977 Journal o f Food Protection, Vol. 77, No. 6, 2014, Pages 977-980 doi: 10.4315/0362-028X. JFP-13-531 Copyright © , International Association for Food Protection
Research Note
Infrared Sensor-Based Aerosol Sanitization System for Controlling Escherichia coli 0157:H7, Salmonella Typhimurium, and Listeria monocytogenes on Fresh Produce SANG-OH KIM ,'f JAE-WON HA,1! KI-HWAN PARK,2 MYUNG-SUB CHUNG,2
and
DONG-HYUN KANG1*
Department o f Food and Animal Biotechnology, Department o f Agricultural Biotechnology, Center fo r Food and Bioconvergence, and Research Institute fo r Agriculture and Life Sciences, Seoul National University, Seoul 151-921, South Korea; and 2Department o f Food and Nutrition, Chung-Ang University, Gyeonggi-do, 456-756, South Korea MS 13-531: Received 9 December 2013/Accepted 31 January 2014
ABSTRACT An economical aerosol sanitization system was developed based on sensor technology for minimizing sanitizer usage, while maintaining bactericidal efficacy. Aerosol intensity in a system chamber was controlled by a position-sensitive device and its infrared value range. The effectiveness of the infrared sensor-based aerosolization (ISA) system to inactivate Escherichia coli 0157:H7, Salmonella Typhimurium, and Listeria monocytogenes on spinach leaf surfaces was compared with conventional aerosolization (full-time aerosol treated), and the amount of sanitizer consumed was determined after operation. Three pathogens artificially inoculated onto spinach leaf surfaces were treated with aerosolized peracetic acid (400 ppm) for 15, 30, 45, and 60 min at room temperature (22 + 2°C). Using the ISA system, inactivation levels of the three pathogens were equal or better than treatment with conventional full-time aerosolization. However, the amount of sanitizer consumed was reduced by ca. 40% using the ISA system. The results of this study suggest that an aerosol sanitization system combined with infrared sensor technology could be used for transportation and storage of fresh produce efficiently and economically as a practical commercial intervention.
Sales of fresh produce, especially fruits and vegetables, have dramatically increased, as consumers have become progressively concerned with health and nutrition in recent years (1,2,13). Increased consumption of fresh produce has contributed to an increasing frequency of illness outbreaks caused by foodbome pathogens (5, 6, 8,10). Fresh produce can become contaminated with foodbome pathogens while growing in fields or orchards or during harvesting, postharvest handling, processing, and distribution (1, 18). Therefore, the sanitization of fresh produce becomes increasingly important for maintaining product quality and microbiological safety (15). Aqueous sanitizers are typically employed for sanitiz ing fresh produce surfaces. However, numerous studies show that aqueous sanitizers are often not effective at reducing pathogenic microorganisms on surfaces of fresh produce, because microorganisms can become attached to injured or inaccessible surfaces (2, 7, 16, 17, 19, 20). Gaseous sanitizers may overcome limitations associated with the aqueous form. Han et al. (9) reported that gaseous chlorine dioxide treatment was shown to be more efficacious than aqueous chlorine dioxide treatment at the same concentration against Listeria monocytogenes Scott A on injured surfaces of green peppers. However, * Author for correspondence. Tel: +82-2-880-4927; Fax: +82-2-883-4928; E-mail: [email protected]. t These authors contributed equally to this work.
gaseous sanitizers have several disadvantages in that a sophisticated apparatus is needed for gas generation, and only a limited number of sanitizers can convert into a gaseous phase (11). Aerosolization is the dispersion of liquid as a fine mist in air. Antimicrobial applications of aerosols are wellknown for controlling foodbome pathogenic microorgan isms (4, 11,12,14). The aerosolization system consists of a simple apparatus and can deliver a diverse array of antimicrobial agents with high penetration power suitable for foods (4, 12). Therefore, aerosolization may be an alternative sanitizer delivery system and could easily be applicable in semitrailers and shipping containers during transportation and storage of fresh produce. However, some problems are associated with conventional aerosolization for practical use, such as condensation of aerosol mist on food surfaces and draining onto chamber floors at the point of saturation. Consequently, it causes an unnecessary waste of sanitizer. Therefore, this study was undertaken to develop a practical aerosol sanitization system combined with infrared sensor technology for minimizing sanitizer usage, while maintaining bactericidal efficacy. The efficacy of infrared sensor-based and conventional aerosolization to inactivate Escherichia coli 0157:H7, Salmonella Typhimurium, and Listeria monocytogenes on spinach leaf surfaces was compared, and the amount of consumed sanitizer was determined after operation.
