical inhibitors and PL treatment, so PL may inhibit the same pathway with the corresponding typical inhibitor (Cui et al., 2018). As seen in Table 2, the superpose rate (DR) of malonic acid + PL treated group was the minimum, meaning PL mainly suppressed the oxidative respiration metabolism of E. coli O157:H7 through TCA pathway. The process of TCA pathway was displayed in Fig. 7. In order to detailedly explored the effect of PL treatment on the TCA pathway of E. coli O157:H7, the detection of the activity of three key enzymes-citrate synthase (CS), isocitrate dehydrogenase (ICDH) and α-ketoglutarate dehydrogenase (α-KGDH) in TCA pathway after different treatment were detected as well. As revealed in Fig. 8A, the activity of CS, ICDH and α-KGDH was impacted after PL treatment. Especially for ICDH and α-KGDH, they were affected largely by 38.10% and 52.63% reduction in activity after PL treatment. Hence, PL can exert influence on the activity of three key enzymes, and then inhibit the energy metabolism of TCA pathway. The changes of metabolism of E. coli O157:H7 after PL treatment were revealed in Fig. 8B. After penetrating into bacteria, the weak fluorescence resazurin can be degraded by various oxidoreductases into high fluorescence resorufin. Hence, it is feasible to feature the metabolism of E. coli O157:H7 by the changes of fluorescence intensity. From Fig. 8B presented, the metabolism capacity of E. coli O157:H7 decreased gradually after PL treatment. It was clearly seen that after 8 h incubation, the metabolism capacity of E. coli O157:H7 decreased to 39.42%. However, in time-killing analysis, the number of bacteria was undetectable after 8 h incubation. This was ascribed to the sublethal state of bacteria. In this condition, the bacteria were nonculturable on
nutrient medium but still maintained their metabolic activity (Ziuzina, Boehm, Patil, Cullen, & Bourke, 2015). 3.9. Detection of free radicals and intracellular ROS Fig. 9A showed the ESR spectrogram of bacterial suspension with or without PL treatment. 5.5-Dimethyl-1-pyrroline N-oxide (DMPO) was added immediately into bacterial suspension after different treatments to trap the free radicals. The ESR spectrum of control group showed that no free radicals were detected when PL was not applied, indicating that DMPO can not be oxidized by ambient environment (Tahara & Okubo, 2014). After PL treatment, obvious peaks in magnetic field (marked with *), featured as quartet were seen in ESR spectrum of PLtreated samples and ascribed to the existence of plentiful of hydroxyl radical (·OH) (Xie et al., 2019). This result demonstrated that hydrogen peroxide can be produced during PL treatment process and gradually disintegrated to hydroxyl radical (Tahara & Okubo, 2014). Furthermore, hydrogen peroxide can be disintegrated to superoxide anion (·O) and perhydroxyl radical (·OOH) as well as revealed by the following equation:
H2 O2 + 2OH− → ⋅O−2 + 2H2 O+ e
O−2 + H+ → ⋅OOH However, only hydroxyl radicals (·OH) were detected in the ESR spectrum right now, that was because DMPO can capture ·OH more readily than ·O and ·OOH and form a spin adduct with a longer lifetime by the following equation: H3C H3C
N
O-
H
.
OH
H3C
OH H3C
N
H
O-
The generated hydroxyl radical (·OH) by PL treatment can attack the lipid bilayer of cell membrane, as well as induce the generation of intracellular reactive oxygen, thus accelerating the death of E. coli 7
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Fig. 8. (A) Effect of PL on the activity of enzymes-citrate synthase (CS), isocitrate dehydrogenase (ICDH) and α-ketoglutarate dehydrogenase (α-KGDH) in TCA pathway. (B) The changes of metabolism of E. Coil O157:H7 after PL treatment.
O157:H7. The intracellular ROS of E. coli O157:H7 were remarkably increased after PL treatment as revealed in Fig. 9B. High concentration of ROS may hinder the function of some organelles within the cells, thus leading to the destruction of cell structure. 3.10. Antibacterial activity of PL in carrot juice and sensory evaluation The antibacterial effect of PL on E. coli O157:H7 in carrot juice was shown in Fig. 10. As the picture presented, the group C (0.5 Hz, 30 pulse numbers, 500 J of single pulse energy) exhibited better antibacterial effect in carrot juices, which sterilized the bacteria to undetectable level after 3 days incubation. In addition, seen from the results of sensory evaluation (Table 3), it implied that the difference of color, taste, flavor and comprehensive acceptability between control and PL-treated group was not significant, indicating that PL have no adverse effect on the quality of carrot juice. Fig. 10. Antibacterial effect of PL against E. Coil O157:H7 in carrot juice.
