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Pulsed light destruction kinetics of L. monocytogenes
Accepted Manuscript Pulsed light destruction kinetics of L. monocytogenes Allison M. Pollock, Anubhav Pratap Singh, Hosahalli S. Ramaswamy, Michael Ngadi PII:
S0023-6438(17)30355-9
DOI:
10.1016/j.lwt.2017.05.040
Reference:
YFSTL 6256
To appear in:
LWT - Food Science and Technology
Received Date: 24 March 2017 Accepted Date: 21 May 2017
Please cite this article as: Pollock, A.M., Pratap Singh, A., Ramaswamy, H.S., Ngadi, M., Pulsed light destruction kinetics of L. monocytogenes, LWT - Food Science and Technology (2017), doi: 10.1016/ j.lwt.2017.05.040. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Pulsed Light Destruction Kinetics of L. monocytogenes
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Allison M. Pollock1, Anubhav Pratap Singh1+, Hosahalli S. Ramaswamy1,* and Michael
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Ngadi2
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Department of Food Science and Agricultural Chemistry, 2 Department of BioResource
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Engineering, McGill University, 21,111 Lakeshore Rd., Sainte Anne de Bellevue, QC, H9X3V9,
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Canada.
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British Columbia, 2205 East Mall, Vancouver, BC, V6T1Z4, Canada.
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Highlights
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Corresponding Author: [email protected]
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Current address: Food, Nutrition and Health, Faculty of Land and Food Systems, University of
Listeria monocytogenes Scott A was found the most resistant strain in this study Pulsed light (PL) effectiveness depended on the voltage and distance from light source For Scott A strain PL induced D-value at the surface of agar plates was 0.9-2.2s High Zv (500 V) indicated a lower dependence of D value on PL voltage Higher D-value (93s) in liquid media suggests the need for thin-profile treatment
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Abstract (200 words) Pulsed light (PL) treatment is an emerging tool for food safety & is increasingly being used
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for sanitization of food and contact surfaces. L. monocytogenes contamination is one of the most
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important microbiological threat to food safety. This study was conducted to assess its resistance
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to PL treatment by establishing the destruction kinetics of 5 strains of L. monocytogenes
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obtained from different marine sources (shrimp, lobster, crab and salmon) on top of surface-agar
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plates or dispensed in liquid-media. L. monocytogenes Scott A strain was found to be the most
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resistant, requiring 20s for 5-log reduction at 800V and 5cm distance from PL source; while the
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strain 323 was least resistant. PL voltage, treatment time and distance from the PL source played
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a significant role in the destruction of L. monocytogenes and a correlation was established to
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estimate lethality contributed by the PL treatment. Results revealed potential of PL treatment to
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sanitize smooth and dry surfaces (D-value of 0.91±0.23s at 800V, and Zv value of 500±24V);
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however, its penetration to liquid samples was poor (D-value of 93±5s). Nevertheless, for liquid-
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media, a thin-profile treatment can be considered. These results will be useful in establishing PL
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treatment for L. monocytogenes and other pathogens.
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Keywords: pulsed light; Listeria monocytogenes; destruction kinetics; surface decontamination;
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food safety.
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1. Introduction
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Pulsed light (PL) treatment is an innovative technological concept that has great potential for
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extending the shelf-life of foods, without a heat treatment step. It is a method of food
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preservation that involves the use of intense and short duration pulses of broad-spectrum "white
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light", where each pulse, or flash, of light lasts a fraction of a second and the intensity of each
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flash is approximately 20,000 times the intensity of sunlight at sea level. Pulsed light is a
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sterilization method in applications were light can access all the important volume and surfaces,
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such as packaging materials (Buchovec et al., 2010), surfaces (Abida et al., 2014; Woodling and
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Muraru, 2007), transparent materials (Cheigh et al., 2013) and pharmaceutical or medical
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products (Elmnasser et al., 2007). However, due to the non-uniform surfaces and opacity of food
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stuffs, PL treatment cannot sterilize these products; however, it can reduce the microbial load of
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such products.
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The antimicrobial effects of UV wavelengths in the PL spectrum are primarily mediated
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through absorption by highly conjugated carbon-to-carbon double-bond systems in proteins and
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nucleic acids (Barbosa-Canovas et al., 2000; Dunn et al., 1995; Heinrich et al., 2016). Chemical
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modifications and cleavage of DNA are two of the several mechanisms of inactivation. It is
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presumed that the impact of PL on proteins, membranes, and other cellular material occurs
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concurrently with nucleic acid destruction. The proposed mechanisms of action of pulsed UV
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light in macromolecules mentioned above are as follows (XENON Corporation, 2015):
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DNA: demonstration of strand breaks and dimer formation in vivo and vitro.
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RNA: single stranded breaks and formation of dimers.
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Proteins: peptide bonds not broken; inactivation of enzyme activity controlled or
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minimized by controlling the delivery of critical parameters.
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The antimicrobial effect of pulsed light is significantly greater than that of conventional UV
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sources or high-intensity mercury vapor lamps (Bohrerova et al., 2008). A few pulsed-light
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flashes destroy high levels of exposed organisms. For example, A. niger, a common bread mold
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relatively resistant to conventional UV, on conventional UV exposure produces only 2.5-4.5 log
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CFU/cm2 reduction after 10 s treatment, whereas with PL, it produces 7 log CFU/cm2 reduction
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in a fraction of a second (Cerny, 1977). However, the works on liquid samples (Dunn et al.,
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1995, 1997a, 1997b; Krishnamurthy et al., 2004, 2007; Pataro et al., 2011; Sauer and Moraru,
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2009) have revealed unsatisfactory decontamination, particularly due to the low penetration of
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UV light. This warrants further research into the thin-profile liquid treatment.
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Many studies have been carried out to evaluate the effects of static UV light treatment of
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food, however there are very few studies for the application of pulsed UV (Dunn et al., 1995,
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1997a, 1997b; Dunn, 1996; Goff et al., 2015; Macgregor et al., 1998; Ozer and Demirci, 2006;
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Rowan et al., 1998; Uesugi et al., 2007; Sauer and Moraru, 2009; Woodling and Muraru, 2007).
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These studies have shown the ability of PL to destroy microorganisms while maintaining food
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quality. Dunn et al. (1997b) reported that a single PL flash at 1-2 J/cm2 can kill 6 log CFU/cm2 of
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bacterial spores They also showed that a total fluence of about 4-6 J/cm2 will kill more than 7-8
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log CFU/cm2 of bacterial or mold spores and more than 9 log CFU/cm2 of vegetative cells. A
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study by Dunn et al. (1995) involving a micro drop assay for a gram-positive pathogen (L.
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monocytogenes), a gram-negative pathogen (E. coli 0157:H7), aerobic bacterial spore (B.
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pumilus), and fungal conidiospores (A. niger) showed no surviving organisms at 105 CFU/cm2
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concentration using PL treatment of a single flash, and 7-9 logs reduction per cm2 using a few
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flashes. In addition, Rowan et al. (1998) concluded that light pulses of low or high UV content
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can reduce counts of L. monocytogenes, S. enteritidis, P. aeroginosa, B. cereus, and S. aureus by
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up to 2 or 6 log orders, respectively. More recently, a study (XENON Corporation, 2015)
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conducted by the XENON Corporation showed the ability Of XENON's SteriPulse-XL 3000
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equipment to kill bacterial endospores of Bacillus subtilis. Hierro et al. (2011) reported that a PL
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treatment of 8.4 J/cm2 reduced L. monocytogenes by 1.78 cfu/cm2 in cooked ham and by
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1.11 cfu/cm2 in bologna and extended its shelf-life by 30 days. Similarly, Ozer and Demirci
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(2006) found a relatively low reduction (1.02 log CFU/cm2) for L. monocytogenes Scott A.
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treatment on salmon skin. Abida et al. (2014) reported 2.7-log reduction of A. niger on
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packaging material at a fluence of 1 J/cm2 fluence, and 6.5 log reduction of E. coli on stainless
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steel for 3 J/cm2 fluence of PL processing.
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While C. botulinum has been of concern for many years, Listeria monocytogenes (a Gram
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positive non-spore forming rod shaped aerobic and facultative anaerobic bacteria) is a relatively
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new concern, particularly for marine foods (Cheigh et al., 2013). It is widespread in nature,
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occurring in soil, vegetation, water, and many animal and plant products (Lovett and Twetd,
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1988). In addition, it can grow over a wide pH, aw, salt and temperature range. The Food and
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Agriculture Organization (FAO, 1999) reported that L. monocytogenes has been isolated from
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fishery products on a regular basis since the late 1980s (FDA, 2001). The data collected from
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these destruction studies are very useful for designing the PL process. However, unfortunately,
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destruction kinetics have generally not been evaluated for PL treatment in either of these study,
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despite the availability of these data. Destruction kinetics is more commonly evaluated in the
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field of food processing (Pratap Singh et al., 2017) and helps to built up the thermal process by
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establishing process lethality. A similar concept can be imported for PL destruction of
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microorganisms. However, to the best of our knowledge, the literature is scarce. Although some
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data is available and an estimate of PL effectiveness can be made, its characterization is still
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important.
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PL technology is still in its infancy and therefore there are a limited number of studies that
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have been carried out on the effect of PL through destruction kinetics. There is almost no
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literature available for PL decontamination using Magnavolt PUV-01 (Model PUV-01,
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Magnavolt Technologies Inc., Plattsburgh, NY, USA), and it is known that the PL apparatus can 5
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significantly affect the amount of inactivation of specific microorganisms (Saikiran et al., 2016).
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Further, to the best of our knowledge, no study evaluates the destruction kinetics or compares the
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resistance of various strains of L. monocytogenes. In accordance, the objectives of this research
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were to characterize PL distribution in Magnavolt PUV-01, study the effectiveness of PL
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treatment using various strains of Listeria monocytogenes, and to evaluate the PL destruction
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kinetics of Listeria monocytogenes on surface of general purpose media and in liquid media.