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J. Food Prot., Vol. 77, No. 6
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M A TER IA LS A N D M E TH O D S Bacterial cultures and cell suspension. Three strains each of Salmonella Typhimurium (ATCC 19585, ATCC 43971, and DT 104), E. coli 0157:H7 (ATCC 35150, ATCC 43889, and ATCC 43890), and L. monocytogenes (ATCC 7644, ATCC 19114, and ATCC 19115) were obtained from the School of Food Science bacterial culture collection of Seoul National University (Korea). Each strain of Salmonella Typhimurium, E. coli 0157:H7, and L. monocytogenes was cultured in 5 ml of tryptic soy broth (Difco, BD, Sparks, MD) at 37°C for 24 h, followed by centrifugation (4,000 x g for 20 min at 4°C), and washing three times with buffered peptone water (Difco, BD). The final pellets were resuspended in 9 ml of buffered peptone water, corresponding to approximately 108 to 109 CFU/ml. Subsequently, suspended pellets of each strain of the three pathogen species were combined to produce a mixed culture cocktail. These multipathogen culture cocktails, consisting of a final concentration of approximately 108 CFU/ml, were used for subsequent experimentation. Sample preparation and inoculation. Commercial spinach was purchased from a local grocery store (Seoul, Korea) on the day prior to the experiment. Spinach leaves (25 g) were separated and placed on sterile aluminum foil in a biosafety hood. Then, 0.1 ml of the three-pathogen cocktail was inoculated by droplet with a micropipettor onto both sides of each spinach leaf at 15 to 20 locations. Inoculated leaves were air dried for 1 h in a biosafety hood with the fan running at room temperature (22 + 2°C). Spot inoculation is more consistent and produces more reproducible results for the inoculation of a known number of pathogen cells on spinach surfaces than does the dipping inoculation method (3). Sanitizer preparation. Peracetic acid (Omega Chemical, Gyeongbuk, Korea) was used as a aqueous sanitizer and diluted according to the manufacturer’s instructions with distilled water to a concentration of 400 ppm. Peracetic acid is a sanitizer well known for effectively reducing pathogenic microorganisms when applied by aerosolization (11, 12, 14). Inoculated spinach leaves (25 g) were positioned horizontally and vertically on a stainless steel rack to mimic actual transportation conditions. The size of aerosolized particles was approximately 5.42 to 11.42 pm. Spraying was performed for a maximum of 60 min to simulate commercial transportation time. ISA system. The overall hardware design for the infrared sensor-based aerosolization (ISA) system is shown in Figure 1. A model glass cabinet (40 by 40 by 40 cm) was used in this study. The cabinet was sealed, and aerosolized mist was routed from a newly built nebulizer (DRWL-2000, Doore Industrial Co., Seoul,
FIGURE 1. Schematic diagram o f infrared sensor-based aerosol sanitization system used in this study. Korea) by means of a 7.0-cm flexible tube through the lid of the cabinet. A position-sensitive device (PSD; GP2Y0A41 module, Sharp Corp., Nara, Japan) was used, which was affixed to the outside surface of the glass chamber to measure aerosol intensity, and the emitting wavelength range of this module was in the near infrared (870 + 70 ran, product specification). A programmable logic controller (MOACON, Comfile Technology, Seoul, Korea) was built into the aerosol sanitization system for the convenience of development. The programmable logic controller gathered infrared values from the PSD and controlled an aerosol generator state in the ISA system. The detailed principle and an application method are shown in Figure 2. The distance between the PSD and object is in inverse proportion to the output voltage (Fig. 2a). Therefore, the output voltage is directly proportional to the aerosol intensity (Fig. 2b). The measured output voltage data of the PSD was converted to a digital number corresponding to the aerosol intensity. The basic aerosol generation algorithm was such that a generator would turn on when the set point of the digital infrared value was out of range and would turn off when the proper infrared value was reached. Bacterial enumeration. After 15, 30, 45, and 60 min of treatment, spinach leaves were transferred into sterile stomacher bags (Labplas Inc., Sainte-Julie, Quebec, Canada) containing 225 ml of sterile Dey/Engley neutralizing broth (detection limit = 10 CFU/g) and homogenized for 2 min using a stomacher (EASY MIX, AES Chemunex, Rennes, France). After homogenization, samples were 10-fold serially diluted in buffered peptone water, and 0.1 ml of sample or diluent was spread plated onto each
FIGURE 2. Aerosol intensity measure ment using a position-sensitive device (PSD).