4. Conclusion affecting the glycometabolism of bacteria by TCA pathway and key enzymes in this pathway. The ESR spectrum indicated that PL can generate the hydroxyl radicals during treatment process, leading to the increase in the amount of intracellular ROS, accelerating the death of bacteria from the outside and the inside attack. Finally, PL (0.5 Hz, 30 pulse numbers, 500 J of single pulse energy) sterilized the bacteria to undetectable level after 3 days incubation in carrot juice, without significant impact on its quality, so PL can be regarded as an effective
In this work, the antibacterial activity and antimicrobial mechanism of PL against E. coli O157:H7 were thoroughly investigated. After PL treatment, the morphology and cell integrity of E. coli O157:H7 were damaged seriously as revealed by TEM and SEM results. The contents of protein, ATP and DNA were reduced by 32.49%, 59.47% and 56% respectively, for E. coli O157:H7 treated by PL. Furthermore, PL can inhibit the activity of some key enzymes and metabolism in E. coli O157:H7. The respiration metabolism test demonstrated that PL mainly
Fig. 9. (A) ESR spectrum of control and PL treated groups. (B) Detection of intracellular ROS after PL treatment in E. Coil O157:H7. The a and b in Fig. 8B meaned significant difference between two samples. 8
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Table 3 Sensory evaluation of carrot juice. Parameters
Control
Color Flavor Taste Comprehensive acceptability
7.23 6.83 5.67 5.32
± ± ± ±
A 0.28 0.12 0.15 0.18
7.19 6.85 5.72 5.24
B ± ± ± ±
0.17 0.27 0.23 0.21
7.32 6.74 5.75 5.38
Garcia, A. B., et al. (2019). Electron spin resonance (ESR) spectroscopy study of cheese treated with accelerated electrons. Food Chemistry, 276, 315–321. Hierro, E., Barroso, E., Hoz, L. D. L., Ordóñez, J. A., Manzano, S., & Fernández, M. (2011). Efficacy of pulsed light for shelf-life extension and inactivation of Listeria monocytogenes on ready-to-eat cooked meat products. Innovative Food Science & Emerging Technologies, 12(3), 275–281. Hu, W., Li, C., Dai, J., Cui, H., & Lin, L. (2019). Antibacterial activity and mechanism of Litsea cubeba essential oil against methicillin-resistant Staphylococcus aureus (MRSA). Industrial Crops and Products, 130, 34–41. Jeon, M.-J., & Ha, J.-W. (2018). Efficacy of UV-A, UV-B, and UV-C irradiation on inactivation of foodborne pathogens in different neutralizing buffer solutions. LWT Food Science and Technology, 98, 591–597. Keklik, N. M., Demirci, A., & Puri, V. M. (2010). Inactivation of Listeria monocytogenes on unpackaged and vacuum-packaged chicken frankfurters using pulsed UV-light. Journal of Food Science, 74(8), 431–439. Koch, F., Wiacek, C., & Braun, P. G. (2019). Pulsed light treatment for the reduction of Salmonella Typhimurium and Yersinia enterocolitica on pork skin and pork loin. International Journal of Food Microbiology, 292, 64–71. Kramer, B., Wunderlich, J., & Muranyi, P. (2015). Pulsed light induced damages inListeria innocuaand Escherichia coli. Journal of Applied Microbiology, 119(4), 999–1010. Kramer, B., Wunderlich, J., & Muranyi, P. (2017). Impact of treatment parameters on pulsed light inactivation of microorganisms on a food simulant surface. Innovative Food Science & Emerging Technologies, 42, 83–90. Lin, L., Wang, X., & Cui, H. (2019a). Synergistic efficacy of pulsed magnetic fields and Litseacubeba essential oil treatment against Escherichia coli O157:H7 in vegetable juices. Food Control, 106, 106686. Lin, L., Wang, X., He, R., & Cui, H. (2019b). Action mechanism of pulsed magnetic field against E. coli O157:H7 and its application in vegetable juice. Food Control, 95, 150–156. Lin, L., Zhu, Y., & Cui, H. (2018). Electrospun thyme essential oil/gelatin nanofibers for active packaging against Campylobacter jejuni in chicken. LWT Food Science and Technology, 97, 711–718. Nicorescu, I., Nguyen, B., Chevalier, S., & Orange, N. (2014). Effects of pulsed light on the organoleptic properties and shelf-life extension of pork and salmon. Food Control, 44, 138–145. Omelon, S., Georgiou, J., & Habraken, W. (2016). A cautionary (spectral) tail: Red-shifted fluorescence by DAPI-DAPI interactions. Biochemistry Society Transaction, 44(1), 46–49. Phillips, J. L., Haggren, W., Thomas, W. J., Ishida-Jones, T., & Adey, W. R. (1992). Magnetic field-induced changes in specific gene transcription. Biochimica et Biophysica Acta, 1132(2), 140–144. Qian, J., Zhou, C., Ma, H., Li, S., Yagoub, E. G. A., & Abdualrahman, M. A. Y. (2016). Biological effect and inactivation mechanism of Bacillus subtilis exposed to pulsed magnetic field: Morphology, membrane permeability and intracellular contents. Food Biophysics, 11(4), 1–7. Tahara, M., & Okubo, M. (2014). Detection of free radicals produced by a pulsed electrohydraulic discharge using electron spin resonance. Journal of Electrostatics, 72(3), 222–227. Wang, H., Zou, D., Xie, K., & Xie, M. (2014). Antibacterial mechanism of fraxetin against Staphylococcus aureus. Molecular Medicine Reports, 10(5), 2341–2345. Watson, A. L., & Chiu, N. H. L. (2016). Fluorometric cell-based assay for β-galactosidase activity in probiotic gram-positive bacterial cells — lactobacillus helveticus. Journal of Microbiological Methods, 128, 58–60. Xie, Y., Jiang, S., Li, M., Guo, Y., Cheng, Y., Qian, H., et al. (2019). Evaluation on the formation of lipid free radicals in the oxidation process of peanut oil. Lwt Food Science and Technology, 104, 24–29. Yang, R. J., Li, S. Q., & Zhang, Q. H. (2010). Effects of pulsed electric fields on the activity of enzymes in aqueous solution. Journal of Food Science, 69(4), 241–248. Zhang, Y., Choi, J. R., & Park, S. J. (2018). Enhancing the heat and load transfer efficiency by optimizing the interface of hexagonal boron nitride/elastomer nanocomposites for thermal management applications. Polymer, 143, 1–9. Zhang, Y., Liu, X., Wang, Y., Jiang, P., & Quek, S. Y. (2016). Antibacterial activity and mechanism of cinnamon essential oil against Escherichia coli and Staphylococcus aureus. Food Control, 59, 282–289. Ziuzina, D., Boehm, D., Patil, S., Cullen, P. J., & Bourke, P. (2015). Cold plasma inactivation of bacterial biofilms and reduction of quorum sensing regulated virulence factors. PLoS One, 10(9), 1–21.
C ± ± ± ±
0.24 0.25 0.19 0.25
7.26 6.89 5.59 5.42
± ± ± ±
0.19 0.16 0.20 0.16
A, B and C indicated different PL treatment (A: 0.5 Hz, 20 pulse numbers, 500 J of single pulse energy. B: 0.5 Hz, 25 pulse numbers, 500 J of single pulse energy. C:0.5 Hz, 30 pulse numbers, 500 J of single pulse energy). There was no significant difference between different samples.