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2. Materials and Methods 2.1.
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A bench-top pulsed UV system fitted with a low-pressure xenon flash lamp (Model PUV-01,
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Magnavolt Technologies Inc., Plattsburgh, NY, USA) was used to subject samples to PL
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treatment (Fig. 1). During PL processing, the electric power is magnified by accumulating
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electrical energy in an energy storage capacitor over relatively long times (for example, a
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decimal fraction of a second) and releasing this stored energy to do work in much shorter times
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(milliseconds or nanoseconds). This results in very high power burst generated during the short
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duty cycle with the expenditure of only modest average power consumption. PL treatments were
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conducted by varying three PL parameters: treatment time (0-600 s), voltage intensity (0-1000
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volts) and distance from the PL source (5-35 cm). All treatments were given 1 pulse per second
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(pps) in this study.
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Pulsed light Equipment
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2.2.
Bacterial strains
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Five strains of Listeria monocytogenes; HPB strains Scott A (smoked salmon isolate), 323
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(shrimp iso1ate), 392 (lobster isolate), 439 (crab isolate) and 976 (smoked salmon isolate) were
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obtained from the Microbial Hazards Bureau (Health Protection Branch (HPB) Health Canada,
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Ottawa, Canada). The cultures were maintained frozen at -20°C (700 µL of a 24 h culture grown
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in TSB (tryptic soy broth) + YE (yeast extract) (Difco Laboratories, Detroit, MI) with 300 µL of
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a 50% (v/v) glycerol solution). A loopful of the above culture was streaked onto tryptic soy agar
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(TSA, Difco Laboratories, Detroit, MI) plates and incubated at 35 oC for 48 h. Isolated colonies
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were then transferred to a tube containing 9 mL of tryptic soy broth (TSB, Difco Laboratories,
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Detroit, MI) supplemented with 0.6% yeast extract (TSB/YE) and incubated for approximately
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12 h at 35°C to give a suspension of approximately 1x109 CFU/mL. Inoculums were prepared
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using this culture which was further diluted with the appropriate volume of 0.1 % peptone water
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to achieve the desired inoculum concentration (101-109 CFU/mL). All sample preparations and
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inoculation were carried out in a Purifier™ Class II Safety Cabinet (Labconco, Model #36205-
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04, Labconco, Kansas, MI, USA) equipped with a HEPA filter to ensure minimal contamination
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of the samples and the surrounding environment as well as the safety of the research personnel.
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2.3.
Relative resistance of different strains of L. monocytogenes to PL treatment
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Room temperature solid tryptic soy agar (TSA, Difco) Petri plates were inoculated by
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spreading 0.1 mL of a 1x107 CFU/mL suspension of L. monocytogenes (Scott A, 323, 392, 439,
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976 and a mix of all five in equal volumes) evenly onto each plate using a sterile hockey stick, to
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give a final inoculum level of 1x106 CFU/plate. Control samples were inoculated in a similar
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fashion with sterile 0.1 % peptone water. The plates were then subjected to the following PL
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treatments under the center of the flash lamp: 600-800 V treatment voltage at 5-15 cm distance
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from the PL source for 20-60 s @ 1 pps. It must be noted that previous results (not shown here)
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concluded that 1 pps was the most efficient pulse frequency (in the range 0.1-10 pps).
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2.4.
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Petri plates were inoculated as described earlier, however only L. monocytogenes Scott A
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were used. Twelve inoculated plates were placed on the treatment tray as shown in Fig. 2 and
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subjected to desired PL treatments (600-800 V treatment voltage at 5-15 cm distance from the
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PL source for 60 s or 60 pulses at 1 pps) to establish the lethality (% kill) of PL treatment on L.
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monocytogenes. 2.5.
Destruction kinetics of L. monocytogenes
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PL treatment lethality determination.
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2.5.1. L. monocytogenes on surface of a solid general purpose media
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Room temperature solidified tryptic soy agar (TSA, Difco) petri plates were inoculated by
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spreading 0.1 mL of varying concentrations of L. monocytogenes Scott A evenly onto each plate
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using a sterile hockey stick. Control samples were inoculated in a similar fashion with 0.1 %
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peptone water. The plates were then subjected to PL treatment at 600-800 V treatment voltage at
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5 cm distance from the PL source for 1-25 s @ 1 pps.
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2.5.2. L. monocytogenes in liquid media in Whirl Pak bags
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A 20 mL aliquot of a 0.1 % peptone water solution containing 108 CFU/mL L.
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monocytogenes Scott A was aseptically transferred to a 2 oz transparent polyethylene sampling
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bag (Whirl Pak, Fisher Scientific, Ottawa, ON, Canada) and heat sealed. Control samples were
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packaged in a similar fashion with 0.1 % peptone water. The 20 mL aliquot represented a sample
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thickness of approximately 7 mm in the sealed Whirl Pak bags. All samples (in duplicate) were
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PL treated at 800 V treatment voltage at 5 cm distance from the PL source for 0-120 s @ 1pps.
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2.6.
Enumeration
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Following PL treatment on solid general purpose media (Sections 2.3-2.4 and 2.5.1), plates
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were incubated at 35°C for 24 h and then enumerated. Following PL treatment in Whirl Pak bags
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(Section 2.5.2), 1 mL aliquots were transferred to tubes containing 9 mL of 0.1 % sterile peptone
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water and further dilutions were made using 0.1 % sterile peptone water. Duplicate samples were
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enumerated by plating 0.1 mL of the appropriate dilutions on tryptic soy agar (TSA, Difco
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Laboratories, Detroit, MI) by the spread plate method and incubating at 35°C for 24 h. For initial
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studies, a qualitative scale classifying the growth on plates into six levels (0-5 as shown in Table
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1) was used to represent growth of L. monocytogenes on petri-plates.
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2.7.
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The PL destruction kinetics of L. monocytogenes were analyzed based on a first-order
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Pulsed Light destruction kinetics.
reaction indicating a logarithmic order of death, and expressed as Eq. (1): =−
(1)
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where N is the number of organisms that survived the PL treatment for time t (min), No is the
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initial number of microorganisms present before PL treatment and k is the reaction rate constant
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(min-1).
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The treatment time, at a given voltage, resulting in 90% destruction of the existing microbial
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population, is referred to as the decimal reduction time (D-value). This was obtained as the
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negative reciprocal slope of the survivor curve (log N/No v/s t curve), and expressed as Eq. (2):
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=−
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where N1 and N2 represent survivor counts at time tl and t2, respectively. Since the survivor
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curve can have a lag period, this must be considered while using the data for practical
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applications.
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The voltage dependence of the kinetic parameters D-value was analyzed by voltage Z-value
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(Zv), which represents the voltage range that results in a 10 foldchange in D-value. Zv was
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determined by the negative reciprocal of the slope of the log (D-values) v/s treatment voltage
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curve, as expressed by Eq. (3):
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where D1 and D2 represent decimal reduction times at voltages V1 and V2, respectively.
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2.8.
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Duplicate observations of same sample for each treatment were carried out and each
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observation was enumerated in duplicated. All the results, where feasible, were presented as
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mean ± standard deviation of 4 observations. Each set of observations were compared using T-
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test by comparing means and differences were considered significant for p<0.05. Where
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significant differences were present, individual treatments were compared using the least
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significant difference (LSD) test. Regression analysis were conducted using Microsoft Excel
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2016 (Microsoft Corporation, USA).
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3. Results and Discussions
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Statistical Analysis
3.1.
Relative resistance of different strains of L. monocytogenes to PL treatment
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The qualitative growth of 5 strains and a mixed culture of L. monocytogenes following PL
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treatment are shown in Table 2. It was important to investigate the possible differences in
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resistance of 5 strains of L. monocytogenes, all from marine sources, to PL treatment. From these
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results, a representative high resistance strain was to be chosen for further studies. T-test & LSD
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results suggested the following order for the resistance of each strain: Scott A ~ Mixture > 976,
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392, 439 > 323.
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It can be seen in Table 2 that strain Scott A and the mixed culture were most resistant to PL
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treatments showing up to 5-log reductions in a 20s treatment at 800 V voltage treatment at 5 cm
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distance from the PL source. On the other hand, there was very little microbial inactivation for
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these strains at 600 V and colonies were too high to be counted as shown, suggesting a strong
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dependence of PL inactivation on the treatment voltage. It can be visualized from Fig. 3, which
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is a sample representation from Table 3, that in contrast to Scott A, Strain 323 from the shrimp
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isolate was least resistant, and thus most susceptible, to the PL treatment. The PL inactivation of
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strains 976, 392 and 439 were between than of 323 and Scott A without any statistical
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differences (p>0.05) between each other. The mixed culture was found less or equally resistant
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as Scott A (p~0.05). This might entail that the growth of the survived Scott A microbes in the
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cocktail was more dominant than other strains inside that cocktail. Being the most resistant
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strain, Scott A was used in all experiments to follow.
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3.2.
Lethality of PL light on L. monocytogenes
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In order to establish the inactivation of L. monocytogenes at each distance from the PL
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source, the average kill of L. monocytgenes was calculated for plates for a 60s at the respective
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treatment voltage and distances from the PL source (Table 3). Fig. 4 represents a model of the
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effect of voltage (600 and 800 V) and distance (5, 10 and 15 cm) from the PL source on the
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percent kill of L. monocytogenes by PL treatment. The regression equation which fitted to the
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data is shown as Eq. (4): % #$ = 132.33 − 0.018 ∗ , − 14.42 ∗ + 0.014 ∗ , ∗ / 0 = 94.85
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(4)
where V is the voltage (V) and D is the distance (cm) from the lamp source.
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From these results, optimum maximum and minimum values were determined for percent
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kill of L. monocytogenes. A maximum value (approx. 100% kill) would be achieved at 800 V
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and 5 cm distance from the PL source. A minimum value (31.3%) would be achieved at 600 V
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and 15 cm distance from the PL source. These finding are in accordance with the fact that the
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lethality of PL treatment increases with increasing light intensity or fluence (FDA, 2000).
