Tendances Récentes Emergentes et Nouvelles Drogues Tendances récentes émergentes et nouvelles drogues Marseille 2009 – juin 2010 Présentation du dispositif TREND Marseille 2010 .7 Les espaces étudiés. 7 1.1 Espace urbain et espace festif. 7 1.2 L'expérimentation d'un élargissement du champ d'observation. 7 Les outils de recueil des données. 8
Doi:10.1016/s0168-1605(03)00158-2International Journal of Food Microbiology 90 (2004) 1 – 8 Inhibition of pathogens on fresh produce by ultraviolet energy Brian R. Yaun*, Susan S. Sumner, Joseph D. Eifert, Joseph E. Marcy Department of Food Science and Technology, Virginia Tech., Blacksburg, VA 24061, USA Received 10 September 2002; received in revised form 20 February 2003; accepted 3 March 2003 Ultraviolet energy at a wavelength of 253.7 nm (UVC) was investigated for its bactericidal effects on the surface of Red Delicious apples, leaf lettuce and tomatoes inoculated with cultures of Salmonella spp. or Escherichia coli O157:H7. Inoculatedsamples were subjected to different doses ranging from 1.5 to 24 mW/cm2 of UVC and enumerated on tryptic soy agar plus 0.05g/l nalidixic acid to determine effective log reductions of microbial populations. UVC applied to apples inoculated with E. coliO157:H7 resulted in the highest log reduction of approximately 3.3 logs at 24 mW/cm2. Lower log reductions were seen ontomatoes inoculated with Salmonella spp. (2.19 logs) and green leaf lettuce inoculated with both Salmonella spp. and E. coliO157:H7 (2.65 and 2.79, respectively). No significant statistical difference ( p>0.05) was seen in the ability of UVC toinactivate a higher population of either Salmonella spp. or E. coli O157:H7 on the surface of green leaf lettuce. No significantdifference was seen among the use of different doses applied to the surface of fresh produce for reduction of E. coli O157:H7 orSalmonella spp. ( p>0.05). The use of UVC may prove to be beneficial in protecting the safety of fruits and vegetables inconjunction with Good Agricultural Practices and Good Manufacturing Practices.
D 2003 Elsevier B.V. All rights reserved.
Keywords: UV; Salmonella; Escherichia coli O157:H7; Apples; Lettuce; Tomato Shigella spp. and Escherichia coli O157:H7 1995). In September 1997, an EPA Scientific Advi- Documented cases of foodborne illness associated sory Panel specifically identified Salmonella, L.
with fresh fruits and vegetables have risen in the last monocytogenes and E. coli O157:H7 as pathogens of public health concern on produce. The panel also 1999, 2000). Major outbreaks involving fresh produce recommended testing five outbreak-related strains in a have been associated with common foodborne patho- cocktail for each pathogen gens such as Salmonella, Listeria monocytogenes, Agency, 1997).
One strategy to minimize the risks involved with the consumption of fresh fruits and vegetablesinvolves either reducing or eliminating external sur-face contamination. Previous attempts used to reduce * Corresponding author. Tel.: +1-540-231-8697; fax: +1-540- surface microbial numbers and prolonging shelf life E-mail address: [email protected] (B.R. Yaun).
of fresh produce include modified atmosphere pack- 0168-1605/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0168-1605(03)00158-2 B.R. Yaun et al. / International Journal of Food Microbiology 90 (2004) 1–8 2. Materials and methods 1992; Bennik et al., 1995), partial processing usingchemical sanitizers, 2.1. Preparation of inoculum 1998; Wei et al., 1995; Beuchat et al., 1998, 2001;Koseki et al., 2001), low-temperature storage A total of five strains each of Salmonella and E. coli et al., and the use of O157:H7 were used in this study. Three strains of E.