sterilization methods after the deeply research of its antimicrobial mechanism against E. coli O157:H7. Declaration of interests 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. Acknowledgement This study was financially supported by National Natural Science Foundation of China (Grant no. 31470594), Natural Science Foundation of Jiangsu Province (Grant no. BK20170070), Hunan Science and Technology Major Project (Grant no. 2016NK1001-3), Jiangsu Province Research Fund (Grant no. NY-013) and Jiangsu University Research Fund (Grant no. 11JDG050). References Bajpai, I., Saha, N., & Basu, B. (2012). Moderate intensity static magnetic field has bactericidal effect on E. Coli and S. Epidermidis on sintered hydroxyapatite. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 100(5), 1206–1217. Batz, M. B., Hoffmann, S., & Morris, J. G. (2012). Ranking the disease burden of 14 pathogens in food sources in the United States using attribution data from outbreak investigations and expert elicitation. Journal of Food Protection, 75(7), 1278–1291. Cantero, G.,., Campanella, C.,., Mateos, S., & Cortés, F. (2006). Topoisomerase II inhibition and high yield of endoreduplication induced by the flavonoids luteolin and quercetin. Mutagenesis, 21(5), 321. Chang, Y. C., Nair, M. G., & Nitiss, J. L. (1995). Metabolites of daidzein and genistein and their biological activities. Journal of Natural Products, 58(12), 1901–1905. Chen, D., Wiertzema, J., Peng, P., Cheng, Y., Liu, J., Mao, Q., et al. (2018). Effects of intense pulsed light on Cronobacter sakazakii inoculated in non-fat dry milk. Journal of Food Engineering, 238, 178–187. Cui, H., Yuan, L., Li, W., & Lin, L. (2017). Antioxidant property of SiO2-eugenol liposome loaded nanofibrous membranes on beef. Food Packaging and Shelf Life, 11, 49–57. Cui, H., Zhang, C., Li, C., & Lin, L. (2018). Antimicrobial mechanism of clove oil on Listeria monocytogenes. Food Control, 94, 140–146. Cui, H., Zhang, X., Zhou, H., Zhao, C., & Lin, L. (2015a). Antimicrobial activity and mechanisms of Salvia sclarea essential oil. Botanical Studies, 56(6), 1–8. Cui, H., Zhao, C., & Lin, L. (2015b). The specific antibacterial activity of liposome-encapsulated Clove oil and its application in tofu. Food Control, 56, 128–134. Escudero, R., Segura, J., Velasco, R., Valhondo, M., Romero de Ávila, M. D., Garcia-
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Antimicrobial mechanism of pulsed light for the control of Escherichia coli O157:H7 and its application in carrot juice
T
Yulin Zhua, Changzhu Lib, Haiying Cuia,∗, Lin Lina,∗∗ a b
School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, China Department of Bioresource, Hunan Academy of Forestry, Changsha, 410007, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Escherichia coli O157:H7 Pulsed light Antimicrobial mechanism Enzyme activity Carrot juice
As a non-thermal sterilization method, pulsed light (PL) has been widely applied in food industries to control the microbial contamination, however little is known about the antimicrobial mechanism of PL. This study deeply investigated the antimicrobial mechanism of PL against Escherichia coli O157:H7 (E. coli O157:H7). The changes of morphology, endoenzyme, entocyte, respiratory metabolism and DNA of E. coli O157:H7 after PL treatment were emphatically studied. TEM and SEM results exhibited that cell integrity was destroyed heavily after PL treatment. Entocyte including proteins, ATP and DNA were reduced remarkably for PL-treated samples. In addition, the activity of ATPase, β-galactosidase, alkaline phosphatase, topoisomerase, as well as the metabolism were inhibited for E. coli O157:H7 with PL treatment. The electron spin resonance spectrum validated the production of hydroxyl radical by PL, thus resulting in the elevation of intracellular reactive oxygen and the acceleration of cell death. Finally, PL displayed better antibacterial activity in carrot juice without adverse effect on its quality.
1. Introduction Vegetable juices are prevailing beverages in the world due to their healthcare functions and abundant nutrition. However, they are vulnerable by different microorganism. Escherichia coli O157:H7 (E. coli O157:H7) is the representative bacteria of life-threatening foodborne pathogen in vegetable juices. E. coli O157:H7 can cause severe clinical symptoms like diarrhea, pyrexia, septicemia, further still induce hemolytic uremic syndrome to human body at all ages, especially for those with lower immunity like baby, elders and patients (Lin, Wang, He, & Cui, 2019b). In the United States, it was reported that 63153 illnesses and 2138 hospitalizations were associated with E. coli O157:H7 infections, which resulted in 20 deaths and 255 million loss in money each year (Batz, Hoffmann, & Morris, 2012). In order to minimize E. coli O157:H7 infections in vegetable juices, general physical and chemical methods are applied to reduce the microorganism load. However, undesired disadvantages such as side effect of some chemical additives, adverse impact to food quality by heat treatment restrain the application of part of the physical and chemical methods (Jeon & Ha, 2018). Pulsed light (PL) is the emerging technology gaining increased attentions as the effective, nonthermal, alternative and simple method to
∗
reduce the risk of microbial infection. Different pulsed light equipment contains several common components, including a flash lamp filled with Xenon gas, a high voltage power supply and a storage capacitor, which the pulsed light is produced by Xenon gas discharge flash lamp converting input electricity into light pulses of intense broad spectrum (100–1100 nm) rich in UV lights (Chen et al., 2018). PL is warranted with excellent antibacterial activity against a broad-spectrum of pathogens. Bactericidal performance of PL has been extensively verified on several food-borne pathogenic bacteria, such as salmonella, Escherichia coli, Listeria monocytogenes and Pseudomonas fluorescens, where reductions of these bacteria acquire from 1 to 3.4 log units (Hierro et al., 2011; Keklik, Demirci, & Puri, 2010; Nicorescu, Nguyen, Chevalier, & Orange, 2014). The efficiency of PL sterilization is closely associated with the input voltage power, the gap between samples and flash lamp, frequency and the number of applied pulses (Kramer, Wunderlich, & Muranyi, 2017). However, it is still not exactly known the antimicrobial mechanism of PL against a series of bacteria, especially on molecular level. Some former researches merely ascribed the germicidal effect of pulsed light to photochemical and photothermal effects, which leading to the damage of cytoderm and leakage of vacuoles (Koch, Wiacek, & Braun, 2019). Herein, the objective of this study was to deeply explore the
Corresponding author. Corresponding author. E-mail addresses: [email protected] (H. Cui), [email protected] (L. Lin).