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The findings suggest that as voltage increased from 600 to 800 V, microbial inactivation of
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L. monocytogenes increased. Also, as the distance from the PL source is increased (from 5 to 15
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cm), the percent kill of L. monocytogenes reduces due to reduction in PL intensity. These
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findings have also shown that in general, as the number of pulses is increased (i.e. increasing the
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treatment time), microbial destruction is increased.
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These results were for Scott A only and agree well with the findings of other researchers,
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such as Ozer and Demirci (2006), working with L. monocytogenes. Ozer and Demirci (2006)
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found only 1.02 log reduction at 8 cm distance with a 60s treatment on raw salmon skin fillets
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for L. monocytogenes Scott A. However, they also agree well with that obtained for other strains
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of L. monocytogenes, as shown in Table 2. Other researchers (Krishnamurthy et al., 2007) have
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found a similar ~ 7-log reduction of S. aureus in milk when treatment time was increased from
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30 to 180 s. PL destruction in any type of micro-organisms follows a similar trend as observed in
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this study. From the results of this study, a PL treatment at 800 V treatment voltage at 5 cm
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distance from the PL source for 60 s @ 1 pps was recommended, whenever possible, to
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maximize microbial destruction.
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3.3.
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Destruction kinetics of L. monocytogenes on surface of solid general purpose media
The survival curves for L. monocytogenes on the surface of the solidified agar plates at 600,
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700 and 800 V are shown in Fig. 5a, which indicates that the destruction was significantly
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influenced (P < 0.05, by comparison of means) by the voltage used and the treatment time. The
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survival curves at higher voltages (800 V) were steeper than at lower voltages (600 V) which
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illustrate that the destruction rate of L. monocytogenes was higher at higher voltages. Gomez-
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Lopez (2005) reported that efficiency of reduction of counts of L. monocytogenes on agar
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surface reduced with decrease in PL intensity, and our results agreed well with their study.
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The decimal reduction times at 800, 700 and 600 V were 0.91±0.23, 1.37±0.31 and
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2.25±0.38 s respectively. It must be noted that D-values were computed by ignoring the initial
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lag period (4-7 s), only after which a first order rate of destruction was observed. The lag times
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and associated D values are summarized in Table 4. For example, at 800V, the associated D
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value is 0.91±0.23 s and the lag is 4.0 ± 0.1 s. So for achieving one decimal reduction in counts,
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the treatment time would be the D value of 0.91 s plus the las of 4 s for a total of 4.91 s.
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However, if 5 decimal reduction are required, the exposure time would be (0.91 x 5) + 4.0 = 8.55
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s. Thus, it is imperative that the associated lag periods must be used along with the respective D
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values to have a meaningful prediction of the PL destruction power.
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Lower D-values are associated with higher voltages demonstrating a higher destruction rate.
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Fig. 5b demonstrates the curve of log (D-value) vs. treatment voltage to compute the voltage
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sensitivity parameter (Zv value). The Zv value of L. monocytogenes was calculated to be 500±24
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V, meaning that for every 500 V increase in treatment voltage, the D-value would be reduced by
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1 log cycle. A reference D-value and z fully describes the pulsed destruction kinetics over the
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range of voltages studies. The z-value of 500 V was relatively high and suggests a rather weak
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dependence on treatment, when the voltage range of only 600-800 V is considered. This is
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advantageous in a way, because even at low power consumption, potentially satisfactory
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inactivation of L. monocytogenes can be obtained by slightly reducing the distance from the PL
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source.
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The low D-values & high Z-value in this experiment show that PL has an incredible potential
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as a surface sanitation method. Surface inactivation of L. monocytogenes was monitored in a
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similar study that reported a 7-log reduction in L. monocytogenes, on the surface of a general
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purpose media, following PL treatment for 512 s at 30 kV and 1 pps (MacGregor et al., 1998).
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The longer treatment time required for the reduction in counts in this study was due to the lower
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stored energy in the pulse generator of 3 J/pulse, versus 12.8 J/pulse utilized in this study. On the
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other hand, Hierro et al. (2011) prescribed a dosage between 3-5s for effective reduction of L.
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monocytogenes on bologna and cooked ham surface, which is in accordance with the
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observations of high effectiveness of PL treatment for surface decontamination of L.
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monocytogenes.
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3.4.
L. monocytogenes in liquid media in liquid media
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The survival curve for L. monocytogenes in liquid media (shown in Fig. 6) depicts a small
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reduction in counts, even after 120 s of treatment at 800 V. The figure demonstrates a good fit of
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data for the first order model (R2 = 0.97) which suggests that L. monocytogenes in Whirl Pak
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bags did follow logarithmic destruction under PL treatment. Due to relatively low inactivation,
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the lag-phase was not apparent for this treatment. A decimal reduction time of 93 ± 5 s resulted
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from the PL treatment, which is quite large and would render PL treatment undesirable &
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uneconomical.
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Thus, it is seen that PL treatment was not effective for volumetric treatment in Whirl Pak
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bags. It must be noted that due to the limited penetration of PL treatment, the samples, which had
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a thickness of approx. 7 mm, were too thick to achieve a reasonable rate of destruction.
310
Secondly, the penetration power of the light gets attenuated exponentially. Difficulty with PL
311
treatment in thick and opaque samples has been well documented by Dunn et al. (1995, 1997a,
312
1997b), Krishnamurthy et al. (2004, 2007), Sauer and Moraru (2009); and more recently by
313
Pataro et al. (2011). Krishnamurthy et al. (2007) showed that the ability of PL to inactivate S.
314
aureus in milk decreased as the sample volume increased from 12-48 mL due to poor penetration
315
capacity of UV light. Although there could have been substantial reduction in counts at the
316
surface, the survivor in the deeper layers would dominate the total counts when mixed. For
317
example, even if 80% of the cells in the light's path get destroyed by several log cycles, leaving
318
the other 20% in the central region unaffected, the overall count will still be in the same
319
logarithmic scale as in the beginning. This means the overall reduction would be less than one
320
log cycle. Obviously, it would be desirable to use the liquid in much thinner profiles so that the
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destruction occurs throughout.
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4. Conclusions This study evaluated the relative resistance of 5 strains of L. monocytogenes and found Scott
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A strain to be the most resistant. Upto 5-log reduction of Scott A strain of L. monocytogenes was
325
obtained within 20s for treatment at 800 V. PL voltage, treatment time and distance from the PL
326
source played a significant role in the destruction of L. monocytogenes. Maximum destruction
327
was achieved at high voltage and minimum distance from the PL source. Destruction kinetics for
328
L. monocytogenes were evaluated in a liquid media, on the surface of a general-purpose media
329
following various PL treatments. High efficiency of PL application was obtained for PL
330
treatment for on surface of agar plates with a D-value of 0.91±0.23 s at 800V, and a Zv of
331
500±24 V. The D-values were sufficiently low to use a few flashes for surface decontamination
332
of microorganisms in a few seconds. On the other hand, the high Zv suggested relatively lower
333
dependence of PL lethality on the PL voltage, as compared to the treatment time. In liquid-media
334
inside whirl pak bags, D-values (93±5 s) were very high, suggesting relatively poor PL
335
penetration into liquid media. The low microbial lethality of PL treatment for liquid samples
336
warrants more research into thin-profile PL treatment of liquids, which can potentially counter
337
this effect of PL. These results shall be useful in establishing PL treatment for L. monocytogenes
338
and other microbes.
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5. Acknowledgments
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This work was supported by funds ministère de l'Agriculture, des Pêcheries et de l'Alimentation du Québec (MAPAQ).
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6. References
343
Abida, J., Rayees, B., & Masoodi, F. A. (2014). Pulsed light technology: a novel method for food
345 346
preservation. International Food Research Journal, 21(3), 839-848.
RI PT
344
Barbosa-Canovas, G. V., Schaffner, D. W., Pierson, M. D., & Zhang, Q. H. (2000). Pulsed light technology. Journal of Food Science, 65(s8), 82–85.
Bohrerova, Z., Shemer, H., Lantis, R., Impellitteri, C. A., & Linden, K. G. (2008). Comparative
348
disinfection efficiency of pulsed and continuous-wave UV irradiation technologies.
349
Water Research, 42(12), 2975-2982.
M AN U
SC
347
350
Buchovec, I., Paskeviciute, E., & Luksiene, Z. (2010). Photosensitization-based inactivation of
351
food pathogen Listeria monocytogenes in vitro and on the surface of packaging material.
352
Journal of Photochemistry and Photobiology B: Biology, 99(1), 9-14. Cheigh, C. I., Hwang, H. J., & Chung, M. S. (2013). Intense pulsed light (IPL) and UV-C
354
treatments for inactivating Listeria monocytogenes on solid medium and seafood’s.
355
Food Research International, 54(1), 745-752.
TE D
353
Hierro, E., Barroso, E., De la Hoz, L., Ordóñez, J. A., Manzano, S., & Fernández, M. (2011).
357
Efficacy of pulsed light for shelf-life extension and inactivation of Listeria
358
monocytogenes on ready-to-eat cooked meat products. Innovative Food Science and
360 361 362 363
AC C
359
EP
356
Emerging Technologies, 12(3), 275-281.
Cerny, G. (1977). Sterilization of packaging materials for aseptic packaging. Verpack- ungsRdsch, Tech-nisch-wiss Beilage, 28(10), 77.
Dunn, J., Ott, T., & Clark, W. (1995). Pulsed-light treatment of food and packaging. Food Technology, 49(9), 95-98.
364
17
ACCEPTED MANUSCRIPT
365 366
Dunn, I. (1996). Pulsed light and pulsed electric field for foods and eggs. Poultry Science, 75, 1133-1136. Dunn, I, Burgess, D., & Leo, F. (1997a). Investigation of pulsed light for terminal sterilization of
368
WFI filled blow/fill/seal polyethylene containers. PDA Journal of Pharmaceutical
369
Science and Technology, 51(3), 111-115.