edible films Effective surface coli O157:H7 and five strains of Salmonella that were decontamination techniques could be employed to isolated from outbreaks associated with raw vegetables reduce the surface load of pathogens. Simply wash- or unpasteurized fruit juices were used. E. coli ing fresh produce with water may not remove O157:H7 (H1730) was isolated from a lettuce-associ- pathogens and other spoilage organisms ated outbreak, E. coli O157:H7 (F4546) from an alfalfa et al., 1998). Traditional detergents are known to sprout-associated outbreak, E. coli O157:H7 (cider) be partially effective in removing pathogens, how- from a cider-related outbreak, E. coli O157:H7 ever, each type of disinfectant varies both in effi- (E0019) from a beef outbreak and E. coli O157:H7 ciency and in allowable maximum concentration (994) from a salami outbreak. Salmonella Montevideo The use of a nonselective was isolated from a tomato-associated outbreak, Sal- treatment for the destruction of pathogens on the monella Agona from an alfalfa sprout-related out- surface of fresh fruits and vegetables would be break, Salmonella Baildon from a lettuce- and toma- desirable. One such alternative process is the use to-associated outbreak, Salmonella Michigan from a of germicidal ultraviolet light at a wavelength of cantaloupe-associated outbreak and Salmonella Gami- 200 – 280 nm (UVC). Treatment with ultraviolet nara from an orange juice-associated outbreak. All energy offers several advantages to food processors serotypes were obtained from the University of Geor- as it does not leave a residue, does not have legal gia from Dr. Larry Beuchat at the Center for Food restrictions and does not require extensive safety Safety and Quality Enhancement (Griffin, GA). All equipment to utilize strains are resistant to 0.05 g/l nalidixic acid.
et al., 1998).
Cultures were maintained at 80 jC in tryptic soy Information regarding the use of UV radiation for broth (TSB) (Becton Dickinson, Sparks, MD) supple- the destruction of pathogens on produce has not been mented with 0.05 g/l of nalidixic acid (ICN Biomed- well documented. Studies by ana- icals, Aurora, OH) (TSBN). Prior to use, cultures were lyzed the effect of UV on inoculated tomatoes for the grown in TSB at 35 jC and were transferred three inhibition of black and gray mold formation. Dose times at 24-h intervals prior to their use in the levels of 1.3 – 40 kJ/m2 were applied to the surface of inoculation. Incubation for 24 h allowed the respec- the fruit. Results from this study supported the pre- tive bacteria to approach the stationary phase of vious work of and found that ripening growth at a concentration of approximately 108 cfu/ was delayed which in turn extended shelf life. Studies ml. Equal aliquots of each individual strain were by found that the UV exposure did vortexed and then aseptically combined into a sterile not affect fruit weight loss in pears.
dilution blank to produce a cocktail of five strains.
Recent Hazard Analysis and Critical Control Points (HACCP) regulations require a 5-log reduction 2.2. Preparation of produce samples of the pertinent pathogen in juice products. The use ofUVC may prove to be useful as a treatment step in Unwaxed Red Delicious apples were obtained HACCP protocols if it is effective at reducing micro- from Virginia Tech's Kentland Research Farm in bial numbers on the surface of fresh fruits and Blacksburg, VA. Tomatoes were obtained from a local vegetables. The overall objective of this study was distributor. Leaf lettuce was obtained from a local to define the UVC dose required to effectively reduce grocery store in Blacksburg, VA. Red Delicious the numbers of antibiotic-resistant strains of Salmo- apples and tomatoes were of uniform size, shape nella and E. coli O157:H7 on the surface of apples, and free of visual defects such as cuts, abrasions lettuce and tomatoes.
and bruises. Apples and tomatoes were stored at 4 B.R. Yaun et al. / International Journal of Food Microbiology 90 (2004) 1–8 jC until use. Approximately 1.5 1.5U outer leaves of ation time in seconds. As intensity was kept constant, green leaf lettuce were excised from a single head of variable exposure times were then employed to allow lettuce and transferred to a sterile petri dish prior to for different doses ranging from 1.5 to 24 mW/cm2 to inoculation. Produce was allowed to equilibrate to be applied to the surface of the produce. Light intensity room temperature (22 jC) for 18 – 24 h prior to was evaluated several times during the experiments to ensure consistent output.
2.3. Produce inoculation Produce was placed on a petri dish in a laminar UVC-treated produce was aseptically transferred to flow biosafety hood, and 100 Al of inoculum at a sterile sampling bag and rinsed with 20 ml of 0.1% approximately 107 cfu/ml was applied in multiple sodium lauryl sulfate (Sigma, St. Louis, MO). Serial spots around the calyx of the apple and blossom stem dilutions in 0.1% peptone (Becton Dickinson) were scar of the tomato taking care not to inoculate either pour-plated with tryptic soy agar (TSA) (Becton Dick- area. The surface of outer leaves of green leaf lettuce inson) supplemented with 0.05 g/l of nalidixic acid was similarly surface-inoculated, taking care to avoid (ICN Biomedicals) (TSAN) or xylose lysine deoxy- the torn edge of the leaf. Produce was allowed to dry cholate agar (Becton Dickinson) supplemented with under the laminar flow hood for a minimum of 30 min 0.05 g/l nalidixic acid (XLDN). Plates were incubated prior to UVC treatment.