Light S cattering
In frared Sensor (PSD)
]|T
upMi Aerosol
C h am b er - > Aerosol Intensity’
(a) Principle
O u tp u t Voltage
(b) Application
J. Food Prot., Vol. 77, No. 6
979
INFRARED SENSOR-BASED AEROSOL SANITIZATION SYSTEM
TABLE 1. Average running times and the amount of consumed sanitizer for each infrared value rangea Consumed sanitizer (ml) Infrared value range
Running time (s/min)
Control (full time) 1,000-2,000 150-2,000 150-1,900
60 50.5 46.4 40.6
30 min 246.67 208.33 172.00 155.00
± ± + +
60 min
16.50 a 20.11 b 16.09 c 18.68 c
465.00 403.67 356.67 282.00
+ ± ± ±
13.00 a 14.15 b 18.58 c 15.87 d
a Vales are means of three replications ± standard deviations. Means with the same letter in the same column are not significantly different (P > 0.05). selective medium. Xylose lysine desoxycholate agar (Difco, BD), sorbitol MacConkey agar (Difco, BD), and Oxford agar base with Bacto Oxford antimicrobic supplement (Difco, BD) were used as selective media for the enumeration of Salmonella Typhimurium, E. coli 0157:H7, and L. monocytogenes, respectively. All agar media were incubated at 37°C for 24 to 48 h before counting. To confirm the identity of the pathogens, random colonies were selected from the enumeration plates and subjected to biochem ical and serological tests. These tests consisted of the Salmonella latex agglutination assay (Oxoid, Ogdensberg, NY), E. coli 0157:H7 latex agglutination assay (RIM, Remel, Lenexa, KS), and API Listeria (bioMerieux, Inc. Hazelwood, MO) for Salmonella Typhimurium, E. coli 0157:H7, and L. monocyto genes, respectively.
2,000 to ascertain the minimum consumption range of the sanitizer. The average running times per minute and the amount of sanitizer consumed for each infrared value range are summarized in Table 1. The lowest amount of consumption was in the 150 to 1,900 value range (ca. 40% savings compared with control) when compared with other infrared value ranges. As we mentioned earlier, a major problem with conventional full-time aerosolization is the condensation of sanitizer on food surfaces and draining onto the chamber floor at the point of saturation. Actually, in our previous aerosol sanitization studies, there was an unnecessary waste of sanitizers when treated with full-time aerosolization for 50 min (4) and 60 min (11) in a sealed model cabinet. However, in the 150 to 1,900 value range (running time, 40.6 s/min), the level of sanitizer condensa tion diminished considerably, and less sanitizer was consumed. Accordingly, we adopted this aerosol intensity (mnning time, 40.6 s/min) for the ISA system used in this study and conducted subsequent pathogen inactivation experiments in this range. To evaluate the disinfection level of the newly developed ISA system, pathogen reductions (log n0/n) were compared with those of conventional aerosolization (full time aerosol treatment using the same chamber). Reductions of Salmonella Typhimurium, E. coli 0157:H7, and L. monocytogenes on spinach leaves during ISA and conven tional aerosolization treatment are shown in Table 2. ISA treatment for 60 min achieved 2.57-, 2.65-, and 2.51-log reductions in E. coli 0157:H7, Salmonella Typhimurium, and L. monocytogenes, respectively, whereas, after 60 min of conventional aerosolization treatment, log reductions of
Statistical analysis. All experiments were repeated three times with duplicate samples. Triplicate data was analyzed by analysis of variance with Duncan’s multiple range test using the Statistical Analysis System (SAS Institute Inc., Cary, NC). The value of P < 0.05 was used to indicate significant differences. RESULTS AND DISCUSSION
In early stages of the experimental design, a humidity sensor was used to control aerosol intensity. However, in a sealed chamber system, aerosol intensity was not found to be correlated with humidity. Next, various sensor modules were evaluated, but ultimately, the PSD was selected. The PSD was developed using infrared sensor technology to detect the room position of objects. We found that PSD output voltage was proportional to aerosol intensity (Fig. 2). Digital infrared value ranges obtained from the PSD were subdivided into 150 to 1,900, 150 to 2,000, and 1,000 to
TABLE 2. Comparison of pathogen reductions following treatment with conventional and infrared sensor-based aerosolization (ISA)a,b Log reduction (log n jn ) E. coli 0157:H7 Treatment time (min)
