Use of Ozone & Plant Antimicrobials for Improving Safety & Quality of Organic Leafy Greens

Non-Technical Summary:<br/>
Yuma, Arizona making them an important source of income to the growers. Between 1996 and 2008, 82 produce related foodborne illness outbreaks occurred in the United States, 28 of which were linked to leafy greens resulting in both illnesses and massive financial losses to producers. Fresh produce is the top-selling item amongst organic foods. Since organic foods are produced without the use of pesticides, antibiotics, and hormones, limited treatment options are available. Hence, there is a need for alternative processing methods to reduce or prevent pathogen contamination. According to the Center for Disease Control and Prevention (2009), of the estimated 3.6 million cases of foodborne illnesses caused by bacteria, non-typhoidal Salmonella was responsible for 1 million (11%) cases. E. coli O157:H7 is another foodborne pathogen of concern in fresh produce. E. coli O157:H7 causes about 73,000 illnesses in the United States annually and results in an added burden of approximately $405 million annual loss associated with industrial productivity and related healthcare costs. The occurrence of several Salmonella and E. coli O157:H7 outbreaks associated with spinach and other leafy greens indicates that these pathogens can withstand current interventions designed to decontaminate the produce before it is packaged for human consumption. Natural plant-derived compounds, such as extracts, spices, and essential oils have been shown to have antimicrobial activity against foodborne pathogens. Potential antimicrobial activity of plant-derived compounds has been demonstrated in fresh produce. Ozone is a powerful antimicrobial because of the damage it causes to bacterial cellular components. Ozone is effective against a range of pathogenic bacteria and does not form harmful byproducts in water, unlike chlorine. The use of edible and food compatible plant antimicrobials and ozone individually and in combinations against S. enterica, E. coli O157:H7 and naturally occurring spoilage bacteria on organic leafy greens will be investigated. The most effective plant antimicrobials and ozone combinations will then be tested on recycled wash water used for washing organic leafy greens. Sensory qualities of organic leafy greens from the most effective treatments will be determined. The effects of the most active treatments on the quality of wash water will be investigated. The most effective treatments will be evaluated on a commercial or semi-commercial scale using a non-pathogenic E. coli strain in flume washers in Yuma. We expect that the proposed technologies could eliminate or reduce the presence of pathogens on organic leafy greens while improving their safety and quality, thereby resulting in an increased consumption of organic leafy greens. The long term goal of this project is to improve the profitability of organic leafy green farms by developing and implementing commercially feasible technology for post-harvest use.
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Approach:<br/>
Salmonella enterica serovar Newport & Escherichia coli O157:H7 will be used for this study. Four types of organic leafy greens (OLG): romaine lettuce, iceberg lettuce, mature bunched spinach, & baby spinach will be used. Plant antimicrobials will include essential oils (oregano oil, cinnamon oil, lemongrass oil, clove oil etc.) & plant extracts (olive, apple, green tea, black tea, white tea, grapeseed, grape pomace, pomegranate, hibiscus etc). Essential oils will be tested at 0.1, 0.3 & 0.5% concentrations & the plant extracts at 1, 3, & 5% concentrations. Objective 1: OLG will be inoculated with S. enterica or E. coli O157:H7 at levels of 4 & 8 log CFU/ml. Each leaf will be subject to a) individual plant antimicrobial treatment b) ozonated water (approx. 5-20 mg/L ozone concentration) & c) combination of an antimicrobial with ozone. Samples will be taken for enumeration of survivors at day 0, 1, & 3 by plating on appropriate media.
<br/>Objective 2: The aforementioned procedure for pathogen inoculation & antimicrobial treatment will be followed without any attempt to eliminate background microflora to assess efficacy of plant antimicrobials individually and in combination in a natural setting. The bacterial populations will be enumerated on Tryptic Soy Agar (TSA), deMan Rogossa and Sharpe agar (MRS) & Pseudomonas isolation agars.
<br/>Objective 3: The recyclability of the proposed antimicrobials will be evaluated against spoilage & pathogenic microorganisms in wash water (WW). Pre-inoculated OLG will be immersed into the treatment solution for 30 sec. Washed OLG & treatment solution will be evaluated for bacterial populations. WW will be reused & tested each time for bacterial population. Tap water aliquots used to wash OLG will be inoculated with S. enterica & E. coli O157:H7 & then be treated with ozone (approx. 5-20 mg/L ozone concentration) & plant compounds to eliminate the pathogens. The drop in pathogen population will be enumerated.
<br/>Objective 4: A consumer panel will be chosen to determine acceptability of OLG using a 9-point hedonic scale (1 = dislike extremely; 9 = like extremely). Sensory attributes tested will be color, flavor, texture, odor & overall acceptability. Objective 5: The effect of ozone on OLG -WW turbidity & biochemical oxygen demand (BOD) will be tested using the nephelometric method and the Winkler method or an oxygen probe, respectively, according to the American Public Health Association protocol. Objective 6: The most effective antimicrobial combinations will be tested on a semi-commercial scale at the Yuma Agricultural Center using non-pathogenic E. coli. Controls used will be hydrogen peroxide & peracetic acid. Data Analysis: Experiments will be performed thrice. ANOVA will be performed on enumerated means post treatment. Significant differences between controls & treatments will be analyzed. Least significant differences (LSD) will be calculated (P<0.05) to identify significant differences among treatment means. Sensory attributes will be analyzed by paired-sample t-tests (P < 0.05).