371
Dunn, J., Bushnell, A., Ott, T., & Clark, W. (1997b). Pulsed white light food processing. Cereal Foods World, 42(7), 510-515.
SC
370
RI PT
367
Elmnasser, N., Guillou, S., Leroi, F., Orange, N., Bakhrouf, A., & Federighi, M. (2007). Pulsed-
373
light system as a novel food decontamination technology: a review. Canadian Journal
374
of Microbiology, 53(7), 813-821.
375
M AN U
372
FAO (1999). FAO expert consultation on the trade impact of Listeria monocytogenes in fish products.
377
http://www.fao.org/docrep/003/x3018e/x3018e00.htm (accessed 04.11.16).
378
Food
and
Agriculture
Organization,
TE D
376
Amherst,
MA,
USA.
FDA (2000). Kinetics of Microbial Inactivation for Alternative Food Processing Technologies. Food
380
http://www.fda.gov/Food/FoodScienceResearch/SafePracticesforFoodProcesses/ucm10
381
0158.htm (accessed. 04.11.16)
383 384 385
Drug
Administration,
Silver
Spring,
MD,
FDA (2001). Processing parameters needed to control pathogens in cold smoked fish. Food and
AC C
382
and
EP
379
Drug
Administration,
Silver
Spring,
MD,
http://www.fda.gov/Food/FoodScienceResearch/SafePracticesforFoodProcesses/ucm09 2182.htm (accessed. 04.11.16)
18
ACCEPTED MANUSCRIPT
386
Heinrich, V., Zunabovic, M., Varzakas, T., Bergmair, J., & Kneifel, W. (2016). Pulsed Light
387
Treatment of Different Food Types with a Special Focus on Meat: A Critical Review.
388
Critical Reviews in Food Science and Nutrition, 56(4), 591-613.
390
Krishnamurthy, K., Demirici, A., & lrudayaraj, J. (2004). Inactivation of Staphylococcus aureus
RI PT
389
by pulsed UV -light sterilization. Journal of Food Protection, 67, 1027-1030.
Krishnamurthy, K., Demirici, A., & lrudayaraj, J. (2007). Inactivation of Staphylococcus aureus
392
in milk using flow-through pulsed UV-light treatment system. Journal of Food
393
Protection, 72 (7), M233-M239.
SC
391
Goff, L. L., Hubert, B., Favennec, L., Villena, I., Ballet, J. J., Agoulon, A., & Gargala, G. (2015).
395
Pilot-Scale Pulsed UV Light Irradiation of Experimentally Infected Raspberries
396
Suppresses Cryptosporidium parvum Infectivity in Immunocompetent Suckling Mice.
397
Journal of Food Protection, 78(12), 2247-2252.
M AN U
394
Lovett, J., & Twetd, R. M. (1988). Listeria. Journal of Food Technology, 42(4), 188.
399
MacGregor, S. I., Rowan, N. I., Macilvaney, L., Anderson, J. G., Fouracre, R. A., Farish, O.
400
(1998). Light inactivation of food-related pathogenic becteria using a pulsed power
401
source. Letters in Applied Microbiology, 27, 67-70.
EP
TE D
398
Ozer, N. P., & Demirci., A. (2006). Inactivation of Escherichia coli O157:H7 and Listeria
403
monocytogenes inoculated on raw salmon fillets by pulsed-UV light treatment.
404
AC C
402
International Journal of Food Science and Technology, 41 (4): 354-360.
405
Pataro, G., Muñoz, A., Palgan, I., Noci, F., Ferrari, G., & Lyng, J. G. (2011). Bacterial
406
inactivation in fruit juices using a continuous flow Pulsed Light (PL) system. Food
407
Research International, 44(6), 1642-1648.
19
ACCEPTED MANUSCRIPT
408
Pratap Singh, A., Singh, A., & Ramaswamy, H. S. (2017). Heat transfer phenomena during
409
thermal processing of liquid particulate mixtures – A Review. Critical Reviews in Food
410
Science and Nutrition, 57(7):1350-1364. Rowan, N., MacGregor, S. J., Anderson, I. G., Fouracre, R. A., McIlvaney, L., & Farish, O.
412
(1998). Pulsed-light inactivation of food-related microorganisms. Applied Environmetal
413
Microbiology, 65(3), 1312-1315.
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Saikiran, K. C. S., Mn, L., & Venkatachalapathy, N. (2016). Different pulsed light systems and
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their application in foods: a review. International Journal of Science, Environment, and
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Technology, 5(3), 1463-1476.
M AN U
SC
414
417
Sauer, A., & Moraru C. I. (2009). Inactivation of E. coli ATCC 25922 and E. coli O157:H7 in
418
apple juice and apple cider using Pulsed Light treatment. Journal of Food Protection,
419
72(5), 937-44
Uesugi, A., Woodling, S. E., & Moraru, C. I. (2007). Inactivation kinetics and factors of
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variability in the Pulsed Light treatment of Listeria innocua cells. Journal of Food
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Protection, 70(11), 2518-2525
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Woodling, S. E., & Moraru, C. I. (2007). Effect of spectral range in surface inactivation of
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Listeria innocua using broad spectrum Pulsed Light. Journal of Food Protection, 70(4),
425
906-916
427 428 429
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XENON Corporation. (2015). Applications of pulsed light for sterilization. XENON Corporation,
Wilmington,
MA,
USA.
http://www.xenoncorp.com/files/5914/5451/5085/Pulsed_Light_Sterilization_Applicati ons.pdf (accessed 04.11.16).
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Table 1: Qualitative scale to represent growth of L. monocytogenes.
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Growth
Description of Growth
Approximate
number
of
5
Lawn of growth
4
Extensive
growth
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colonies TNTCa (distinct >300
3
Heavy growth (countable)
2
Heavy growth (countable)
1
Light growth
~10
0
No growth
0
>300 ~30
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TNTC = too high to count
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colonies)
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Table 2: Growth (on a scale of 1 to 5 – refer Table 1) of 5 strains and a mixed culture of L.
436
monocytogenes isolated from marine sources and pulsed treated at various combinations of
437
treatment voltage, treatment time and distance from the PL source. Distance Treatment Treatment Growth
source
Time (s)
(V)
Mixture
Scott A
976
(cm)
40
1
60
1
20
0.5
392
of all 5 strains
0.5
0.5
0.5
0
1.5
0
0.5
0.5
0.5
0.5
0
0
0.5
0.5
0.5
1
0.5
0
0.5
40
0.5
0
0
0
0
0
60
0
0
0
0
0
0
2
1
1
1
1
2
1
0
0
0.5
0
1
60
0
0
0
0.5
0.5
1
20
0.5
0.5
0
0.5
0
0.5
40
0
0
0.5
0
0
0
60
0
0
0
0
0
0
20
5
4
3
4
4
5
40
1
1
0
1
1
1
60
1
1
0.5
0.5
1
1
800
600
20
10 800
600
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15
0.5
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439
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600
20
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from PL Voltage
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800
20
1
1
1
0.5
1
1
40
1
1
0
0.5
0
0.5
60
0
0.5
0
0.5
0
0
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438 439
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440
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442
Table 3: Percent kill of L. monocytogenes at various treatment voltages and distances from
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the PL source.
800
A (%)a
5
93±5
10
58±3
15
33±8
5
100±0
10
89±5
15 a
68±7
Values reported as mean±sd
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600
(cm)
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Treatment Voltage Distance from PL source Average Kill of L. monocytogenes Scott
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446
Table 4: Calculation of decimal reduction times (D-values) of L. monocytogenes on the
447
surface of solidified agar plates. D-value following lag
Lag time
(V)
period (s)a
(s)a
800
0.91±0.23
4±1
700
1.37±0.31
5±1
600
2.25±0.38
7±2
reduction (s)1 4.91
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PL exposure time for n decirnal reductions = (D x n) + lag time
a
Values reported as mean±sd
449
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9.25
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Total time for one 4-value
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452 453
Fig. 1. Schematic diagram of the PL apparatus.
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Fig. 2. Position of Petri plates on PL chamber treatment tray during spatial power
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distribution analysis.
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Inactivation of individual and cocktail of 5 strains Scott A
976
323
439
392
Mixture of 5 strains
5 4
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Growth Scale from Table 1
6
3 2 1 0 600V - 40s
600V - 60s
800V - 20s
800V - 40s
800V - 60s
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600V - 20s
Treatment Intensity
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Fig. 3. Sample representation of growth of 5 strains and a mixed culture of L.
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monocytogenes isolated from marine sources and PL treated at 600 V and 15 cm distance
463
from the PL source.
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120
80
5 cm 7 cm
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Percent Kil (%)l
100
60
9 cm
11 cm
40
13 cm 15 cm
0 600
620
640
660
680
700
720
Treatment Voltage (V)
740
760
780
800
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466
Fig. 4. Effect of voltage and distance from the PL source on the percent kill of L.
467
monocytogenes by PL treatment
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a. PL Survivor Curve 10 8
y = -0.7267x + 11.48
7
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Survivors (log CFU/ml)
9
6
800 V
5 4
y = -0.4443x + 11.47
y = -1.1x + 10.967
3
600 V
2 1 5
10
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0 0
700 V
15
20
Time (s)
30
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b. Voltage Sensitivity Curve 0.4 0.35 0.3
y = -0.002x + 1.5272 zv = 1/0.002 = 500 V
0.2 0.15 0.1 0.05 0
-0.1
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log (D-values)
0.25
400
600
800
1000
Voltage (V)
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Fig. 5: a) Survivor and b) voltage sensitivity curves for L. monocytogenes on the surface of
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general purpose media
473
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Survivor Curve in whirl-pak bags 9
7 6
y = -0.0107x + 8.2893 R2 = 0.97 D value = 1/0.0107 = 93s
5
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Survivors (log CFU/ml))
8
4 3 2
0 0
20
60
80
TIme (s)
100
120
140
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Fig. 6: Survivor curve for L. monocytogenes in a 2-oz whirl pak bag.