0 15 30 45 60
Conventional
0.00 0.97 1.94 2.03 2.29
+ ± ± ± ±
0.00 a a 0.30 b a 0.41 ca 0.22 c a 0.29 c a
Salmonella Typhimurium ISA
0.00 1.16 2.27 2.31 2.57
± ± ± ± ±
Conventional
0.00 a a 0.00 0.25 b a 1.02 0.40 c a 1.92 0.15 c a 2.29 0.21 c a 2.47
+ ± + ± ±
0.00 a a 0.23 b a 0.09 c a 0.14 d a 0.29 d a
ISA
0.00 1.42 2.44 2.54 2.65
± + ± + ±
0.00 a a 0.38 b a 0.07 c b 0.02 d b 0.08 d a
L. monocytogenes Conventional
0.00 0.92 1.89 2.01 2.49
+ ± + + ±
0.00 a a 0.20 b a 0.27 c a 0.16 c a 0.17 d a
ISA
0.00 + 0.00 a a 1.03 ± 0.32 b a 1.89 ± 0.16 c a 2.22 ± 0 .1 0 Da 2.51 ± 0.36 d a
“ Values are means of three replications ± standard deviations. Means with the same capital letter in the same column are not significantly different (P > 0.05). Means with the same lowercase letter in the same row are not significantly different (P > 0.05). ISA, newly developed aerosolization system that controlled the aerosol intensity using an infrared sensor. The aerosol intensity for pathogen inactivation studies was fixed in the 150 to 1,900 infrared value range (running time, 40.6 s/min)
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2.29, 2.47, and 2.49, respectively, were observed in E. coli 0157:H7, Salmonella Typhimurium, and L. monocytogenes (Table 2). Overall, ISA treatment reduced the three pathogens slightly more than conventional aerosolization treatment at all treatment time intervals, while consuming less sanitizer. Especially in Salmonella Typhimurium, there were significantly (P < 0.05) greater reductions for the ISA as opposed to the conventional method at 30 and 45 min (Table 2). In other words, the sporadic treatment of aerosolized sanitizer showed more effective antimicrobial results on spinach leaves than those of full-time treatment. In conclusion, because aerosol intensity was controlled with the PSD, the amount of consumed sanitizer could be reduced by ca. 40%, while inactivation levels of pathogens were not different from or better than treatment with conventional full-time aerosolization. However, because we used a laboratory-scale instrument (Fig. 1) as a preliminary step to investigate the feasibility of utilizing an infrared sensor for enhancing aerosol sanitization on a pilot or commercial scale, the amount of treated sample (25 g) and the length of treatment (60 min) cannot be translated to commercial application. Also, the infrared value range of the PSD needs to be optimized when applied to an industrial-scale container. Therefore, far-ranging studies involving more varieties of fresh produce, sanitizers, and treatment times in a larger model cabinet are required to develop this new aerosol sanitization system as a practical commercial intervention.
5. 6.
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15.
ACKNOWLEDGMENTS This research was supported by R&D Convergence Center Support Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea. This research was also supported by a grant (10162KFDA995) from the Korea Food & Drug Administration in 2012.
16.
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Beuchat, L. R. 1996. Pathogenic microorganisms associated with fresh produce. J. Food Prot. 59:204—216. Beuchat, L. R. 199S. Surface decontamination of fruits and vegetables eaten raw: a review. Document WHO/FSF/FOS/98.2. World Health Organization, Geneva. Beuchat, L. R., J. M. Farber, E. H. Garrett, L. J. Harris, M. E. Parish, T. V. Suslow, and F. F. Busta. 2001. Standardization of a method to determine the efficacy of sanitizers in inactivating human pathogenic microorganisms on raw fruits and vegetables. J. Food Prot. 64:10791084. Choi, M. R., S. Y. Lee, K. H. Park, M. S. Chung, S. R. Ryu, and D. H. Kang. 2012. Effect of aerosolized malic acid against Listeria
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monocytogenes, Salmonella Typhimurium, and Escherichia coli 0157:H7 on spinach and lettuce. Food Control 24:171-176. De Roever, C. 1998. Microbiological safety evaluations and recommendations on fresh produce. Food Control 9:321-347. Doyle, M. P., and M. C. Erickson. 2008. Summer meeting 2007—the problems with fresh produce: an overview. J. Appl. Microbiol. 105: 317-330. Foschino, R., I. Nervegena, A. Motta, and A. Galli. 1998. Bactericidal activity of chlorine dioxide against Escherichia coli in water and on hard surfaces. J. Food Prot. 61:668-672. Francis, G. A., C. Thomas, and D. O’Beime. 