Journal of Applied Science and Agriculture. The Efficacy of Ozonated Water on Reduction of Four Food Borne Pathogens and Microbial Quality of Lettuce

Journal of Applied Science and Agriculture, 8(7) December 20, Pages: 42-40 AENSI Journals Journal of Applied Science and Agriculture Journal home page: www.aensiweb.com/jasa/index.html The Efficacy of Ozonated Water on Reduction of Four Food Borne Pathogens and Microbial Quality of Lettuce M. Bahreini, 2 E. Jahed, F. Lotfian, 4 A. Mousavi Khaneghah, M. Ghaderifarah Department of Food Science and Technology Ferdowsi University of Mashhad, Mashhad, Iran. 2 Department of Food Science and Technology Ferdowsi University of Mashhad, Mashhad, Iran. Department of Food Science and Technology, Varamin-Pishva Branch, Islamic Azad University, Varamin, Iran. 4 Department of Food Science and Technology, Islamic Azad University, Science and Research Branch, Tehran, Iran. Department of Food and Drug, Hamedan University of Medical Sciences, Hamedan, Iran. ARTICLE INFO Article history: Received 7 October 20 Received in revised form 2 November 20 Accepted 28November 20 Available online 2 February 204 Keywords: Ozone, fresh-cut vegetables, E. coli O7:H7, Salmonella, Disinfection lettuce ABSTRACT Background: Ozone is strong oxidant and potent disinfecting agent. There are numerous application areas of ozone in food industry. While there are many investigations on the application of ozone in food industry, relatively little information is available on the potential of ozone to reduce microbial populations in fresh-cut fruits and vegetables and the visual quality of lettuce. Objective: In this study, ozonated water was applied at five concentrations (0.6, 0.8,.2,.6 and 2 ppm) for four exposure times (,, and min) on natural microflora and E. coli O7:H7 and Salmonella inoculated of lettuce. The reduction in the total bacterial count, yeast and mold, total coliform, as well as Lactic acid bacteria counts were examined. Results: The promising results indicated the efficiency of ozonated water to reduce the microbial populations on lettuce. In the best condition, the inoculated samples of E. coli O7:H7 and Salmonella on lettuce were decreased further than 2 log cfu/g. The results showed the population of aerobic mesophilic bacteria, yeasts/ molds, total coliforms and lactic acid bacteria were decreased to.4, 0.94,.94 and. log cfu/g, respectively Conclusion: Reduce the microbial load of disinfectants in packing vegetables have been accepted and has been proven to improve quality and increase the half-life of fresh produce packaging should be used as a disinfection process. Wash vegetables with water, not only have great impact in reducing the pollution load. 20 AENSI Publisher All rights reserved. To Cite This Article: M. Bahreini, E. Jahed, F. Lotfian, A. Mousavi Khaneghah, M. Ghaderifarah., The Efficacy of Ozonated Water on Reduction of Four Food Borne Pathogens and Microbial Quality of Lettuce. J. Appl. Sci. & Agric., 8(7): 42-40, 20 INTRODUCTION In recent years, a lot of growth in fruit and vegetable packaging industry (annually around %) occurred. Several factors are involved in this rapid growth, one of which increased demand by consumers for their good properties in human health and comforts were produced and consumed (Rico et al, 2007). This has caused the number of infectious disease epidemic that is caused by eating contaminated products is high. Disease prevention and control organizations in America, has announced new products to consumers in the years 987-97 and 992-988 in each year has doubled and one product that is responsible for many epidemics are eating salads (De Roever, 998). For example, eating salad and lettuce on contaminated in 2006 in Argentina and in 998 an epidemic in America with Escherichia coli O7: H7 occurred (Ackers et al, 998; Hilborn et al, 999; Leotta et al, 2006; Wright et al, 2000). Escherichia coli as an indicator of fecal contamination in food is known. These bacteria by toxicogenic and enterohemorrhagic strains Fresh fruits and vegetables can be contaminated and cause. Epidemics (Ackers et al, 998; Leotta et al, 2006; Gorny, 2006). So today, with special attention to fresh vegetables packaging, products to be produced without risk to the public health community. Suitable conditions for growth and survival of pathogenic microbes cause increased pollution levels in these fresh vegetables packed products. Studies have shown that the E. coli bacteria (O7: H7) and Listeria monocytogenes can be connected to the surface of cut lettuce leaves and or to enter into the tissue and the leakage of nutrients from the cells lettuce, feed and grow and stay safe from environmental stresses (Hilborn et al, 999). Many factors leading to increased microbial contamination of these products (Beuchat et al, 997; Heaton et al, 2008; Narciso et al, 2006; Waller et al, 2002). Before harvest pollution sources are included soil, water, animals, insects and hands of workers. After harvest pollution sources are included workers, equipment, transportation equipment, systems of production and distribution. Most fruits and vegetables after harvest to remove soil, waste, residues and microbial pesticides are washed from their surface. Remove this waste product Corresponding Author: M. Bahreini, Department of Food Science and Technology Ferdowsi University of Mashhad, Mashhad, Iran. E-mail: mbahreini@um.ac.ir