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S0023-6438(17)30355-9
DOI:
10.1016/j.lwt.2017.05.040
Reference:
YFSTL 6256
To appear in:
LWT - Food Science and Technology
Received Date: 24 March 2017 Accepted Date: 21 May 2017
Please cite this article as: Pollock, A.M., Pratap Singh, A., Ramaswamy, H.S., Ngadi, M., Pulsed light destruction kinetics of L. monocytogenes, LWT - Food Science and Technology (2017), doi: 10.1016/ j.lwt.2017.05.040. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Pulsed Light Destruction Kinetics of L. monocytogenes
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Allison M. Pollock1, Anubhav Pratap Singh1+, Hosahalli S. Ramaswamy1,* and Michael
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Ngadi2
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Department of Food Science and Agricultural Chemistry, 2 Department of BioResource
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Engineering, McGill University, 21,111 Lakeshore Rd., Sainte Anne de Bellevue, QC, H9X3V9,
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Canada.
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British Columbia, 2205 East Mall, Vancouver, BC, V6T1Z4, Canada.
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Highlights
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Corresponding Author: [email protected]
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Current address: Food, Nutrition and Health, Faculty of Land and Food Systems, University of
Listeria monocytogenes Scott A was found the most resistant strain in this study Pulsed light (PL) effectiveness depended on the voltage and distance from light source For Scott A strain PL induced D-value at the surface of agar plates was 0.9-2.2s High Zv (500 V) indicated a lower dependence of D value on PL voltage Higher D-value (93s) in liquid media suggests the need for thin-profile treatment
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Abstract (200 words) Pulsed light (PL) treatment is an emerging tool for food safety & is increasingly being used
22
for sanitization of food and contact surfaces. L. monocytogenes contamination is one of the most
23
important microbiological threat to food safety. This study was conducted to assess its resistance
24
to PL treatment by establishing the destruction kinetics of 5 strains of L. monocytogenes
25
obtained from different marine sources (shrimp, lobster, crab and salmon) on top of surface-agar
26
plates or dispensed in liquid-media. L. monocytogenes Scott A strain was found to be the most
27
resistant, requiring 20s for 5-log reduction at 800V and 5cm distance from PL source; while the
28
strain 323 was least resistant. PL voltage, treatment time and distance from the PL source played
29
a significant role in the destruction of L. monocytogenes and a correlation was established to
30
estimate lethality contributed by the PL treatment. Results revealed potential of PL treatment to
31
sanitize smooth and dry surfaces (D-value of 0.91±0.23s at 800V, and Zv value of 500±24V);
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however, its penetration to liquid samples was poor (D-value of 93±5s). Nevertheless, for liquid-
33
media, a thin-profile treatment can be considered. These results will be useful in establishing PL
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treatment for L. monocytogenes and other pathogens.
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Keywords: pulsed light; Listeria monocytogenes; destruction kinetics; surface decontamination;
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food safety.
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1. Introduction
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Pulsed light (PL) treatment is an innovative technological concept that has great potential for
39
extending the shelf-life of foods, without a heat treatment step. It is a method of food
40
preservation that involves the use of intense and short duration pulses of broad-spectrum "white
41
light", where each pulse, or flash, of light lasts a fraction of a second and the intensity of each
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flash is approximately 20,000 times the intensity of sunlight at sea level. Pulsed light is a
43
sterilization method in applications were light can access all the important volume and surfaces,
44
such as packaging materials (Buchovec et al., 2010), surfaces (Abida et al., 2014; Woodling and
45
Muraru, 2007), transparent materials (Cheigh et al., 2013) and pharmaceutical or medical
46
products (Elmnasser et al., 2007). However, due to the non-uniform surfaces and opacity of food
47
stuffs, PL treatment cannot sterilize these products; however, it can reduce the microbial load of
48
such products.
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The antimicrobial effects of UV wavelengths in the PL spectrum are primarily mediated
50
through absorption by highly conjugated carbon-to-carbon double-bond systems in proteins and
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nucleic acids (Barbosa-Canovas et al., 2000; Dunn et al., 1995; Heinrich et al., 2016). Chemical
52
modifications and cleavage of DNA are two of the several mechanisms of inactivation. It is
53
presumed that the impact of PL on proteins, membranes, and other cellular material occurs
54
concurrently with nucleic acid destruction. The proposed mechanisms of action of pulsed UV
55
light in macromolecules mentioned above are as follows (XENON Corporation, 2015):
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DNA: demonstration of strand breaks and dimer formation in vivo and vitro.
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RNA: single stranded breaks and formation of dimers.
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Proteins: peptide bonds not broken; inactivation of enzyme activity controlled or
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minimized by controlling the delivery of critical parameters.
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The antimicrobial effect of pulsed light is significantly greater than that of conventional UV
61
sources or high-intensity mercury vapor lamps (Bohrerova et al., 2008). A few pulsed-light
62
flashes destroy high levels of exposed organisms. For example, A. niger, a common bread mold
63
relatively resistant to conventional UV, on conventional UV exposure produces only 2.5-4.5 log
64
CFU/cm2 reduction after 10 s treatment, whereas with PL, it produces 7 log CFU/cm2 reduction
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in a fraction of a second (Cerny, 1977). However, the works on liquid samples (Dunn et al.,
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1995, 1997a, 1997b; Krishnamurthy et al., 2004, 2007; Pataro et al., 2011; Sauer and Moraru,
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2009) have revealed unsatisfactory decontamination, particularly due to the low penetration of
68
UV light. This warrants further research into the thin-profile liquid treatment.
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Many studies have been carried out to evaluate the effects of static UV light treatment of
70
food, however there are very few studies for the application of pulsed UV (Dunn et al., 1995,
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1997a, 1997b; Dunn, 1996; Goff et al., 2015; Macgregor et al., 1998; Ozer and Demirci, 2006;
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Rowan et al., 1998; Uesugi et al., 2007; Sauer and Moraru, 2009; Woodling and Muraru, 2007).
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These studies have shown the ability of PL to destroy microorganisms while maintaining food
74
quality. Dunn et al. (1997b) reported that a single PL flash at 1-2 J/cm2 can kill 6 log CFU/cm2 of
75
bacterial spores They also showed that a total fluence of about 4-6 J/cm2 will kill more than 7-8
76
log CFU/cm2 of bacterial or mold spores and more than 9 log CFU/cm2 of vegetative cells. A
77
study by Dunn et al. (1995) involving a micro drop assay for a gram-positive pathogen (L.
78
monocytogenes), a gram-negative pathogen (E. coli 0157:H7), aerobic bacterial spore (B.
79
pumilus), and fungal conidiospores (A. niger) showed no surviving organisms at 105 CFU/cm2
80
concentration using PL treatment of a single flash, and 7-9 logs reduction per cm2 using a few
81
flashes. In addition, Rowan et al. (1998) concluded that light pulses of low or high UV content
82
can reduce counts of L. monocytogenes, S. enteritidis, P. aeroginosa, B. cereus, and S. aureus by
83
up to 2 or 6 log orders, respectively. More recently, a study (XENON Corporation, 2015)
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conducted by the XENON Corporation showed the ability Of XENON's SteriPulse-XL 3000
85
equipment to kill bacterial endospores of Bacillus subtilis. Hierro et al. (2011) reported that a PL
86
treatment of 8.4 J/cm2 reduced L. monocytogenes by 1.78 cfu/cm2 in cooked ham and by
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1.11 cfu/cm2 in bologna and extended its shelf-life by 30 days. Similarly, Ozer and Demirci
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(2006) found a relatively low reduction (1.02 log CFU/cm2) for L. monocytogenes Scott A.
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treatment on salmon skin. Abida et al. (2014) reported 2.7-log reduction of A. niger on
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packaging material at a fluence of 1 J/cm2 fluence, and 6.5 log reduction of E. coli on stainless
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steel for 3 J/cm2 fluence of PL processing.
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While C. botulinum has been of concern for many years, Listeria monocytogenes (a Gram
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positive non-spore forming rod shaped aerobic and facultative anaerobic bacteria) is a relatively
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new concern, particularly for marine foods (Cheigh et al., 2013). It is widespread in nature,
95
occurring in soil, vegetation, water, and many animal and plant products (Lovett and Twetd,
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1988). In addition, it can grow over a wide pH, aw, salt and temperature range. The Food and
97
Agriculture Organization (FAO, 1999) reported that L. monocytogenes has been isolated from
98
fishery products on a regular basis since the late 1980s (FDA, 2001). The data collected from
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these destruction studies are very useful for designing the PL process. However, unfortunately,
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destruction kinetics have generally not been evaluated for PL treatment in either of these study,
101
despite the availability of these data. Destruction kinetics is more commonly evaluated in the
102
field of food processing (Pratap Singh et al., 2017) and helps to built up the thermal process by
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establishing process lethality. A similar concept can be imported for PL destruction of
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microorganisms. However, to the best of our knowledge, the literature is scarce. Although some
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data is available and an estimate of PL effectiveness can be made, its characterization is still
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important.
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PL technology is still in its infancy and therefore there are a limited number of studies that
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have been carried out on the effect of PL through destruction kinetics. There is almost no
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literature available for PL decontamination using Magnavolt PUV-01 (Model PUV-01,
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Magnavolt Technologies Inc., Plattsburgh, NY, USA), and it is known that the PL apparatus can 5
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significantly affect the amount of inactivation of specific microorganisms (Saikiran et al., 2016).
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Further, to the best of our knowledge, no study evaluates the destruction kinetics or compares the
113
resistance of various strains of L. monocytogenes. In accordance, the objectives of this research
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were to characterize PL distribution in Magnavolt PUV-01, study the effectiveness of PL
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treatment using various strains of Listeria monocytogenes, and to evaluate the PL destruction
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kinetics of Listeria monocytogenes on surface of general purpose media and in liquid media.
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2. Materials and Methods 2.1.