1999. The microbio logical safety of minimally processed vegetables. Int. J. Food Sci. Technol. 34:1-22. Han, Y„ R. H. Linton, S. S. Nielsen, and P. E. Nelson. 2001. Reduction of Listeria monocytogenes on green peppers (Capsicum annuum L.) by gaseous and aqueous chlorine dioxide and water washing and its growth at 78°C. J. Food Prot. 64:1730-1738. Nguyen-the, C., and F. Carlin. 1994. The microbiology of minimally processed fresh fruits and vegetables. Crit. Rev. Food Sci. Nutr. 34: 371^401. Oh, S. W., G. I. Dancer, and D. H. Kang. 2005. Efficacy of aerosolized peroxyacetic acid as a sanitizer of lettuce leaves. J. Food Prot. 68:1743-1747. Oh, S. W., P. M. Gray, R. H. Dougherty, and D. H. Kang. 2005. Aerosolization as novel sanitizer delivery system to reduce foodbome pathogens. Lett. Appl. Microbiol. 41:56-60. Park, E. J., E. Alexander, G. A. Taylor, R. Costa, and D. H. Kang. 2008. Effect of electrolyzed water for reduction of foodbome pathogens on lettuce and spinach. J. Food Sci. 73:268-272. Park, S. H., H. L. Cheon, K. H. Park, M. S. Chung, S. H. Choi, S. R. Ryu, and D. H. Kang. 2012. Inactivation of biofilm cells of foodbome pathogen by aerosolized sanitizers. Int. J. Food Microbiol. 154:130134. Rahman, S. M. E., T. Ding, and D. H. Oh. 2010. Inactivation effect of newly developed low concentration electrolyzed water and other sanitizers against microorganisms on spinach. Food Control 21: 1383-1387. Sapers, G. M. 2001. Efficacy of washing and sanitizing methods for disinfection of fresh fruit and vegetable products. Food Technol. Biotechnol. 39:305—311. Seo, K. H., and J. F. Frank. 1999. Attachment of Escherichia coli 0157:H7 to lettuce leaf surface and bacterial viability in response to chlorine treatment as demonstrated by using confocal scanning laser microscopy. J. Food Prot. 62:3—9. Takeuchi, K., C. M. Matute, A. N. Hassan, and J. F. Frank. 2000. Comparison of the attachment of Escherichia coli 0157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Pseudomonas fluorescens to lettuce leaves. J. Food Prot. 63:1433-37. Weissinger, W. R., W. Chantarapanont, and L. R. Beuchat. 2000. Survival and growth of Salmonella baildon in shredded lettuce and diced tomatoes, and effectiveness of chlorinated water as a sanitizer. Int. J. Food Microbiol. 62:123-131. Zhang, S., and J. M. Farber. 1996. The effects of various disinfectants against Listeria monocytogenes on fresh-cut vegetables. Food Microbiol. 13:311—321.
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Research Note
Infrared Sensor-Based Aerosol Sanitization System for Controlling Escherichia coli 0157:H7, Salmonella Typhimurium, and Listeria monocytogenes on Fresh Produce SANG-OH KIM ,'f JAE-WON HA,1! KI-HWAN PARK,2 MYUNG-SUB CHUNG,2
and
DONG-HYUN KANG1*
Department o f Food and Animal Biotechnology, Department o f Agricultural Biotechnology, Center fo r Food and Bioconvergence, and Research Institute fo r Agriculture and Life Sciences, Seoul National University, Seoul 151-921, South Korea; and 2Department o f Food and Nutrition, Chung-Ang University, Gyeonggi-do, 456-756, South Korea MS 13-531: Received 9 December 2013/Accepted 31 January 2014
ABSTRACT An economical aerosol sanitization system was developed based on sensor technology for minimizing sanitizer usage, while maintaining bactericidal efficacy. Aerosol intensity in a system chamber was controlled by a position-sensitive device and its infrared value range. The effectiveness of the infrared sensor-based aerosolization (ISA) system to inactivate Escherichia coli 0157:H7, Salmonella Typhimurium, and Listeria monocytogenes on spinach leaf surfaces was compared with conventional aerosolization (full-time aerosol treated), and the amount of sanitizer consumed was determined after operation. Three pathogens artificially inoculated onto spinach leaf surfaces were treated with aerosolized peracetic acid (400 ppm) for 15, 30, 45, and 60 min at room temperature (22 + 2°C). Using the ISA system, inactivation levels of the three pathogens were equal or better than treatment with conventional full-time aerosolization. However, the amount of sanitizer consumed was reduced by ca. 40% using the ISA system. The results of this study suggest that an aerosol sanitization system combined with infrared sensor technology could be used for transportation and storage of fresh produce efficiently and economically as a practical commercial intervention.