at 35 jC for 24 h. TSAN was chosen in order to aid inthe recovery of injured cells and was used for all 2.4. Ultraviolet chamber products except tomatoes. Sufficient backgroundmicroflora on tomatoes necessitated the use of a The chamber utilized for the UVC irradiation of selective and differential media. As a result, XLDN plates was fabricated in the Virginia Tech Department was used for enumeration of Salmonella from the of Food Science and Technology. The chamber is surface of tomatoes. Confirmation was performed for approximately 40U long and contains a single G36T6 Salmonella on XLD agar and on API 20E test strips Model 4136 germicidal light unit that emits 253.7-nm (Biomerieux, Hazelwood, MO). E. coli O157:H7 was UV light (Fuller Ultraviolet, Frankfort, IL). The light confirmed on Sorbitol MacConkey Agar (Becton source is suspended on a chain and may be moved to Dickinson) and with the use of a Visual Immunopre- either increase or decrease intensity as desired by the cipitate Assay (Biocontrol, Bellevue, WA).
operator. The interior is lined with a reflective foil(Solar Bright, Fuller Ultraviolet) designed to increase 2.7. Statistical design the UVC intensity and to minimize any shadowingeffect on irregularly shaped samples. Access is Trials were replicated at least five times with two through a hinged bifold door. The UVC dose was samples for each ultraviolet dose plus two positive and measured using a dosimeter calibrated to read specif- one uninoculated control, all of which were analyzed ically at 253.7 nm (Spectronics, Westbury, NY). The in duplicate at each sampling interval. Survival data meter was calibrated and standardized by the manu- were treated according to Chick's Law as log (Ns/No) facturer before the study.
where Ns is the density of survivors and No is the initialconcentration of bacteria, which was calculated from 2.5. Ultraviolet treatment the average recovery on the positive control samples.
Data presented are the average log reduction of greater Samples were randomized and individually sub- than 10 trials with the standard error of the mean.
jected to different doses of UVC light. UVC intensity Means and standard error were calculated from a was determined prior to treatment by measuring the commercial spreadsheet (Microsoft Excel, Redmond, output of the light (mW/s/cm2), and the applied dosage WA). Data were subjected to Tukey's Honestly Sig- was calculated from D = L(T) where D = applied dos- nificant Difference in SAS, Version 8 (SAS Institute, age, L = applied intensity in mW/s/cm2 and T = irradi- Cary, NC) to determine if there were significant differ- B.R. Yaun et al. / International Journal of Food Microbiology 90 (2004) 1–8 ences ( p < 0.05) between mean log reductions for each cut edges of lettuce, whereas Salmonella typhimurium attached equally to either the cut edge or the intactsurface. From the data presented, there is no statisticaldifference exhibited in the log reduction between 3. Results and discussion Salmonella or E. coli O157:H7 ( p>0.05) on the sur-face of green leaf lettuce. Although this study did not In order to account for background microflora, the investigate preferential attachment, results suggest pathogenic strains utilized in this study were resistant that the equivalent doses of UVC will inactivate to nalidixic acid. A study by similar numbers of both Salmonella and E. coli demonstrated that antibiotic-resistant and antibiotic- O157:H7 cocktails. Further, there is no statistical susceptible strains of Listeria, Salmonella and E. coli difference among the doses applied for significant grew identically under laboratory conditions when reduction of Salmonella and E. coli O157:H7 on the variables such as pH, water activity and temperature surface of leaf lettuce ( p>0.05). The use of UVC was were altered. All treatments resulted in at least a 99% seen to be more effective at reducing microbial reduction of the selected pathogens on the surface of populations of E. coli O157:H7 than the use of 20 Red Delicious apples, green leaf lettuce and tomatoes.