426 M. Bahreini et al, 20 Journal of Applied Science and Agriculture, 8(7) December 20, Pages: 42-40 with immersion in water containing one or more disinfectant is performed (Sapers, 2006). This washing causes the microbial load of vegetables, especially the factors causing corruption is low and half-shelf life increased. One of the disinfectants that are used for this purpose is sodium hypochlorite. This material impact on reducing corruption and pathogenic bacteria in later stages of the harvest is well studied and known. Sodium hypochlorite disinfectants among the most consumed fresh fruit and vegetables is in the packaging industry but Its use for the production of harmful and toxic byproducts such as trihalomethanes and haloacetic acids which are mutagenic and carcinogenic is reduced (Artes et al, 2009; Betts et al, 200; Kim et al, 999). Recently, many studies on ozone as a disinfectant suitable for use in packaging fresh fruits and vegetables are done and has proved to be a deterrent effect on a wide range of microbes. (Kim et al, 999). Ozone disinfection of water and nutrients in the amounts used (Guzel-Seydim et al, 2004). One of the advantages of using ozone is the precursor to the amount of oxygen that exists in the environment and Another advantage is that no harmful compounds in the washing process does not produce and gaseous and liquid forms can be used depending on the type of product. In this study, of ozontated water was used to replace sodium hypochlorite and its effects on reduce natural microflora lettuce and E. coli O7: H7 and Salmonella inoculated on it and its effect on visual quality of lettuce was evaluated. Methodology: Sample Preparation: Fresh lettuce from the vegetable shop was bought by the city of Mashhad and was transported to the laboratory. Damaged and spoiled external leaves were separated and the mud stuck to them was removed. For better immersion in disinfectant solution, leaves were cut into smaller pieces. Ozonated Water Supply: Our ozone instrument ozone machine model (AS-200M) Iran Ozone water company that was supplying the device to a device for making oxygen, pure oxygen supply was connected with an output of 8 grams per hour. Ozone had been produced by ozone generator (AS-200 M) Iran Ozone Water Company that for feeding pure oxygen is connected to an oxygen produce machine its output was 8 grams per hour. Ozone levels based on the amount of oxygen input to the instrument set and the amount of ozone ppm with portable colorimetric method using kits No.4 DPD was measured. Antimicrobial effects of ozontaed water on lettuce: First, turn on the ozone generator and ozone saturated water was allowed to be And then inoculated and inoculated lettuce without giving ozone was poured in the tank And after periods of,, and minutes, giving up some of the lettuce from the Ozone and % sodium thiosulfate solution was to neutralize the residual ozone and the sanitary conditions were placed in the basket to dry. Before the effects of ozone on bacteria, its effect on lettuce was evaluated. Lettuce is due to the small thickness of the vegetables are tender, why was the effect of different concentrations of ozone on lettuce Add lettuce and after ozone was kept in the refrigerator for a week to determine the extent of damage caused on it. Based on sensory changes and the appearance of lettuce, a suitable concentration of ozone was chosen and then the appropriate concentration of the disinfecting effect of ozone was used Microbial tests were conducted on them. Procurement and preparation of bacteria: Standard Bacteria E. coli strain PTCC 99 and ATCC 489, E. coli O7: H7 strain NCTC 2900 and ATCC 0, the bacteria Salmonella typhimurium strain NCTC 202 and Salmonella enterica under enterica strain ATCC 4028 and Staphylococcus aureus strains PTCC 4 and ATCC 292 lyophilized stoke fungi and bacteria that form of industrial and infectious collection center in Iran, Organization of Scientific and Industrial Research of Iran had purchased or were prepared as friendly. In trypti case soy agar environments and 7 ºC temperature was recovery. Then bacteria In the In trypti case soy agar medium bacteria were cultured to confirm purity. Bacteria In the logarithmic phase: Of pure from bacterial colonies in trypti case soy agar removed and in the In trypti case soy broth medium for 8 h cultured and then at a wavelength of 620 nm turbidity measured by spectrophotometry and when the turbidity of the McFarland was in the range of a half pipe was used for insemination. In addition to standard bacteria, the E. coli O7: H7 and Salmonella isolated from food grown in the laboratory as well as above and inoculated with bacteria was used in standard. Pathogenic bacteria inoculated to lettuce: After minutes of each bacteria were centrifuged and the supernatant was discarded In 000 rpm and the resulting precipitate was washed twice with physiological serum and then was dissolved in physiological