∗∗
https://doi.org/10.1016/j.foodcont.2019.106751 Received 14 June 2019; Received in revised form 2 July 2019; Accepted 3 July 2019 Available online 09 July 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
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2.4.2. Effect of PL on entocyte of E. coli O157:H7 2.4.2.1. Detection of protein content. Briefly, the E. coli O157:H7 with or without PL treatment were centrifuged at 4000 rpm for 10 min, washed twice with sterile PBS, then collecting the precipitated bacteria. After resuspension, the bacterial suspension was crushed in ice bath by an ultrasonic sonifier (300 W, 10 min, 1.1 s of ultrasonic interval). Finally, the protein content of different samples was detected by a bicinchoninic acid (BCA) test box (Lin, Wang, & Cui, 2019a).
antibacterial mechanism of pulsed light against E. coli O157:H7 on molecular level. The effect of PL on the cytomembrane, morphology, endoenzyme, genetic material, metabolism, respiratory metabolism and proteins of E. coli O157:H7 was investigated throughout the whole research. Besides, the application of PL on the control of E. coils O157:H7 in carrot juice was investigated as well. 2. Materials and methods
2.4.2.2. Leakage of protein from E. coli O157:H7. The leakages of protein from E. coli O157:H7 were monitored by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Generally, the bacteria with or without PL treatment were rapidly centrifuged at 5000 rpm, 4 °C for 8 min, the precipitated bacteria were collected, washed with PBS and resuspended in 1 mL of sterile water. Subsequently, the different bacterial suspensions were sonicated at 300 W for 10 min to disrupt the cell. After that, the collected samples were mixed with sample buffer (250 mmol/L Tris-HCl (pH 6.8), SDS (10%, w/v), bromophenol blue (0.5%, w/v), glycerine (50%, v/v) and β-mercaptoethanol (5%, v/v)) at the ratio of 4:1 and boiled for 8 min. Finally, the mixtures were centrifuged at 8000 rpm, 4 °C for 10 min and the supernatant were collected to conduct the SDS-PAGE (Lin, Zhu, & Cui, 2018).
2.1. Materials The strains of E. coli O157:H7 CICC 21530 were purchased from China Center of Industrial Culture Collection (Beijing, China), and incubated in nutrient broth (NB) at 37 °C for 24 h to obtain the log-phase bacteria. Pulsed light equipment was assembled in Jiangsu University (Zhenjiang, Jiangsu). Other agents used in this study were provided by the local supplier without further purification. 2.2. Pulsed light treatment The prepared bacterial samples were placed in the lab-scale PL chamber, which contained two xenon lamp tubes and a sample table. PL treatment was performed at the following conditions: (A) 0.5 Hz, 10 pulse numbers, 500 J of single pulse energy. (B) 0.5 Hz, 15 pulse numbers, 500 J of single pulse energy. (C) 0.5 Hz, 20 pulse numbers, 500 J of single pulse energy. (D) 0.5 Hz, 25 pulse numbers, 500 J of single pulse energy. The bacterial samples were fixed at a 10 cm distance from xenon lamp tubes.
2.4.2.3. Measurement of ATP content. The log phase bacteria were centrifuged at 6000 rpm, 4 °C for 10 min to collect the precipitated bacteria, then washed 3 times with PBS (0.03 M, pH 7.2) and finally resuspended in 2 mL of PBS (0.03 M, pH 7.2). After above steps, the bacteria suspension was suffered from PL treatment, another sample without PL treatment was regarded as control. All samples were centrifuged again (6000 rpm, 4 °C, 10 min) to collect the precipitation. The precipitated bacteria were dissolved in 500 μL lysozyme and 500 μL TE buffer solutions, followed by treating with an ultrasonic sonifier (300 W, 10 min, 1.1 s of ultrasonic interval) to break the cell. Ultimately, the ATP contents of different samples were measured by a ATP detection kit (Cui, Zhang, Zhou, Zhao, & Lin, 2015a).
2.3. Time-killing analysis of PL against E. coli O157:H7 The bacteriocidal activity of PL against E. coli O157:H7 was tested by the plate counting method (Cui, Zhang, Li, & Lin, 2018). In brief, after different PL treatment at the time interval of 0, 1, 2, 4 and 8 h at 37 °C, each bacterial suspension (105–106 CFU/mL) in phosphate buffer (PBS, 0.03 M, pH 7.2) was taken out and serially diluted for ten times. The residual quantity of bacteria was detected by pouring 100 μL of each diluent onto nutrient agar plates and incubated at 37 °C for 24 h. The bacterial suspension without PL treatment was served as the control.
2.4.2.4. Detection of DNA content. The changes of DNA contents of E. Coil O157:H7 with or without PL treatment were analyzed by a microultraviolet spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Initially, the DNA of different samples was extracted by a DNA Kit (TIANGEN Biotechnology, Beijing, China). After extraction, all samples were put into quartzose cuvettes and measured at 260 nm to compare the changes of DNA contents. In addition, a laser scanning confocal microscope (LSCM) (TCS SP5 II, Leica, Germany) was applied to visually observe the changes of DNA of different samples (Cui, Zhao, & Lin, 2015b). In short, the bacterial suspensions with or without PL treatment were added with equal volume of 4′, 6-diamidino-2-phenylindole (DAPI, 10 μg/mL) and reacted in dark for 10 min. After reaction, a bit of the mixture was dropwise added on the microscope slide for LSCM observation.
2.4. Antibacterial mechanism of PL against E. coli O157:H7 2.4.1. Effect of PL on the morphology of E. coli O157:H7 2.4.1.1. Scanning electron microscope (SEM). The morphology of E. coli O157:H7 with different treatment was monitored by a scanning electron microscope (FEI instrument, Inc., Hillsboro, OR, USA). Different bacteriological samples were fixed overnight in 2.5% glutaraldehyde solution (in 0.1 M phosphate buffer, pH 7.0) at 4 °C. Then samples were dehydrated in the following graded series of ethanol solutions: 50, 70, 80, and 90%, and three times with 100% (Zhang, Choi, & Park, 2018). Dried samples were mounted onto brass slip using conducting resin, and then sputter-coated with gold. Subsequently, they were examined at a 10 kV electron velocity to obtain final images.
2.4.3. Effect of PL treatment on the endoenzyme of E. coli O157:H7 2.4.3.1. Adenosine triphosphatese (ATPase). As for the detection of ATPase activity, an ATPase assay kit was employed to determine the absorbance at 660 nm using an ultraviolet spectrophotometer (Agilent Cary 60, Agilent Technologies Co Ltd, USA).
2.4.1.2. Transmission electron microscopy (TEM). The changes of cell integrity of E. coli O157:H7 after PL treatment was observed by a transmission electron microscopy (TEM) (Cui, Yuan, Li, & Lin, 2017). The E. coli O157:H7 without PL treatment was regarded as the control. All bacteriological samples were centrifuged at 6000 rpm for 10 min, washed 3 times with PBS (0.03 M, pH7.2) and collected. A copper mesh was immersed into the bacterial suspension, then drying for 30 min. After that, the copper mesh loaded bacteria were stained by phosphotungstic acid for TEM observation.