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A bench-top pulsed UV system fitted with a low-pressure xenon flash lamp (Model PUV-01,
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Magnavolt Technologies Inc., Plattsburgh, NY, USA) was used to subject samples to PL
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treatment (Fig. 1). During PL processing, the electric power is magnified by accumulating
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electrical energy in an energy storage capacitor over relatively long times (for example, a
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decimal fraction of a second) and releasing this stored energy to do work in much shorter times
124
(milliseconds or nanoseconds). This results in very high power burst generated during the short
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duty cycle with the expenditure of only modest average power consumption. PL treatments were
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conducted by varying three PL parameters: treatment time (0-600 s), voltage intensity (0-1000
127
volts) and distance from the PL source (5-35 cm). All treatments were given 1 pulse per second
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(pps) in this study.
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2.2.
Bacterial strains
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Five strains of Listeria monocytogenes; HPB strains Scott A (smoked salmon isolate), 323
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(shrimp iso1ate), 392 (lobster isolate), 439 (crab isolate) and 976 (smoked salmon isolate) were
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obtained from the Microbial Hazards Bureau (Health Protection Branch (HPB) Health Canada,
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Ottawa, Canada). The cultures were maintained frozen at -20°C (700 µL of a 24 h culture grown
134
in TSB (tryptic soy broth) + YE (yeast extract) (Difco Laboratories, Detroit, MI) with 300 µL of
135
a 50% (v/v) glycerol solution). A loopful of the above culture was streaked onto tryptic soy agar
136
(TSA, Difco Laboratories, Detroit, MI) plates and incubated at 35 oC for 48 h. Isolated colonies
137
were then transferred to a tube containing 9 mL of tryptic soy broth (TSB, Difco Laboratories,
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Detroit, MI) supplemented with 0.6% yeast extract (TSB/YE) and incubated for approximately
139
12 h at 35°C to give a suspension of approximately 1x109 CFU/mL. Inoculums were prepared
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using this culture which was further diluted with the appropriate volume of 0.1 % peptone water
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to achieve the desired inoculum concentration (101-109 CFU/mL). All sample preparations and
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inoculation were carried out in a Purifier™ Class II Safety Cabinet (Labconco, Model #36205-
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04, Labconco, Kansas, MI, USA) equipped with a HEPA filter to ensure minimal contamination
144
of the samples and the surrounding environment as well as the safety of the research personnel.
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2.3.
Relative resistance of different strains of L. monocytogenes to PL treatment
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Room temperature solid tryptic soy agar (TSA, Difco) Petri plates were inoculated by
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spreading 0.1 mL of a 1x107 CFU/mL suspension of L. monocytogenes (Scott A, 323, 392, 439,
148
976 and a mix of all five in equal volumes) evenly onto each plate using a sterile hockey stick, to
149
give a final inoculum level of 1x106 CFU/plate. Control samples were inoculated in a similar
150
fashion with sterile 0.1 % peptone water. The plates were then subjected to the following PL
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treatments under the center of the flash lamp: 600-800 V treatment voltage at 5-15 cm distance
152
from the PL source for 20-60 s @ 1 pps. It must be noted that previous results (not shown here)
153
concluded that 1 pps was the most efficient pulse frequency (in the range 0.1-10 pps).
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2.4.
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Petri plates were inoculated as described earlier, however only L. monocytogenes Scott A
156
were used. Twelve inoculated plates were placed on the treatment tray as shown in Fig. 2 and
157
subjected to desired PL treatments (600-800 V treatment voltage at 5-15 cm distance from the
158
PL source for 60 s or 60 pulses at 1 pps) to establish the lethality (% kill) of PL treatment on L.
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monocytogenes. 2.5.
Destruction kinetics of L. monocytogenes
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PL treatment lethality determination.
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2.5.1. L. monocytogenes on surface of a solid general purpose media
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Room temperature solidified tryptic soy agar (TSA, Difco) petri plates were inoculated by
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spreading 0.1 mL of varying concentrations of L. monocytogenes Scott A evenly onto each plate
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using a sterile hockey stick. Control samples were inoculated in a similar fashion with 0.1 %
165
peptone water. The plates were then subjected to PL treatment at 600-800 V treatment voltage at
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5 cm distance from the PL source for 1-25 s @ 1 pps.
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2.5.2. L. monocytogenes in liquid media in Whirl Pak bags
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A 20 mL aliquot of a 0.1 % peptone water solution containing 108 CFU/mL L.
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monocytogenes Scott A was aseptically transferred to a 2 oz transparent polyethylene sampling
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bag (Whirl Pak, Fisher Scientific, Ottawa, ON, Canada) and heat sealed. Control samples were
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packaged in a similar fashion with 0.1 % peptone water. The 20 mL aliquot represented a sample
172
thickness of approximately 7 mm in the sealed Whirl Pak bags. All samples (in duplicate) were
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PL treated at 800 V treatment voltage at 5 cm distance from the PL source for 0-120 s @ 1pps.
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2.6.
Enumeration
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Following PL treatment on solid general purpose media (Sections 2.3-2.4 and 2.5.1), plates
176
were incubated at 35°C for 24 h and then enumerated. Following PL treatment in Whirl Pak bags
177
(Section 2.5.2), 1 mL aliquots were transferred to tubes containing 9 mL of 0.1 % sterile peptone
178
water and further dilutions were made using 0.1 % sterile peptone water. Duplicate samples were
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enumerated by plating 0.1 mL of the appropriate dilutions on tryptic soy agar (TSA, Difco
180
Laboratories, Detroit, MI) by the spread plate method and incubating at 35°C for 24 h. For initial
181
studies, a qualitative scale classifying the growth on plates into six levels (0-5 as shown in Table
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1) was used to represent growth of L. monocytogenes on petri-plates.
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2.7.
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The PL destruction kinetics of L. monocytogenes were analyzed based on a first-order
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Pulsed Light destruction kinetics.
reaction indicating a logarithmic order of death, and expressed as Eq. (1): =−
(1)
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where N is the number of organisms that survived the PL treatment for time t (min), No is the
188
initial number of microorganisms present before PL treatment and k is the reaction rate constant
189
(min-1).
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The treatment time, at a given voltage, resulting in 90% destruction of the existing microbial
191
population, is referred to as the decimal reduction time (D-value). This was obtained as the
192
negative reciprocal slope of the survivor curve (log N/No v/s t curve), and expressed as Eq. (2):
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=−
=
(2)
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where N1 and N2 represent survivor counts at time tl and t2, respectively. Since the survivor
195
curve can have a lag period, this must be considered while using the data for practical
196
applications.
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The voltage dependence of the kinetic parameters D-value was analyzed by voltage Z-value
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(Zv), which represents the voltage range that results in a 10 foldchange in D-value. Zv was
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determined by the negative reciprocal of the slope of the log (D-values) v/s treatment voltage
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curve, as expressed by Eq. (3):
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(2)
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where D1 and D2 represent decimal reduction times at voltages V1 and V2, respectively.
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2.8.
205
Duplicate observations of same sample for each treatment were carried out and each
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observation was enumerated in duplicated. All the results, where feasible, were presented as
207
mean ± standard deviation of 4 observations. Each set of observations were compared using T-
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test by comparing means and differences were considered significant for p<0.05. Where
209
significant differences were present, individual treatments were compared using the least
210
significant difference (LSD) test. Regression analysis were conducted using Microsoft Excel
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2016 (Microsoft Corporation, USA).
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3. Results and Discussions
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Statistical Analysis
3.1.
Relative resistance of different strains of L. monocytogenes to PL treatment
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The qualitative growth of 5 strains and a mixed culture of L. monocytogenes following PL
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treatment are shown in Table 2. It was important to investigate the possible differences in
216
resistance of 5 strains of L. monocytogenes, all from marine sources, to PL treatment. From these
217
results, a representative high resistance strain was to be chosen for further studies. T-test & LSD
218
results suggested the following order for the resistance of each strain: Scott A ~ Mixture > 976,
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392, 439 > 323.
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It can be seen in Table 2 that strain Scott A and the mixed culture were most resistant to PL
221
treatments showing up to 5-log reductions in a 20s treatment at 800 V voltage treatment at 5 cm
222
distance from the PL source. On the other hand, there was very little microbial inactivation for
223
these strains at 600 V and colonies were too high to be counted as shown, suggesting a strong
224
dependence of PL inactivation on the treatment voltage. It can be visualized from Fig. 3, which
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is a sample representation from Table 3, that in contrast to Scott A, Strain 323 from the shrimp
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isolate was least resistant, and thus most susceptible, to the PL treatment. The PL inactivation of
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strains 976, 392 and 439 were between than of 323 and Scott A without any statistical
228
differences (p>0.05) between each other. The mixed culture was found less or equally resistant
229
as Scott A (p~0.05). This might entail that the growth of the survived Scott A microbes in the
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cocktail was more dominant than other strains inside that cocktail. Being the most resistant
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strain, Scott A was used in all experiments to follow.
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3.2.
Lethality of PL light on L. monocytogenes
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In order to establish the inactivation of L. monocytogenes at each distance from the PL
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source, the average kill of L. monocytgenes was calculated for plates for a 60s at the respective
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treatment voltage and distances from the PL source (Table 3). Fig. 4 represents a model of the
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236
effect of voltage (600 and 800 V) and distance (5, 10 and 15 cm) from the PL source on the
237
percent kill of L. monocytogenes by PL treatment. The regression equation which fitted to the
238
data is shown as Eq. (4): % #$ = 132.33 − 0.018 ∗ , − 14.42 ∗ + 0.014 ∗ , ∗ / 0 = 94.85
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(4)
where V is the voltage (V) and D is the distance (cm) from the lamp source.
241
From these results, optimum maximum and minimum values were determined for percent
242
kill of L. monocytogenes. A maximum value (approx. 100% kill) would be achieved at 800 V
243
and 5 cm distance from the PL source. A minimum value (31.3%) would be achieved at 600 V
244
and 15 cm distance from the PL source. These finding are in accordance with the fact that the
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lethality of PL treatment increases with increasing light intensity or fluence (FDA, 2000).