427 M. Bahreini et al, 20 Journal of Applied Science and Agriculture, 8(7) December 20, Pages: 42-40 serum and the turbidity with a spectrophotometer in 0. OD was set In the turbidity of bacterial populations between 8-9 it was. After the opacity determination, ml of each bacterium to liters of distilled water was added and a homogeneous then 00 g of leaves, roughly the same size (about g) previously washed with a mild detergent and dry was added to water containing pathogenic bacteria and two minutes remained in suspension until the bacteria are connected to the lettuce leaves. And the hand was gently stirred until all parts of the leaf is in contact with the bacterial suspension. The lettuce leaves in health conditions of the suspension was removed and dried under a hood. The preparation and dilution of bacteria in specific environments and cultures of bacteria were counted. Microbial tests: After the drying and weighing grams of lettuce treated with 90 ml of buffered peptone saline in a blender and mix for 2 minutes In buffered peptone saline dilutions of the top ten to 4 were prepared and then taken microliters of each dilution In a medium that was previously part of the petri was divided with a marker, was placed and allowed to fully absorb the environments and then was placed In oven until the bacteria grow. Of the plates count agar medium, YGC agar, lactose agar and MRS agar violet red bile agar for the cultivation of aerobic mesophilic bacteria, molds and yeasts, coliforms bacteria, lactic acid bacteria was used. Each of which the temperature of 0 C º for 48-24 hour period, 2 C º for - days, 7C º for 24 hours and 0C º for 48-24 hours were incubated E. coli O7: H7 bacteria in the Sf Sym tlvt Mac Kanky agar medium and for 24 hours at 7C º was incubated. RESULTS AND DISCUSSION Ozonated water impact on the natural flora in lettuce: Effect of different concentrations of ozonated water and different treatment times on the natural flora of lettuce and E. coli bacteria O7: H7 and Salmonella were inoculated into which the results are presented in Table To determine the effective concentration of ozone, ozone at different concentrations at various times was on the lettuce and lettuce for a week in the refrigerator were treated. And every day of the appearance and sensory properties were investigated. Concentrations used were as follows 0.2 ppm, 0.4 ppm, 0.6 ppm, 0.8 ppm,.2 ppm,.6 ppm, 2 ppm, 2. ppm and ozone concentrations under pressure to create high. Higher concentrations of 2 ppm, even in short periods of reduced damage to lettuce appearance and thus were not suitable for use. Concentrations 0.2 ppm and 0.4 ppm in terms of appearance and sensory properties of a problem and would also be used even in times of more than minutes but had a great impact in eliminating bacteria. Sensory and physical properties of the results showed that the concentrations 0.6 ppm to 2 ppm up to -minute maximum negative impact on sensory properties and no apparent lettuce. And can be used as a disinfectant, but the concentration 2. ppm to the negative impact on lettuce leaves with concentrations in the 2 ppm and.6,.2, 0.8 0.6, 0.4 continued. The highest concentration of ozonated water up to.4 logarithmic cycle, was able to reduce the population of aerobic mesophilic bacteria. At higher concentrations of between and minute periods, there was no significant difference between periods and minutes, but significant differences were observed in all concentrations. At different concentrations between the two concentrations 0.6 ppm and 0.8 ppm reduction of aerobic mesophilic bacteria were not significantly different. The effect of ozonated water on the mold and yeast population was very low and at best less than one cycle of mold and yeast population decreased logarithmically (0.94 log cfu/g). The concentration of ozonated water treatment increased the amount of time and reduce mold and yeast population would increase, but its impact was not too much. Between concentrations. 