2.4.3.2. β-galactosidase. The E. coli O157:H7 with or without PL treatment were centrifuged and washed to collect the precipitated bacteria. Then, 0.5 mL of each lysozyme (10 mg/mL), TE buffer and PBS were added to resuspend the bacteria. The bacteria suspensions were treated by an ultrasonic sonifier (300 W, 10 min, 1.1 s of ultrasonic interval) to break the cell. After that, 0.5 mL of 10 mg/mL onitrophenyl-β-D-galactoside (ONPG) and 1000 μL of β-galactosidase 2
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was 100 G, static field was 3460 G, frequency was 9.85 GHz and power was 19.10 Mw (Escudero et al., 2019).
reaction buffer were added into 0.5 mL of the above bacteria suspensions. The absorbances of different samples at 405 nm were monitored by an ultraviolet spectrophotometer to compare the changes of β-galactosidase activity (Cui et al., 2018).
2.4.5.2. Formation of intracellular ROS induced by PL treatment. The detection of intracellular ROS after PL treatment was performed by the aid of fluorescence probe of 2′,7′-dichlorofluorescin diacetate (DCFHDA). After centrifugation and rinse, the collected E. coli O157:H7 were resuspended in DCFH-DA solutions (10 μmol/L) to reach the final concentration of 2 × 108 CFU/mL and incubated in dark at 37 °C for 30 min to allow the penetration of DCFH-DA into bacteria. After incubation, the mixture was washed three times to remove the free DCFH-DA, then suffered from PL treatment. The sample without PL treatment was served as the control. The fluorescence intensity of different samples was recorded by a microplate reader (Infinite F50, Thermo Fisher Co., USA) at the excitation wavelength of 488 nm and emission wavelength of 525 nm (Kramer, Wunderlich, & Muranyi, 2015).
2.4.3.3. Alkaline phosphatase (AKP). The AKP activity of different samples was measured by the AKP detection kit using an ultraviolet spectrophotometer (Agilent Cary 60, Agilent Technologies Co Ltd, USA) to detect the absorbance at 520 nm. 2.4.3.4. Topoisomerase (Topo). The TopoⅠand TopoⅡwere extracted from E. coli O157:H7. The activity of TopoⅠand TopoⅡ was measured using pBR 322 DNA relaxation reaction by the following procedure: 0.5 μL pBR 322 DNA and different volume of Topo with or without PL treatment were poured into 20 μL of reaction buffer to react at 37 °C for 30 min. After that, the reaction was terminated by addition of 2 μL of sodium dodecyl sulfate (10%, w:v) and 0.5 μL of proteinase K (20 mg/ mL) for 30 min at 37 °C. Finally, the mixtures containing different samples were loaded onto agarose gel (0.8%, w:v) and performed at 115 V for 30 min (Wang, Zou, Xie, & Xie, 2014).
2.5. Application of PL treatment on the control of E. coli O157:H7 in carrot juice The carrot was cut into pieces and squeezed to obtain vegetable stock solution. Next, the stock solution was mixed with sterile water at the ratio of 1:9 to simulate carrot juice. After sterilization by an autoclave, the carrot juice was inoculated with 105–106 CFU/mL of E. coli O157:H7, followed by treating with PL at the following conditions: (A) 0.5 Hz, 20 pulse numbers, 500 J of single pulse energy. (B) 0.5 Hz, 25 pulse numbers, 500 J of single pulse energy. (C) 0.5 Hz, 30 pulse numbers, 500 J of single pulse energy. The residual amount of E. coli O157:H7 in carrot juices after PL treatment was monitored everyday for 4 days at 4 °C by plate counting method. The E. coli O157:H7 inoculated carrot juice without PL treatment was served as the control.
2.4.4. Effect of PL treatment on the vitality of E. Coil O157:H7 2.4.4.1. Oxidative respiratory metabolism. The effect of PL treatment on the oxidative respiratory metabolism of E. coli O157:H7 was measured by typical inhibitor method (Hu, Li, Dai, Cui, & Lin, 2019). Initially, the dissolved oxygen content of initial solutions (3.6 mL of 0.03 M, pH 7.2 PBS, 0.4 mL of 1% glucose, 1 mL of 106 CFU/mL bacteria) after exposure to air for 5 min was determined by a dissolved oxygen meters (INESA instrument CO., LTD., Shanghai, Chian) and marked as R0. Then the initial solutions were added with individual three kinds of inhibitors (iodoacetic acid, malonic acid and sodium phosphate) or treated with PL to determine the dissolved oxygen content and marked as R1. Next, the initial solutions were treated with respective inhibitor in combination with PL to determine the dissolved oxygen content and marked as R2. Inhibiting rate (IR) and superpose rate (DR) were calculated by the following equation.
IR = DR =
2.6. Sensory evaluation After different PL treatment (A, B and C as described in section 2.5), the carrot juices were stored at 4 °C for 2 days, and the qualities of carrot juices were evaluated in terms of their color, taste, flavor and comprehensive acceptability by 10 untrained students using a 9-point hedonic scale (1 meaned extremely dislike, 9 meaned extremely like).
R 0 − R1 × 100% R0 R1 − R2 × 100% R1
2.7. Statistical analysis
After determining the major respiratory metabolic pathway that PL played role in, the activity of some critical enzymes in this pathway was detected as well by corresponding kit.
All experiments were performed at least for three times to obtain the value of average ± standard deviation (SD). The significance analysis was tested by One-way analysis of variance (ANOVA) of SPSS software at the level of P < 0.05.
2.4.4.2. Metabolism. The metabolic level of bacteria with different treatments was measured by resazurin. In general, the bacterial suspensions with or without PL treatment were added with 100 μg/ ml of resazurin solution and placed in a shaker for 2 h in the dark. Then, the cultured bacterial suspension was centrifuged at 10,000 rpm for 4 min, and the supernatant was collected and detected under a fluorescent microplate reader at excitation wavelength of 560 nm and emission wavelength of 590 nm.
3. Results and discussion 3.1. Time-killing analysis In the time-killing analysis (Fig. 1), it could be seen that the population of bacteria in control group remained stable during 8 h incubation. In comparison with control group, the antibacterial activity of group A and group B was slight. However, in group C, the amount of E. coli O157:H7 decreased from 5.56 to 1.02 log CFU/mL within 4 h incubation, still further decreased to undetectable level after 8 h reaction. This result indicated that 0.5 Hz, 20 pulse numbers, 500 J of single pulse energy of PL treatment was enough for the sterilization of E. coli O157:H7. Thus we selected this condition for the following study of antibacterial mechanism.