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The findings suggest that as voltage increased from 600 to 800 V, microbial inactivation of
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L. monocytogenes increased. Also, as the distance from the PL source is increased (from 5 to 15
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cm), the percent kill of L. monocytogenes reduces due to reduction in PL intensity. These
249
findings have also shown that in general, as the number of pulses is increased (i.e. increasing the
250
treatment time), microbial destruction is increased.
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These results were for Scott A only and agree well with the findings of other researchers,
252
such as Ozer and Demirci (2006), working with L. monocytogenes. Ozer and Demirci (2006)
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found only 1.02 log reduction at 8 cm distance with a 60s treatment on raw salmon skin fillets
254
for L. monocytogenes Scott A. However, they also agree well with that obtained for other strains
255
of L. monocytogenes, as shown in Table 2. Other researchers (Krishnamurthy et al., 2007) have
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found a similar ~ 7-log reduction of S. aureus in milk when treatment time was increased from
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30 to 180 s. PL destruction in any type of micro-organisms follows a similar trend as observed in
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this study. From the results of this study, a PL treatment at 800 V treatment voltage at 5 cm
259
distance from the PL source for 60 s @ 1 pps was recommended, whenever possible, to
260
maximize microbial destruction.
261 262
3.3.
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Destruction kinetics of L. monocytogenes on surface of solid general purpose media
The survival curves for L. monocytogenes on the surface of the solidified agar plates at 600,
264
700 and 800 V are shown in Fig. 5a, which indicates that the destruction was significantly
265
influenced (P < 0.05, by comparison of means) by the voltage used and the treatment time. The
266
survival curves at higher voltages (800 V) were steeper than at lower voltages (600 V) which
267
illustrate that the destruction rate of L. monocytogenes was higher at higher voltages. Gomez-
268
Lopez (2005) reported that efficiency of reduction of counts of L. monocytogenes on agar
269
surface reduced with decrease in PL intensity, and our results agreed well with their study.
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The decimal reduction times at 800, 700 and 600 V were 0.91±0.23, 1.37±0.31 and
271
2.25±0.38 s respectively. It must be noted that D-values were computed by ignoring the initial
272
lag period (4-7 s), only after which a first order rate of destruction was observed. The lag times
273
and associated D values are summarized in Table 4. For example, at 800V, the associated D
274
value is 0.91±0.23 s and the lag is 4.0 ± 0.1 s. So for achieving one decimal reduction in counts,
275
the treatment time would be the D value of 0.91 s plus the las of 4 s for a total of 4.91 s.
276
However, if 5 decimal reduction are required, the exposure time would be (0.91 x 5) + 4.0 = 8.55
277
s. Thus, it is imperative that the associated lag periods must be used along with the respective D
278
values to have a meaningful prediction of the PL destruction power.
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Lower D-values are associated with higher voltages demonstrating a higher destruction rate.
280
Fig. 5b demonstrates the curve of log (D-value) vs. treatment voltage to compute the voltage
281
sensitivity parameter (Zv value). The Zv value of L. monocytogenes was calculated to be 500±24
282
V, meaning that for every 500 V increase in treatment voltage, the D-value would be reduced by
283
1 log cycle. A reference D-value and z fully describes the pulsed destruction kinetics over the
284
range of voltages studies. The z-value of 500 V was relatively high and suggests a rather weak
285
dependence on treatment, when the voltage range of only 600-800 V is considered. This is
286
advantageous in a way, because even at low power consumption, potentially satisfactory
287
inactivation of L. monocytogenes can be obtained by slightly reducing the distance from the PL
288
source.
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The low D-values & high Z-value in this experiment show that PL has an incredible potential
290
as a surface sanitation method. Surface inactivation of L. monocytogenes was monitored in a
291
similar study that reported a 7-log reduction in L. monocytogenes, on the surface of a general
292
purpose media, following PL treatment for 512 s at 30 kV and 1 pps (MacGregor et al., 1998).
293
The longer treatment time required for the reduction in counts in this study was due to the lower
294
stored energy in the pulse generator of 3 J/pulse, versus 12.8 J/pulse utilized in this study. On the
295
other hand, Hierro et al. (2011) prescribed a dosage between 3-5s for effective reduction of L.
296
monocytogenes on bologna and cooked ham surface, which is in accordance with the
297
observations of high effectiveness of PL treatment for surface decontamination of L.
298
monocytogenes.
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3.4.
L. monocytogenes in liquid media in liquid media
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The survival curve for L. monocytogenes in liquid media (shown in Fig. 6) depicts a small
301
reduction in counts, even after 120 s of treatment at 800 V. The figure demonstrates a good fit of
302
data for the first order model (R2 = 0.97) which suggests that L. monocytogenes in Whirl Pak
303
bags did follow logarithmic destruction under PL treatment. Due to relatively low inactivation,
304
the lag-phase was not apparent for this treatment. A decimal reduction time of 93 ± 5 s resulted
305
from the PL treatment, which is quite large and would render PL treatment undesirable &
306
uneconomical.
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Thus, it is seen that PL treatment was not effective for volumetric treatment in Whirl Pak
308
bags. It must be noted that due to the limited penetration of PL treatment, the samples, which had
309
a thickness of approx. 7 mm, were too thick to achieve a reasonable rate of destruction.
310
Secondly, the penetration power of the light gets attenuated exponentially. Difficulty with PL
311
treatment in thick and opaque samples has been well documented by Dunn et al. (1995, 1997a,
312
1997b), Krishnamurthy et al. (2004, 2007), Sauer and Moraru (2009); and more recently by
313
Pataro et al. (2011). Krishnamurthy et al. (2007) showed that the ability of PL to inactivate S.
314
aureus in milk decreased as the sample volume increased from 12-48 mL due to poor penetration
315
capacity of UV light. Although there could have been substantial reduction in counts at the
316
surface, the survivor in the deeper layers would dominate the total counts when mixed. For
317
example, even if 80% of the cells in the light's path get destroyed by several log cycles, leaving
318
the other 20% in the central region unaffected, the overall count will still be in the same
319
logarithmic scale as in the beginning. This means the overall reduction would be less than one
320
log cycle. Obviously, it would be desirable to use the liquid in much thinner profiles so that the
321
destruction occurs throughout.
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4. Conclusions This study evaluated the relative resistance of 5 strains of L. monocytogenes and found Scott
324
A strain to be the most resistant. Upto 5-log reduction of Scott A strain of L. monocytogenes was
325
obtained within 20s for treatment at 800 V. PL voltage, treatment time and distance from the PL
326
source played a significant role in the destruction of L. monocytogenes. Maximum destruction
327
was achieved at high voltage and minimum distance from the PL source. Destruction kinetics for
328
L. monocytogenes were evaluated in a liquid media, on the surface of a general-purpose media
329
following various PL treatments. High efficiency of PL application was obtained for PL
330
treatment for on surface of agar plates with a D-value of 0.91±0.23 s at 800V, and a Zv of
331
500±24 V. The D-values were sufficiently low to use a few flashes for surface decontamination
332
of microorganisms in a few seconds. On the other hand, the high Zv suggested relatively lower
333
dependence of PL lethality on the PL voltage, as compared to the treatment time. In liquid-media
334
inside whirl pak bags, D-values (93±5 s) were very high, suggesting relatively poor PL
335
penetration into liquid media. The low microbial lethality of PL treatment for liquid samples
336
warrants more research into thin-profile PL treatment of liquids, which can potentially counter
337
this effect of PL. These results shall be useful in establishing PL treatment for L. monocytogenes
338
and other microbes.
339
5. Acknowledgments
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This work was supported by funds ministère de l'Agriculture, des Pêcheries et de l'Alimentation du Québec (MAPAQ).
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6. References
343
Abida, J., Rayees, B., & Masoodi, F. A. (2014). Pulsed light technology: a novel method for food
345 346
preservation. International Food Research Journal, 21(3), 839-848.
RI PT
344
Barbosa-Canovas, G. V., Schaffner, D. W., Pierson, M. D., & Zhang, Q. H. (2000). Pulsed light technology. Journal of Food Science, 65(s8), 82–85.
Bohrerova, Z., Shemer, H., Lantis, R., Impellitteri, C. A., & Linden, K. G. (2008). Comparative
348
disinfection efficiency of pulsed and continuous-wave UV irradiation technologies.
349
Water Research, 42(12), 2975-2982.
M AN U
SC
347
350
Buchovec, I., Paskeviciute, E., & Luksiene, Z. (2010). Photosensitization-based inactivation of
351
food pathogen Listeria monocytogenes in vitro and on the surface of packaging material.
352
Journal of Photochemistry and Photobiology B: Biology, 99(1), 9-14. Cheigh, C. I., Hwang, H. J., & Chung, M. S. (2013). Intense pulsed light (IPL) and UV-C
354
treatments for inactivating Listeria monocytogenes on solid medium and seafood’s.
355
Food Research International, 54(1), 745-752.
TE D
353
Hierro, E., Barroso, E., De la Hoz, L., Ordóñez, J. A., Manzano, S., & Fernández, M. (2011).
357
Efficacy of pulsed light for shelf-life extension and inactivation of Listeria
358
monocytogenes on ready-to-eat cooked meat products. Innovative Food Science and
360 361 362 363
AC C
359
EP
356
Emerging Technologies, 12(3), 275-281.
Cerny, G. (1977). Sterilization of packaging materials for aseptic packaging. Verpack- ungsRdsch, Tech-nisch-wiss Beilage, 28(10), 77.
Dunn, J., Ott, T., & Clark, W. (1995). Pulsed-light treatment of food and packaging. Food Technology, 49(9), 95-98.
364
17
ACCEPTED MANUSCRIPT
365 366
Dunn, I. (1996). Pulsed light and pulsed electric field for foods and eggs. Poultry Science, 75, 1133-1136. Dunn, I, Burgess, D., & Leo, F. (1997a). Investigation of pulsed light for terminal sterilization of
368
WFI filled blow/fill/seal polyethylene containers. PDA Journal of Pharmaceutical
369
Science and Technology, 51(3), 111-115.