6 ppm or 2 ppm ozonated water at all times, there was no significant difference in treatment. The results of this study, Coliforms bacteria were more sensitive to ozonated water and were more reduced than other bacteria. Coli forms of population reduction in the maximum ozonated water concentration in ppm 2 water were minutes.94 logarithmic cycles decreased. Concentrations in.2 ppm or.6 ppm at and ppm 2 min and the concentration at all times there was a significant difference. On ozonated water Coliforms of bacteria was less effective than lactic acid bacteria and lactic acid bacteria population in the best conditions to. cycle logarithmic decrease (2 ppm concentration and time minutes). Between concentrations.6 ppm or 2 ppm was not observed significant differences between different times, but there was a significant difference between treatments at all concentrations. The effect of ozonated water pathogenic bacteria inoculated on to lettuce: Concentration of ozonated water in ppm 2 and minutes was able to arrange up to.94 and 2.2 logarithmic cycles of Escherichia coli and E. coli O7: H7 inoculated on cut lettuce. Results showed that the treatment time increases the more ozone is between and minute periods, there were significant differences in all concentrations (Table2). These two bacteria were more sensitive to ozone than normal flora. The effect of ozonated water on Salmonella population was more than two previous pathogenic bacteria and could, at best, to 2.4 cycle logarithmic reduce Salmonella population. Salmonella and other bacteria tested with the same concentration decreased the most. Between concentrations. 6 ppm or 2 ppm was observed, but significant

428 M. Bahreini et al, 20 Journal of Applied Science and Agriculture, 8(7) December 20, Pages: 42-40 differences between treatments at and minutes there was a significant difference in different concentrations. Staphylococcus aureus bacteria, the bacteria were more sensitive and showed the greatest decrease in population. Staphylococcus aureus at concentrations of ozone for minutes to nearly ppm 2 cycle logarithmic (log cfu/g) was reduced. According to the results obtained between different concentrations and treatment times, there was a significant difference. Table : The effect of ozonated water on reduce bacteria population (log cfu/g). Bactria time 0.6 ppm 0.8 ppm.2 ppm.6 ppm 2 ppm (min) aerobic mesophilic bacteria *B0/2 d** B0/8c A0/74d C0/2d B0/72b AB0/86cd C0/2c B0/72b A0/97c B0/98b A/08ab B/8a B/2 a A/8b A/48a Mold and yeast Coliform lactic acid bacteria A0/8c B0/06a C0/08a B0/a C0/2a C0/42b C0/6b D0/9a C/06a C0/07c C0/bc B0/b A0/97bc B0/c 0/9bc BC B0/29ab B0/4a BC0/66b AB/2a C/a C/a BC0/09c C0/07c B0/b Ba A/0b A0/28c B0/c A0/6b A0/92a AB0/84b B/0b AB/6a AB/7a B0/b B0/4b Aa C0/6c B/0a * Latin capital letters (A to C) the difference between the mean each column to show each bacteria. ** Latin small letters (a to d) the difference between the mean each row to show each bacteria E. coli O7: H7 B0/7b C0/4b A0/a B/ab A/c A0/49b A0/68a A0/8a B0/69c AB/08b BC/4a B/6a A0/8c A0/7bc A0/8ab Ba A/4a A0/c A/6b A0/7b A0/94a A0/98d A/c A/67b A/94a A0/c A0/72c Ab A/a Table 2: The effect of ozonated water pathogenic bacteria inoculated on to lettuce. Pathogenic Bactria* time (min) 0/6 ppm 0/8 ppm /2 ppm /6 ppm 2 ppm * C0/b** BC0/4b AB0/8c C/c B0/B B/0A C/0a 0/4c Bc A0/7Bc /0Ab B C/a ABc B/2b B/72a C/c b B/72 A 2/2a Salmonella C0/48c BC0/6c B0/78c A/c B0/69c A0/97b A0/94bc A/8c Bb Bb B/8b A/82b *Each population was inoculated with bacteria(6log cfu/g) * Latin capital letters (A to C) the difference between the mean each column to show each bacteria. ** Latin small letters (a to d) the difference between the mean each row to show each bacteria. C/a B/7a B/8a A2/22a B B/46 b B/2 B/6b B/46 Conclusion: Reduce the microbial load of disinfectants in packing vegetables have been accepted and has been proven to improve quality and increase the half-life of fresh produce packaging should be used as a disinfection process. Wash vegetables with water, not only have great impact in reducing the pollution load. According to research done by various researchers has been proven that water can wash with 0.2 to. logarithmic cycle of pathogenic bacteria from the surface of vegetables (Behrsing et al, 2000; Sapers et al, 2006; Walle, 2002). But the results obtained in ideal conditions for each sample as a washing solution prepared with distilled water and rinsed several times in the industry that such practice is not possible. So continuous use of water for washing and rinsing is necessary to reduce the number of disinfectants are used to reduce waste production and increasing number of pathogenic microbes from the surface of certain types of vegetables are removed or killed. On the other hand is known that microbes can be attached to the surfaces of vegetables and plants are protected in this connection in order to protect themselves from environmental stress. These areas include openings, cut parts, parts of the plant surface is cracked or wounds and fluff. In addition, other factors such as hydrophobic interactions, the surface charge of bacteria, extracellular polysaccharides are fimbriae or vegetables and fruits are also involved in the binding of bacteria to surfaces (Solomon et al, 2002). Other studies have also shown that microbial growth in vegetables and fruits can be a result of biofilm formation (Burnett et al, 200).