2.4.5. Detection of free radicals and intracellular reactive oxygen species (ROS) 2.4.5.1. The species of free radicals generated by PL. The detection of free radicals during PL treatment was implemented by the electron spin resonance (ESR) (A300 10/12, Bruker Co., LTD., German). The bacterial suspensions (108 CFU/mL, 80 μL) after PL treatment were immediately mixed with 20 μL of 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO, Aladdin, China) and removed into a capillary, then placed in the cavity of the ESR instrument. The bacterial suspension without PL treatment was regarded as the control. The parameters of ESR instrument were set as follows: center field was 3510 G, sweep width
3.2. Morphological changes of E. coli O157:H7 The morphological changes of E. coli O157:H7 with or without PL 3
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Fig. 1. Time killing analysis of PL against E. Coil O157:H7.
Fig. 2. SEM pictures of (A) control (B) PL-treated E. Coil O157:H7. TEM pictures of (C) control (D) PL-treated E. Coil O157:H7. The white arrows indicated the malformed bacteria, and the yellow arrows indicated the sunken bacteria.
4
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Fig. 3. (A) SDS-PAGE picture of E. Coil O157:H7 with different treatment. C meaned control group, T indicated the bacteria treated with PL. (B) Changes of protein concentration of E. Coil O157:H7 after different treatment. (C) Changes of ATP content of E. Coil O157:H7 after different treatment. The a and b in Fig. 2B and C meaned significant difference between two samples.
Georgiou, & Habraken, 2016). As seen from Fig. 3B, the protein concentration of PL-treated bacteria was 241 μg/mL. Compared with 357 μg/mL of control group, 32.49% reduction in protein quantity after PL treatment was achieved. This conclusion was consistent with SDSPAGE result. As revealed in Fig. 3A, the protein bands of PL-treated E. coli O157:H7 were light and dim than control group, indicating the leakage of proteins after PL treatment. The reason may be ascribed to the destructive effect of PL to the cell structure of E. coli O157:H7, leading to the leakage of intracellular proteins (Qian et al., 2016).
treatment were revealed by TEM and SEM. Seen from Fig. 2A, the untreated E. coli O157:H7 exhibited bacilliform morphology and the surface looked plump and intact. Conversely, the PL-treated bacteria (Fig. 2B) were irregular and shrinking, with some hollowness exhibited on the surface. What's more, sunken and malformed bacteria were observed as well after PL treatment. TEM pictures showed the similar trend with SEM results. As seen in Fig. 2C, the untreated bacteria were intact, unbroken and smooth, with integrated cell membrane and slippy surface. In contrast, the PL-treated bacteria (Fig. 2D) were incomplete and broken. The phospholipid bilayer of bacteria after PL treatment was deformed and destroyed heavily, resulting in the leakage of entocyte in cells, which accelerated the apoptosis of E. coli O157:H7. TEM and SEM results demonstrated that PL treatment played an destructive effect on the cytomembrane and altered the cell structure of E. coli O157:H7, which can be regarded as the antimicrobial mechanism of PL against E. coli O157:H7 (Bajpai, Saha, & Basu, 2012).
3.4. Detection of ATP content The changes of ATP content were revealed in Fig. 3C. ATP is the energy factor in bacterial cells, providing power and energy to the metabolism and vitality of E. coli O157:H7. The ATP content was decreased by 59.47% after PL treatment, compared with the samples without PL treatment. Yang, Li, and Zhang (2010) found the similar tendency for the ATP contents of bacteria after pulsed electric fields treatment. In conclusion, PL treatment significantly reduced the ATP content of E. coli O157:H7, thus impacting the vitality and survival of bacteria.
3.3. Protein concentration and protein leakage Protein is the most important biomacromolecule that exists widely in various organelles and cytoplasm of bacterial cell, acting as different roles such as catalyzator and proppant to support the structure and vitality of the living cells (Zhang, Liu, Wang, Jiang, & Quek, 2016). The release of intracellular proteins and changes of protein content can be regarded as the indication for the integrality of cell structure (Omelon,
3.5. Changes of DNA DNA is the most important genetic substance that controls the
Fig. 4. (A) Changes of DNA content of E. Coil O157:H7 after different treatment. The a and b meaned significant difference between two samples. (B) CLSM pictures of control and PL-treated groups. 5
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Fig. 5. (A) Detection of different ATPase activity of E. Coil O157:H7 after different treatment. (B) The changes of β-galactosidase and AKP activity of E. Coil O157:H7 after different treatment. The a and b in Fig. 4A and B meaned significant difference between two samples. Fig. 6. Effect of PL treatment on TopoⅠand TopoⅡ in E. Coil O157:H7. The a,b, c and d indicated that the PBR 322DNA was treated with different amount (0, 2, 4 and 6 μL) of topoisomerase. The A, B, C and D indicated that the PBR 322DNA was reacted with different amount (0, 2, 4 and 6 μL) of PL-treated topoisomerase. Form Ⅰ, supercoiled DNA. Form Ⅱ, linear DNA or open circular DNA.
hydrolyze the lactose into galactose and glucose, therefore providing the energy and carbon sources for the vitality of bacteria (Watson & Chiu, 2016). ONPG is the analogue of lactose. It can be hydrolyzed to Onitrophenol under the action of β-galactosidase, and O-nitrophenol showed the absorption peak at 405 nm. Hence, the difference of β-galactosidase activity can be reflected by the changes of absorbance at 405 nm. As seen in Fig. 5B, the OD405nm value of PL-treated bacteria decreased by 36% compared with control group, indicating reduced activity of β-galactosidase after PL treatment. AKP is the enzyme exists between cytomembrane and cytoderm. As for the AKP activity (Fig. 5B), the PL-treated bacteria was 0.38 U/mg prot, compared with 0.21 U/mg prot of control group, which achieved 44.74% reduction in activity after PL treatment. This result indicated that PL treatment inhibited the activity and expression of AKP.
proliferation, growth and heredity. In normal conditions, DNA can not be detected outside the bacterial cells, unless the cell structure was destroyed by irresistible force. Fig. 4A revealed that DNA content decreased by 56% after PL treatment, when compared with control group. Phillips, Haggren, Thomas, Ishida-Jones, and Adey (1992) ascribed this phenomenon to the formation of genetically-destructive rings, hence resulting in the reduction and leakage of DNA. DAPI is the fluorochrome that can penetrate the cytomembrane and bind with DNA of bacteria (Omelon et al., 2016). The fluorescent pictures of DNA of E. coli O157:H7 with different treatment were shown in Fig. 4B. The pictures showed that the fluorescence intensity of control group was higher than PL-treated group, which demonstrated less DNA content of E. coli O157:H7 after PL treatment. The results obtained above indicated that PL treatment can accelerate the leakage of DNA from the damaged cell membrane or impede the synthesis of DNA of E. coli O157:H7, thereby DNA content of PL-treated group was much lower than E. coli O157:H7 without any treatment.