371
Dunn, J., Bushnell, A., Ott, T., & Clark, W. (1997b). Pulsed white light food processing. Cereal Foods World, 42(7), 510-515.
SC
370
RI PT
367
Elmnasser, N., Guillou, S., Leroi, F., Orange, N., Bakhrouf, A., & Federighi, M. (2007). Pulsed-
373
light system as a novel food decontamination technology: a review. Canadian Journal
374
of Microbiology, 53(7), 813-821.
375
M AN U
372
FAO (1999). FAO expert consultation on the trade impact of Listeria monocytogenes in fish products.
377
http://www.fao.org/docrep/003/x3018e/x3018e00.htm (accessed 04.11.16).
378
Food
and
Agriculture
Organization,
TE D
376
Amherst,
MA,
USA.
FDA (2000). Kinetics of Microbial Inactivation for Alternative Food Processing Technologies. Food
380
http://www.fda.gov/Food/FoodScienceResearch/SafePracticesforFoodProcesses/ucm10
381
0158.htm (accessed. 04.11.16)
383 384 385
Drug
Administration,
Silver
Spring,
MD,
FDA (2001). Processing parameters needed to control pathogens in cold smoked fish. Food and
AC C
382
and
EP
379
Drug
Administration,
Silver
Spring,
MD,
http://www.fda.gov/Food/FoodScienceResearch/SafePracticesforFoodProcesses/ucm09 2182.htm (accessed. 04.11.16)
18
ACCEPTED MANUSCRIPT
386
Heinrich, V., Zunabovic, M., Varzakas, T., Bergmair, J., & Kneifel, W. (2016). Pulsed Light
387
Treatment of Different Food Types with a Special Focus on Meat: A Critical Review.
388
Critical Reviews in Food Science and Nutrition, 56(4), 591-613.
390
Krishnamurthy, K., Demirici, A., & lrudayaraj, J. (2004). Inactivation of Staphylococcus aureus
RI PT
389
by pulsed UV -light sterilization. Journal of Food Protection, 67, 1027-1030.
Krishnamurthy, K., Demirici, A., & lrudayaraj, J. (2007). Inactivation of Staphylococcus aureus
392
in milk using flow-through pulsed UV-light treatment system. Journal of Food
393
Protection, 72 (7), M233-M239.
SC
391
Goff, L. L., Hubert, B., Favennec, L., Villena, I., Ballet, J. J., Agoulon, A., & Gargala, G. (2015).
395
Pilot-Scale Pulsed UV Light Irradiation of Experimentally Infected Raspberries
396
Suppresses Cryptosporidium parvum Infectivity in Immunocompetent Suckling Mice.
397
Journal of Food Protection, 78(12), 2247-2252.
M AN U
394
Lovett, J., & Twetd, R. M. (1988). Listeria. Journal of Food Technology, 42(4), 188.
399
MacGregor, S. I., Rowan, N. I., Macilvaney, L., Anderson, J. G., Fouracre, R. A., Farish, O.
400
(1998). Light inactivation of food-related pathogenic becteria using a pulsed power
401
source. Letters in Applied Microbiology, 27, 67-70.
EP
TE D
398
Ozer, N. P., & Demirci., A. (2006). Inactivation of Escherichia coli O157:H7 and Listeria
403
monocytogenes inoculated on raw salmon fillets by pulsed-UV light treatment.
404
AC C
402
International Journal of Food Science and Technology, 41 (4): 354-360.
405
Pataro, G., Muñoz, A., Palgan, I., Noci, F., Ferrari, G., & Lyng, J. G. (2011). Bacterial
406
inactivation in fruit juices using a continuous flow Pulsed Light (PL) system. Food
407
Research International, 44(6), 1642-1648.
19
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Pratap Singh, A., Singh, A., & Ramaswamy, H. S. (2017). Heat transfer phenomena during
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thermal processing of liquid particulate mixtures – A Review. Critical Reviews in Food
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Science and Nutrition, 57(7):1350-1364. Rowan, N., MacGregor, S. J., Anderson, I. G., Fouracre, R. A., McIlvaney, L., & Farish, O.
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(1998). Pulsed-light inactivation of food-related microorganisms. Applied Environmetal
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Microbiology, 65(3), 1312-1315.
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Saikiran, K. C. S., Mn, L., & Venkatachalapathy, N. (2016). Different pulsed light systems and
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their application in foods: a review. International Journal of Science, Environment, and
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Technology, 5(3), 1463-1476.
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Sauer, A., & Moraru C. I. (2009). Inactivation of E. coli ATCC 25922 and E. coli O157:H7 in
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apple juice and apple cider using Pulsed Light treatment. Journal of Food Protection,
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72(5), 937-44
Uesugi, A., Woodling, S. E., & Moraru, C. I. (2007). Inactivation kinetics and factors of
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variability in the Pulsed Light treatment of Listeria innocua cells. Journal of Food
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Protection, 70(11), 2518-2525
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Woodling, S. E., & Moraru, C. I. (2007). Effect of spectral range in surface inactivation of
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Listeria innocua using broad spectrum Pulsed Light. Journal of Food Protection, 70(4),
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906-916
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XENON Corporation. (2015). Applications of pulsed light for sterilization. XENON Corporation,
Wilmington,
MA,
USA.
http://www.xenoncorp.com/files/5914/5451/5085/Pulsed_Light_Sterilization_Applicati ons.pdf (accessed 04.11.16).
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Table 1: Qualitative scale to represent growth of L. monocytogenes.
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Growth
Description of Growth
Approximate
number
of
5
Lawn of growth
4
Extensive
growth
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colonies TNTCa (distinct >300
3
Heavy growth (countable)
2
Heavy growth (countable)
1
Light growth
~10
0
No growth
0
>300 ~30
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TNTC = too high to count
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colonies)
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Table 2: Growth (on a scale of 1 to 5 – refer Table 1) of 5 strains and a mixed culture of L.
436
monocytogenes isolated from marine sources and pulsed treated at various combinations of
437
treatment voltage, treatment time and distance from the PL source. Distance Treatment Treatment Growth
source
Time (s)
(V)
Mixture
Scott A
976
(cm)
40
1
60
1
20
0.5
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of all 5 strains
0.5
0.5
0.5
0
1.5
0
0.5
0.5
0.5
0.5
0
0
0.5
0.5
0.5
1
0.5
0
0.5
40
0.5
0
0
0
0
0
60
0
0
0
0
0
0
2
1
1
1
1
2
1
0
0
0.5
0
1
60
0
0
0
0.5
0.5
1
20
0.5
0.5
0
0.5
0
0.5
40
0
0
0.5
0
0
0
60
0
0
0
0
0
0
20
5
4
3
4
4
5
40
1
1
0
1
1
1
60
1
1
0.5
0.5
1
1
800
600
20
10 800
600
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15
0.5
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20
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from PL Voltage
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800
20
1
1
1
0.5
1
1
40
1
1
0
0.5
0
0.5
60
0
0.5
0
0.5
0
0
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Table 3: Percent kill of L. monocytogenes at various treatment voltages and distances from
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the PL source.
800
A (%)a
5
93±5
10
58±3
15
33±8
5
100±0
10
89±5
15 a
68±7
Values reported as mean±sd
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600
(cm)
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Treatment Voltage Distance from PL source Average Kill of L. monocytogenes Scott
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Table 4: Calculation of decimal reduction times (D-values) of L. monocytogenes on the
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surface of solidified agar plates. D-value following lag
Lag time
(V)
period (s)a
(s)a
800
0.91±0.23
4±1
700
1.37±0.31
5±1
600
2.25±0.38
7±2
reduction (s)1 4.91
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PL exposure time for n decirnal reductions = (D x n) + lag time
a
Values reported as mean±sd
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9.25
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Total time for one 4-value
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Fig. 1. Schematic diagram of the PL apparatus.
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Fig. 2. Position of Petri plates on PL chamber treatment tray during spatial power
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distribution analysis.
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Inactivation of individual and cocktail of 5 strains Scott A
976
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439
392
Mixture of 5 strains
5 4
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Growth Scale from Table 1
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3 2 1 0 600V - 40s
600V - 60s
800V - 20s
800V - 40s
800V - 60s
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600V - 20s
Treatment Intensity
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Fig. 3. Sample representation of growth of 5 strains and a mixed culture of L.
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monocytogenes isolated from marine sources and PL treated at 600 V and 15 cm distance
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from the PL source.
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120
80
5 cm 7 cm
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Percent Kil (%)l
100
60
9 cm
11 cm
40
13 cm 15 cm
0 600
620
640
660
680
700
720
Treatment Voltage (V)
740
760
780
800
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Fig. 4. Effect of voltage and distance from the PL source on the percent kill of L.
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monocytogenes by PL treatment
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a. PL Survivor Curve 10 8
y = -0.7267x + 11.48
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Survivors (log CFU/ml)
9
6
800 V
5 4
y = -0.4443x + 11.47
y = -1.1x + 10.967
3
600 V
2 1 5
10
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0 0
700 V
15
20
Time (s)
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b. Voltage Sensitivity Curve 0.4 0.35 0.3
y = -0.002x + 1.5272 zv = 1/0.002 = 500 V
0.2 0.15 0.1 0.05 0
-0.1
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400
600
800
1000
Voltage (V)
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Fig. 5: a) Survivor and b) voltage sensitivity curves for L. monocytogenes on the surface of
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general purpose media
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Survivor Curve in whirl-pak bags 9
7 6
y = -0.0107x + 8.2893 R2 = 0.97 D value = 1/0.0107 = 93s
5
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Survivors (log CFU/ml))
8
4 3 2
0 0
20
60
80
TIme (s)
100
120
140
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Fig. 6: Survivor curve for L. monocytogenes in a 2-oz whirl pak bag.
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