Sales of fresh produce, especially fruits and vegetables, have dramatically increased, as consumers have become progressively concerned with health and nutrition in recent years (1,2,13). Increased consumption of fresh produce has contributed to an increasing frequency of illness outbreaks caused by foodbome pathogens (5, 6, 8,10). Fresh produce can become contaminated with foodbome pathogens while growing in fields or orchards or during harvesting, postharvest handling, processing, and distribution (1, 18). Therefore, the sanitization of fresh produce becomes increasingly important for maintaining product quality and microbiological safety (15). Aqueous sanitizers are typically employed for sanitiz ing fresh produce surfaces. However, numerous studies show that aqueous sanitizers are often not effective at reducing pathogenic microorganisms on surfaces of fresh produce, because microorganisms can become attached to injured or inaccessible surfaces (2, 7, 16, 17, 19, 20). Gaseous sanitizers may overcome limitations associated with the aqueous form. Han et al. (9) reported that gaseous chlorine dioxide treatment was shown to be more efficacious than aqueous chlorine dioxide treatment at the same concentration against Listeria monocytogenes Scott A on injured surfaces of green peppers. However, * Author for correspondence. Tel: +82-2-880-4927; Fax: +82-2-883-4928; E-mail: [email protected]. t These authors contributed equally to this work.
gaseous sanitizers have several disadvantages in that a sophisticated apparatus is needed for gas generation, and only a limited number of sanitizers can convert into a gaseous phase (11). Aerosolization is the dispersion of liquid as a fine mist in air. Antimicrobial applications of aerosols are wellknown for controlling foodbome pathogenic microorgan isms (4, 11,12,14). The aerosolization system consists of a simple apparatus and can deliver a diverse array of antimicrobial agents with high penetration power suitable for foods (4, 12). Therefore, aerosolization may be an alternative sanitizer delivery system and could easily be applicable in semitrailers and shipping containers during transportation and storage of fresh produce. However, some problems are associated with conventional aerosolization for practical use, such as condensation of aerosol mist on food surfaces and draining onto chamber floors at the point of saturation. Consequently, it causes an unnecessary waste of sanitizer. Therefore, this study was undertaken to develop a practical aerosol sanitization system combined with infrared sensor technology for minimizing sanitizer usage, while maintaining bactericidal efficacy. The efficacy of infrared sensor-based and conventional aerosolization to inactivate Escherichia coli 0157:H7, Salmonella Typhimurium, and Listeria monocytogenes on spinach leaf surfaces was compared, and the amount of consumed sanitizer was determined after operation.
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KIM ET AL.
M A TER IA LS A N D M E TH O D S Bacterial cultures and cell suspension. Three strains each of Salmonella Typhimurium (ATCC 19585, ATCC 43971, and DT 104), E. coli 0157:H7 (ATCC 35150, ATCC 43889, and ATCC 43890), and L. monocytogenes (ATCC 7644, ATCC 19114, and ATCC 19115) were obtained from the School of Food Science bacterial culture collection of Seoul National University (Korea). Each strain of Salmonella Typhimurium, E. coli 0157:H7, and L. monocytogenes was cultured in 5 ml of tryptic soy broth (Difco, BD, Sparks, MD) at 37°C for 24 h, followed by centrifugation (4,000 x g for 20 min at 4°C), and washing three times with buffered peptone water (Difco, BD). The final pellets were resuspended in 9 ml of buffered peptone water, corresponding to approximately 108 to 109 CFU/ml. Subsequently, suspended pellets of each strain of the three pathogen species were combined to produce a mixed culture cocktail. These multipathogen culture cocktails, consisting of a final concentration of approximately 108 CFU/ml, were used for subsequent experimentation. Sample preparation and inoculation. Commercial spinach was purchased from a local grocery store (Seoul, Korea) on the day prior to the experiment. Spinach leaves (25 g) were separated and placed on sterile aluminum foil in a biosafety hood. Then, 0.1 ml of the three-pathogen cocktail was inoculated by droplet with a micropipettor onto both sides of each spinach leaf at 15 to 20 locations. Inoculated leaves were air dried for 1 h in a biosafety hood with the fan running at room temperature (22 + 2°C). Spot inoculation is more consistent and produces