3.7. Topoisomerase DNA topoisomerase is a key enzyme in cell nucleus of bacteria, which can not only repair the topological structure of DNA, but also affect the physiological metabolism including genetic recombination, transcription and repair of nucleic acid (Chang, Nair, & Nitiss, 1995). Its main function is the catalyzing of breaking and unwinding of DNA. TopoⅠand TopoⅡ, as two main topoisomerases in bacterial cells, can unwinding the pBR 322 DNA from superhelical structure (Form Ⅰ) to linear or open circular form (Form Ⅱ) (Cantero, Campanella, Mateos, & Cortés, 2006). The effect of PL on the activity of TopoⅠand TopoⅡwas investigated by the enzyme-mediated supercoiled pBR322 relaxation. As revealed in Fig. 6, the band of superhelical form (Form Ⅰ) of pBR322 relaxation (a) became dim and light after addition of different volume of TopoⅠand TopoⅡextracting solutions (b, c, d), indicating better unwinding effect of the extracted enzyme solutions to superhelical DNA.
3.6. Effect of PL treatment on ATPase, β-galactosidase and AKP ATPase is the spherical protein which is important for energy metabolism of bacteria. Its catalytic activity depends on the native configuration and conformation of active sites and surrounding proteins. The changes of ATPase activity of E. coli O157:H7 with different treatment were shown in Fig. 5A. As the picture revealed, the activity of Na+ K+ ATPase, Ca2+ ATPase and Mg2+ ATPase of bacteria treated with PL decreased by 64.26%, 51.07% and 26.50% respectively, as compared with the E. coli O157:H7 without PL treatment. This result indicated that PL may change the structure and conformation of protein, thus resulting in the reduction of ATPase activity. β-galactosidase is a hydrolase exists in the cytoplasm. It can 6
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Table 1 The inhibiting rate (IR) of iodoacetic acid, malonic acid, sodium phosphate and PL. Inhibitors
R0/μmol O2 (g.min)−1
R1/μmol O2 (g.min)−1
IR (%)
Iodoacetic acid Malonic acid Sodium phosphate PL
0.64 0.58 0.61 0.59
0.52 0.41 0.57 0.37
18.75 29.31 6.56 37.29
Table 2 The superpose rate (DR) of PL with different inhibitors. Inhibitors
R2/μmol O2 (g.min)−1
DR (%)
Iodoacetic acid + PL Malonic acid + PL Sodium phosphate + PL
0.41 0.39 0.45
21.15 4.88 26.67
However, After addition of PL-treated TopoⅠand TopoⅡ(B,C,D), the unwinding effect to superhelical DNA (Form Ⅰ) was not apparent compared with TopoⅠand TopoⅡ without PL treatment. The results indicated that PL affected the activity of TopoⅠand TopoⅡ, thereby impacting the metabolism of nucleic acid and bacterial growth. 3.8. Oxidative respiration metabolism and metabolism Fig. 7. The tricarboxylic acid cycle pathway.
The metabolism of glucose mainly rely on three pathways in bacterial cells, including embden-meyerhof-parnas (EMP) pathway, tricarboxylic acid cycle (TCA) and hexose monophosphate pathway (HMP) pathway. Iodoacetic acid, malonic acid and sodium phosphate are the typical inhibitors of EMP, TCA and HMP pathways respectively. The inhibiting rate (IR) and superpose rate (DR) were shown in Table 1 and Table 2. As the table presented, the PL possessed the highest IR than iodoacetic acid, malonic acid and sodium phosphate, indicating PL possessed of better inhibiting ability to the metabolic pathways of E. coli O157:H7. Superpose rate (DR) was used to determine the pathway that PL mainly imposed effort on. Smaller superpose rate (DR) meaned weaker cooperative effect between typical inhibitors and PL treatment, so PL may inhibit the same pathway with the corresponding typical inhibitor (Cui et al., 2018). As seen in Table 2, the superpose rate (DR) of malonic acid + PL treated group was the minimum, meaning PL mainly suppressed the oxidative respiration metabolism of E. coli O157:H7 through TCA pathway. The process of TCA pathway was displayed in Fig. 7. In order to detailedly explored the effect of PL treatment on the TCA pathway of E. coli O157:H7, the detection of the activity of three key enzymes-citrate synthase (CS), isocitrate dehydrogenase (ICDH) and α-ketoglutarate dehydrogenase (α-KGDH) in TCA pathway after different treatment were detected as well. As revealed in Fig. 8A, the activity of CS, ICDH and α-KGDH was impacted after PL treatment. Especially for ICDH and α-KGDH, they were affected largely by 38.10% and 52.63% reduction in activity after PL treatment. Hence, PL can exert influence on the activity of three key enzymes, and then inhibit the energy metabolism of TCA pathway. The changes of metabolism of E. coli O157:H7 after PL treatment were revealed in Fig. 8B. After penetrating into bacteria, the weak fluorescence resazurin can be degraded by various oxidoreductases into high fluorescence resorufin. Hence, it is feasible to feature the metabolism of E. coli O157:H7 by the changes of fluorescence intensity. From Fig. 8B presented, the metabolism capacity of E. coli O157:H7 decreased gradually after PL treatment. It was clearly seen that after 8 h incubation, the metabolism capacity of E. coli O157:H7 decreased to 39.42%. However, in time-killing analysis, the number of bacteria was undetectable after 8 h incubation. This was ascribed to the sublethal state of bacteria. In this condition, the bacteria were nonculturable on
nutrient medium but still maintained their metabolic activity (Ziuzina, Boehm, Patil, Cullen, & Bourke, 2015). 3.9. Detection of free radicals and intracellular ROS Fig. 9A showed the ESR spectrogram of bacterial suspension with or without PL treatment. 5.5-Dimethyl-1-pyrroline N-oxide (DMPO) was added immediately into bacterial suspension after different treatments to trap the free radicals. The ESR spectrum of control group showed that no free radicals were detected when PL was not applied, indicating that DMPO can not be oxidized by ambient environment (Tahara & Okubo, 2014). After PL treatment, obvious peaks in magnetic field (marked with *), featured as quartet were seen in ESR spectrum of PLtreated samples and ascribed to the existence of plentiful of hydroxyl radical (·OH) (Xie et al., 2019). This result demonstrated that hydrogen peroxide can be produced during PL treatment process and gradually disintegrated to hydroxyl radical (Tahara & Okubo, 2014). Furthermore, hydrogen peroxide can be disintegrated to superoxide anion (·O) and perhydroxyl radical (·OOH) as well as revealed by the following equation:
H2 O2 + 2OH− → ⋅O−2 + 2H2 O+ e
O−2 + H+ → ⋅OOH However, only hydroxyl radicals (·OH) were detected in the ESR spectrum right now, that was because DMPO can capture ·OH more readily than ·O and ·OOH and form a spin adduct with a longer lifetime by the following equation: H3C H3C
N
O-
H
.