429 M. Bahreini et al, 20 Journal of Applied Science and Agriculture, 8(7) December 20, Pages: 42-40 The studies demonstrated that pathogenic microbes such as Listeria monocytogenes, E. coli O7: H7 and Yersinia enterocolitica is naturally found in fruits and vegetables are contaminated. And if you have sufficient time and suitable environmental conditions, can grow in these products and their population of approximately 7 cfu / g kill. (Velazquez et al, 2009). Roughly the same population is inoculated on vegetables in the study. Due to these issues alone cannot wash with water from the surface of microbes attached vegetables, washed and removed, then it is necessary that the use of disinfectants to kill bacteria attached and removed. Ozone as a disinfectant in the packaged foods industry has gained importance because the impact time is short and the process does not produce any harmful product, the addition of harmful compounds such as pesticides, which is one of the hazardous chemical agents are used in the cultivation of vegetables and fruits are also destroyed (Guzel-Seydim et al, 2004). The studies determined that increasing the concentration of ozone impact on the natural flora and pathogenic microbes will be more. Natural micro lettuce less pathogenic bacteria was affected by ozone. Lettuce treated with ozonated water to reduce the.4, 0.94,.94 and. logarithmic cycle of aerobic mesophilic bacteria, respectively, molds and yeasts, bacteria and lactic acid bacteria in general form. Treated with ozonated water molds and yeasts showed more resistance and less reduction than in other microbes. Pathogenic bacteria more sensitive to ozone and were up to.94, 2.2, 2.4 and 2.9 cycle logarithmic order of the population of E. coli, E. coli O7: H7 and Salmonella, and Staphylococcus aureus inoculated to lettuce decreased the sodium hypochlorite and other compounds with similar or better antiseptic effect ([Abadiasa et al, 20; Behrsing et al, 2000; Crowe et al, 2007; Ukuku, 2006; Velazquez et al, 2009). Several reports in connection with ozone effects against pathogenic bacteria inoculated on lettuce there. Selma and colleagues [Selma et al, 2008].reported that concentrations of ozone gas, 20000 ppm could be up to /6, 0/7 and / logarithmic cycle of Coli forms of bacteria, mold and yeast and lactic acid bacteria on the surface of cut cantaloupe and Salmonella populations inoculated fruit surface depending on the moisture level of.4 to 2.8 cycle logarithmic decrease. Rodgers and colleagues (Rodgers et al, 2004) showed that Listeria monocytogenes inoculated into lettuce when treated ozone ppm to minutes was reduced to about logarithmic cycles. Boyer and colleagues (Boyer et al, 2007) reported the use of ozone for min ppm impact in reducing the Listeria monocytogenes inoculated on lettuce but effectively coli O7: H7 reduced. Because of these differences is that ozone effects are influenced by several factors. Varieties of lettuce, microbes studied the initial concentration of inoculated bacteria, the physiological states of bacterial cells and methods of ozone production are important factors that are causing the difference in treatment effect. Two factors also influence the concentration of ozone and release it on appearance and sensory properties are lettuce. Ölmez and Akbas (Ölmez et al, 2009). Reported that ozone level 2 ppm will improve the quality and appearance of lettuce 2. ppm properties affect its appearance and reduces quality of life of fresh lettuce and brown color and its which ultimately will lead to decreased half-life lasting product. Reduce the apparent quality of lettuce with increasing ozone concentration could be due to its high oxidation power can cause damage to lettuce tissue damage, possibly because of increased activity of the enzyme phenylalanine ammonia lyase is (Koseki et al, 2006). Previous studies indicated that increased activity of the enzyme phenylalanine ammonia lyase appearance and sensory properties of reduced shelf life of lettuce and lettuce in half by the process (Lopez-Galvez et al, 996). 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