more reproducible results for the inoculation of a known number of pathogen cells on spinach surfaces than does the dipping inoculation method (3). Sanitizer preparation. Peracetic acid (Omega Chemical, Gyeongbuk, Korea) was used as a aqueous sanitizer and diluted according to the manufacturer’s instructions with distilled water to a concentration of 400 ppm. Peracetic acid is a sanitizer well known for effectively reducing pathogenic microorganisms when applied by aerosolization (11, 12, 14). Inoculated spinach leaves (25 g) were positioned horizontally and vertically on a stainless steel rack to mimic actual transportation conditions. The size of aerosolized particles was approximately 5.42 to 11.42 pm. Spraying was performed for a maximum of 60 min to simulate commercial transportation time. ISA system. The overall hardware design for the infrared sensor-based aerosolization (ISA) system is shown in Figure 1. A model glass cabinet (40 by 40 by 40 cm) was used in this study. The cabinet was sealed, and aerosolized mist was routed from a newly built nebulizer (DRWL-2000, Doore Industrial Co., Seoul,
FIGURE 1. Schematic diagram o f infrared sensor-based aerosol sanitization system used in this study. Korea) by means of a 7.0-cm flexible tube through the lid of the cabinet. A position-sensitive device (PSD; GP2Y0A41 module, Sharp Corp., Nara, Japan) was used, which was affixed to the outside surface of the glass chamber to measure aerosol intensity, and the emitting wavelength range of this module was in the near infrared (870 + 70 ran, product specification). A programmable logic controller (MOACON, Comfile Technology, Seoul, Korea) was built into the aerosol sanitization system for the convenience of development. The programmable logic controller gathered infrared values from the PSD and controlled an aerosol generator state in the ISA system. The detailed principle and an application method are shown in Figure 2. The distance between the PSD and object is in inverse proportion to the output voltage (Fig. 2a). Therefore, the output voltage is directly proportional to the aerosol intensity (Fig. 2b). The measured output voltage data of the PSD was converted to a digital number corresponding to the aerosol intensity. The basic aerosol generation algorithm was such that a generator would turn on when the set point of the digital infrared value was out of range and would turn off when the proper infrared value was reached. Bacterial enumeration. After 15, 30, 45, and 60 min of treatment, spinach leaves were transferred into sterile stomacher bags (Labplas Inc., Sainte-Julie, Quebec, Canada) containing 225 ml of sterile Dey/Engley neutralizing broth (detection limit = 10 CFU/g) and homogenized for 2 min using a stomacher (EASY MIX, AES Chemunex, Rennes, France). After homogenization, samples were 10-fold serially diluted in buffered peptone water, and 0.1 ml of sample or diluent was spread plated onto each
FIGURE 2. Aerosol intensity measure ment using a position-sensitive device (PSD).
Light S cattering
In frared Sensor (PSD)
]|T
upMi Aerosol
C h am b er - > Aerosol Intensity’
(a) Principle
O u tp u t Voltage
(b) Application
J. Food Prot., Vol. 77, No. 6
979
INFRARED SENSOR-BASED AEROSOL SANITIZATION SYSTEM
TABLE 1. Average running times and the amount of consumed sanitizer for each infrared value rangea Consumed sanitizer (ml) Infrared value range
Running time (s/min)
Control (full time) 1,000-2,000 150-2,000 150-1,900
60 50.5 46.4 40.6
30 min 246.67 208.33 172.00 155.00
± ± + +
60 min
16.50 a 20.11 b 16.09 c 18.68 c
465.00 403.67 356.67 282.00
+ ± ± ±
13.00 a 14.15 b 18.58 c 15.87 d
a Vales are means of three replications ± standard deviations. Means with the same letter in the same column are not significantly different (P > 0.05). selective medium. Xylose lysine desoxycholate agar (Difco, BD), sorbitol MacConkey agar (Difco, BD), and Oxford agar base with Bacto Oxford antimicrobic supplement (Difco, BD) were used as selective media for the enumeration of Salmonella Typhimurium, E. coli 0157:H7, and L. monocytogenes, respectively. All agar media were incubated at 37°C for 24 to 48 h before counting. To confirm the identity of the pathogens, random colonies were selected from the enumeration plates and subjected to biochem ical and serological tests. These tests consisted of the Salmonella latex agglutination assay (Oxoid, Ogdensberg, NY), E. coli 0157:H7 latex agglutination assay (RIM, Remel, Lenexa, KS), and API Listeria (bioMerieux, Inc. Hazelwood, MO) for Salmonella Typhimurium, E. coli 0157:H7, and L. monocyto genes, respectively.