OH
H3C
OH H3C
N
H
O-
The generated hydroxyl radical (·OH) by PL treatment can attack the lipid bilayer of cell membrane, as well as induce the generation of intracellular reactive oxygen, thus accelerating the death of E. coli 7
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Fig. 8. (A) Effect of PL on the activity of enzymes-citrate synthase (CS), isocitrate dehydrogenase (ICDH) and α-ketoglutarate dehydrogenase (α-KGDH) in TCA pathway. (B) The changes of metabolism of E. Coil O157:H7 after PL treatment.
O157:H7. The intracellular ROS of E. coli O157:H7 were remarkably increased after PL treatment as revealed in Fig. 9B. High concentration of ROS may hinder the function of some organelles within the cells, thus leading to the destruction of cell structure. 3.10. Antibacterial activity of PL in carrot juice and sensory evaluation The antibacterial effect of PL on E. coli O157:H7 in carrot juice was shown in Fig. 10. As the picture presented, the group C (0.5 Hz, 30 pulse numbers, 500 J of single pulse energy) exhibited better antibacterial effect in carrot juices, which sterilized the bacteria to undetectable level after 3 days incubation. In addition, seen from the results of sensory evaluation (Table 3), it implied that the difference of color, taste, flavor and comprehensive acceptability between control and PL-treated group was not significant, indicating that PL have no adverse effect on the quality of carrot juice. Fig. 10. Antibacterial effect of PL against E. Coil O157:H7 in carrot juice.
4. Conclusion affecting the glycometabolism of bacteria by TCA pathway and key enzymes in this pathway. The ESR spectrum indicated that PL can generate the hydroxyl radicals during treatment process, leading to the increase in the amount of intracellular ROS, accelerating the death of bacteria from the outside and the inside attack. Finally, PL (0.5 Hz, 30 pulse numbers, 500 J of single pulse energy) sterilized the bacteria to undetectable level after 3 days incubation in carrot juice, without significant impact on its quality, so PL can be regarded as an effective
In this work, the antibacterial activity and antimicrobial mechanism of PL against E. coli O157:H7 were thoroughly investigated. After PL treatment, the morphology and cell integrity of E. coli O157:H7 were damaged seriously as revealed by TEM and SEM results. The contents of protein, ATP and DNA were reduced by 32.49%, 59.47% and 56% respectively, for E. coli O157:H7 treated by PL. Furthermore, PL can inhibit the activity of some key enzymes and metabolism in E. coli O157:H7. The respiration metabolism test demonstrated that PL mainly
Fig. 9. (A) ESR spectrum of control and PL treated groups. (B) Detection of intracellular ROS after PL treatment in E. Coil O157:H7. The a and b in Fig. 8B meaned significant difference between two samples. 8
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Table 3 Sensory evaluation of carrot juice. Parameters
Control
Color Flavor Taste Comprehensive acceptability
7.23 6.83 5.67 5.32
± ± ± ±
A 0.28 0.12 0.15 0.18
7.19 6.85 5.72 5.24
B ± ± ± ±
0.17 0.27 0.23 0.21
7.32 6.74 5.75 5.38
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C ± ± ± ±
0.24 0.25 0.19 0.25
7.26 6.89 5.59 5.42
± ± ± ±
0.19 0.16 0.20 0.16
A, B and C indicated different PL treatment (A: 0.5 Hz, 20 pulse numbers, 500 J of single pulse energy. B: 0.5 Hz, 25 pulse numbers, 500 J of single pulse energy. C:0.5 Hz, 30 pulse numbers, 500 J of single pulse energy). There was no significant difference between different samples.
sterilization methods after the deeply research of its antimicrobial mechanism against E. coli O157:H7. Declaration of interests 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. Acknowledgement This study was financially supported by National Natural Science Foundation of China (Grant no. 31470594), Natural Science Foundation of Jiangsu Province (Grant no. BK20170070), Hunan Science and Technology Major Project (Grant no. 2016NK1001-3), Jiangsu Province Research Fund (Grant no. NY-013) and Jiangsu University Research Fund (Grant no. 11JDG050). References Bajpai, I., Saha, N., & Basu, B. (2012). Moderate intensity static magnetic field has bactericidal effect on E. Coli and S. Epidermidis on sintered hydroxyapatite. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 100(5), 1206–1217. Batz, M. B., Hoffmann, S., & Morris, J. G. (2012). Ranking the disease burden of 14 pathogens in food sources in the United States using attribution data from outbreak investigations and expert elicitation. Journal of Food Protection, 75(7), 1278–1291. Cantero, G.,., Campanella, C.,., Mateos, S., & Cortés, F. (2006). Topoisomerase II inhibition and high yield of endoreduplication induced by the flavonoids luteolin and quercetin. Mutagenesis, 21(5), 321. Chang, Y. C., Nair, M. G., & Nitiss, J. L. (1995). Metabolites of daidzein and genistein and their biological activities. Journal of Natural Products, 58(12), 1901–1905. Chen, D., Wiertzema, J., Peng, P., Cheng, Y., Liu, J., Mao, Q., et al. (2018). Effects of intense pulsed light on Cronobacter sakazakii inoculated in non-fat dry milk. Journal of Food Engineering, 238, 178–187. Cui, H., Yuan, L., Li, W., & Lin, L. (2017). Antioxidant property of SiO2-eugenol liposome loaded nanofibrous membranes on beef. Food Packaging and Shelf Life, 11, 49–57. Cui, H., Zhang, C., Li, C., & Lin, L. (2018). Antimicrobial mechanism of clove oil on Listeria monocytogenes. Food Control, 94, 140–146. Cui, H., Zhang, X., Zhou, H., Zhao, C., & Lin, L. (2015a). Antimicrobial activity and mechanisms of Salvia sclarea essential oil. Botanical Studies, 56(6), 1–8. Cui, H., Zhao, C., & Lin, L. (2015b). The specific antibacterial activity of liposome-encapsulated Clove oil and its application in tofu. Food Control, 56, 128–134. Escudero, R., Segura, J., Velasco, R., Valhondo, M., Romero de Ávila, M. D., Garcia-
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