ppm chlorine 200 ppm chlorine Lettuce was inoculated with an average of 5.51 and acidic electrolyzed water log10 cfu/lettuce for E. coli O157:H7 and 5.39 log10 cfu/lettuce for Salmonella spp. As seen in Tomatoes were inoculated with an average of 3.32 2, both Salmonella and E. coli O157:H7 show similar log10 cfu/tomato for Salmonella spp. UVC was less logarithmic reductions when treated with the same effective at reducing populations of Salmonella on the doses of ultraviolet light. Both organisms required a surface of tomatoes when compared to the other dose of approximately 6 mW/cm2 to achieve a 2-log produce types. depicts maximum log reductions reduction in initial numbers on the surface of leaf of 2.19 log10 cfu/tomato at doses of 24 mW/cm2. No lettuce. Maximum log reductions on green leaf lettuce significant statistical difference was seen among doses for Salmonella and E. coli O157:H7 seen at a dose of applied for reduction of Salmonella on the surface of 24 mW/cm2 were 2.65 and 2.79 logs, respectively.
tomatoes ( p>0.05). Preliminary studies indicated depicts the log reductions of both organisms on background microflora on tomatoes at approximately the surface of green leaf lettuce. 103 cfu/tomato. Initial experiments indicated that used confocal scanning laser microscopy to determine TSAN would be insufficient to exclude the normal that E. coli O157:H7 has a preferential attachment to background flora on tomatoes. Additional studies Fig. 1. Mean log reductions and standard error of Salmonella spp. on the surface of leaf lettuce by ultraviolet light at 253.7 nm (UVC).
R2 = 0.74.
B.R. Yaun et al. / International Journal of Food Microbiology 90 (2004) 1–8 Fig. 2. Mean log reductions and standard error of E. coli O157:H7 on the surface of leaf lettuce by ultraviolet light at 253.7 nm (UVC).
R2 = 0.79.
were performed on XLD before settling on XLDN to toes may be due to the use of a selective media which limit the effect of the high level of background would hinder recovery of injured cells, the presence of contaminates. All Salmonella cultures utilized in these a food grade wax applied by the processor on the experiments were positive for hydrogen sulfide pro- samples or to the competition by resident microflora.
duction which aided in colony identification. Atypical UVC was more effective at reducing populations of isolates, which were hydrogen sulfide-negative colo- Salmonella on tomatoes than 320 ppm chlorine nies, were identified as Pseudomonas aeroginosa. A and 2000 ppm chlorine possible explanation for the lower recovery on toma- et al., 1998). However, it was less effective than the Fig. 3. Mean log reductions and standard error of E. coli O157:H7 and Salmonella spp. on the surface of leaf lettuce by ultraviolet light at 253.7nm (UVC). E. coli O157:H7 R2 = 0.79; Salmonella spp. R2 = 0.74.
B.R. Yaun et al. / International Journal of Food Microbiology 90 (2004) 1–8 Fig. 4. Mean log reductions and standard error of Salmonella on the surface of tomatoes by ultraviolet light at 253.7 nm (UVC). R2 = 0.77.
use of a produce wash or coating 9 mW/cm2, whereas the same dose on lettuce only with an edible film of hydroxypropyl methylcellulose resulted in approximately a 2.2-log kill. A maximum log reduction was seen at 24 mW/cm2 with approx- Apples were inoculated with an average of 4.07 imately a 3.3-log reduction in cellular numbers. How- log10 cfu/apple of E. coli O157:H7. The effect of ever, no significant difference was seen in the UVC on E. coli O157:H7 cells inoculated onto the effective log kill over the range of doses applied to surface of unwaxed Red Delicious apples is depicted the surface of Red Delicious apples for the reduction in In contrast to the results from the lettuce of E. coli O157:H7. Alternative techniques utilized by samples, UVC was more effective at reducing micro- other researchers on apples have resulted in similar bial populations of E. coli O157:H7 on the surface of log reductions. Dipping in acetic acid resulted in a 3- apples. A 3-log reduction was seen at doses exceeding log reduction of E. coli O157:H7 Fig. 5. Mean log reductions and standard error of E. coli O157:H7 on the surface of Red Delicious apples by ultraviolet light at 253.7 nm(UVC). R2 = 0.82.
B.R. Yaun et al. / International Journal of Food Microbiology 90 (2004) 1–8 A solution of 6% hydrogen peroxide reduced numbers of Salmonella Chester by 3 – 4 logs from the surface ofapples and a 3-log reduction This research was supported by the USDA was achieved by exposure to 1.1 mg/l of free chlorine CSREES Special Research Grants Program, Food Safety (USDA/CSREES #99-34382-8463).
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