2,000 to ascertain the minimum consumption range of the sanitizer. The average running times per minute and the amount of sanitizer consumed for each infrared value range are summarized in Table 1. The lowest amount of consumption was in the 150 to 1,900 value range (ca. 40% savings compared with control) when compared with other infrared value ranges. As we mentioned earlier, a major problem with conventional full-time aerosolization is the condensation of sanitizer on food surfaces and draining onto the chamber floor at the point of saturation. Actually, in our previous aerosol sanitization studies, there was an unnecessary waste of sanitizers when treated with full-time aerosolization for 50 min (4) and 60 min (11) in a sealed model cabinet. However, in the 150 to 1,900 value range (running time, 40.6 s/min), the level of sanitizer condensa tion diminished considerably, and less sanitizer was consumed. Accordingly, we adopted this aerosol intensity (mnning time, 40.6 s/min) for the ISA system used in this study and conducted subsequent pathogen inactivation experiments in this range. To evaluate the disinfection level of the newly developed ISA system, pathogen reductions (log n0/n) were compared with those of conventional aerosolization (full time aerosol treatment using the same chamber). Reductions of Salmonella Typhimurium, E. coli 0157:H7, and L. monocytogenes on spinach leaves during ISA and conven tional aerosolization treatment are shown in Table 2. ISA treatment for 60 min achieved 2.57-, 2.65-, and 2.51-log reductions in E. coli 0157:H7, Salmonella Typhimurium, and L. monocytogenes, respectively, whereas, after 60 min of conventional aerosolization treatment, log reductions of
Statistical analysis. All experiments were repeated three times with duplicate samples. Triplicate data was analyzed by analysis of variance with Duncan’s multiple range test using the Statistical Analysis System (SAS Institute Inc., Cary, NC). The value of P < 0.05 was used to indicate significant differences. RESULTS AND DISCUSSION
In early stages of the experimental design, a humidity sensor was used to control aerosol intensity. However, in a sealed chamber system, aerosol intensity was not found to be correlated with humidity. Next, various sensor modules were evaluated, but ultimately, the PSD was selected. The PSD was developed using infrared sensor technology to detect the room position of objects. We found that PSD output voltage was proportional to aerosol intensity (Fig. 2). Digital infrared value ranges obtained from the PSD were subdivided into 150 to 1,900, 150 to 2,000, and 1,000 to
TABLE 2. Comparison of pathogen reductions following treatment with conventional and infrared sensor-based aerosolization (ISA)a,b Log reduction (log n jn ) E. coli 0157:H7 Treatment time (min)
0 15 30 45 60
Conventional
0.00 0.97 1.94 2.03 2.29
+ ± ± ± ±
0.00 a a 0.30 b a 0.41 ca 0.22 c a 0.29 c a
Salmonella Typhimurium ISA
0.00 1.16 2.27 2.31 2.57
± ± ± ± ±
Conventional
0.00 a a 0.00 0.25 b a 1.02 0.40 c a 1.92 0.15 c a 2.29 0.21 c a 2.47
+ ± + ± ±
0.00 a a 0.23 b a 0.09 c a 0.14 d a 0.29 d a
ISA
0.00 1.42 2.44 2.54 2.65
± + ± + ±
0.00 a a 0.38 b a 0.07 c b 0.02 d b 0.08 d a
L. monocytogenes Conventional
0.00 0.92 1.89 2.01 2.49
+ ± + + ±
0.00 a a 0.20 b a 0.27 c a 0.16 c a 0.17 d a
ISA
0.00 + 0.00 a a 1.03 ± 0.32 b a 1.89 ± 0.16 c a 2.22 ± 0 .1 0 Da 2.51 ± 0.36 d a
“ Values are means of three replications ± standard deviations. Means with the same capital letter in the same column are not significantly different (P > 0.05). Means with the same lowercase letter in the same row are not significantly different (P > 0.05). ISA, newly developed aerosolization system that controlled the aerosol intensity using an infrared sensor. The aerosol intensity for pathogen inactivation studies was fixed in the 150 to 1,900 infrared value range (running time, 40.6 s/min)
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KIM ET AL.
2.29, 2.47, and 2.49, respectively, were observed in E. coli 0157:H7, Salmonella Typhimurium, and L. monocytogenes (Table 2). Overall, ISA treatment reduced the three pathogens slightly more than conventional aerosolization treatment at all treatment time intervals, while consuming less sanitizer. Especially in Salmonella Typhimurium, there were significantly (P < 0.05) greater reductions for the ISA as opposed to the conventional method at 30 and 45 min (Table 2). In other words, the sporadic treatment of aerosolized sanitizer showed more effective antimicrobial results on spinach leaves than those of full-time treatment. In conclusion, because aerosol intensity was controlled with the PSD, the amount of consumed sanitizer could be reduced by ca. 40%, while inactivation levels of pathogens were not different from or better than treatment with conventional full-time aerosolization. However, because we used a laboratory-scale instrument (Fig. 1) as a preliminary step to investigate the feasibility of utilizing an infrared sensor for enhancing aerosol sanitization on a pilot or commercial scale, the amount of treated sample (25 g) and the length of treatment (60 min) cannot be translated to commercial application. Also, the infrared value range of the PSD needs to be optimized when applied to an industrial-scale container. Therefore, far-ranging studies involving more varieties of fresh produce, sanitizers, and treatment times in a larger model cabinet are required to develop this new aerosol sanitization system as a practical commercial intervention.
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ACKNOWLEDGMENTS This research was supported by R&D Convergence Center Support Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea. This research was also supported by a grant (10162KFDA995) from the Korea Food & Drug Administration in 2012.
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