Progress 07/01/19 to 02/29/20
Outputs
Target Audience:
Nothing Reported
Changes/Problems:During the execution of this task, ES encountered three major challenges. The first was adapting traditional sanitization standards to allow a direct comparison with ozone. The adaptation of the standard is discussed in Objective 1. The second challenge was disentangling a multitude of combined simultaneous effects observed in washing of produce, including: simultaneous shedding and inactivation, and a blend of oxygen and ozone impacts. The approach taken to solve the issue was a multi-layered series of control studies as discussed in Objectives 2 and 3. The third challenge was ensuring sufficient exploration of the large parameter space presented by the variables "ozone concentration" and "contact time" in produce washing. Once we had identified that a 0.5 ppm concentration of ozone nanobubbles inactivated a sufficiently large number of pathogens, we were able to fix concentration at that level and use the lower end of industry contact time of 60 seconds for assessment. What opportunities for training and professional development has the project provided?Although the research was not intended to provide training and professional development opportunities, we note that training activities have occurred via the mentoring of our student technician by En Solución's VP of Food Safety. The student technician performed hundreds of experiments under the guidance of the VP of Food Safety and other En Solución staff members, and she is now exploring future career options in the analytical chemistry and microbiology fields. How have the results been disseminated to communities of interest?No results have been disseminated as of the date of this report; however, En Solución is writing up peer-review articles for broader dissemination within the community. En Solución is an invited participant on United Fresh Produce Association's Food Safety Council where meeting agendas include discussion on new technologies and alternative treatments. Additionally, En Solución has routine communication regarding nanobubble potential and progress with leaders in the food safety community including the VP of Food Safety for the Produce Marketing Association and the VP of Food Safety and Technology of United Fresh Produce Association. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact. This project aimed to address the existential threat facing growers and processors of fresh produce that their fruits and vegetables could cause the illness or death of a consumer. Postharvest wash is a critical control point in fresh produce processing for reducing or eliminating pathogens and other field-acquired contaminants that can result in foodborne illness outbreaks. En Solución's food safety team has worked to validate the effectiveness of nanobubbles as a postharvest intervention by conducting a series of microbial experiments to determine the impact of gas-filled nanobubbles on pathogen neutralization. These efforts will ultimately lead to the production of innovative technology with the potential to make fresh produce safer in a way that is more effective, cost-competitive, and better for workers and the environment. To meet the project aim, En Solución (ES) established certain objectives in its project initiation application. In support of these objectives, ES conducted over 450 discrete tests. Final results indicate that ozone nanobubbles have great promise as an effective sanitizer for (i) reducing pathogen count of common foodborne bacteria strains in contaminated wash water, (ii) reducing pathogens on the surface of leafy greens, (iii) positively impacting the shelf-life of tested produce, (iv) minimizing worker safety hazard concerns through limited outgassing, and (v) positively impacting disposal effluent concerns through the natural decay of ozone. From the Objective 2 and Objective 3 results discussed below, we can more specifically assert that ozone nanobubbles have potential to not only reduce pathogen load on produce surfaces but can reduce cross-contamination of other produce during the postharvest wash process. Whether in nanobubble form or dissolved ozone, these studies indicate that ozone is a power sanitizer with log 3 and greater reductions in heavily contaminated wash water and a half log on the product itself. These results importantly illustrate the potential for ozone nanobubbles to supplement or replace the current postharvest wash techniques that regularly fail to prevent foodborne illness outbreaks and costly product recalls. Objectives. Throughout the grant performance period, progress was made on Objectives 1-5 as follows: Objective 1: In connection with Objection 1, ES used a modified ASTM E2315 study to determine the impact that nanobubble solutions of various gases had upon pathogen populations. The objective of these experiments was to determine whether nanobubbles formed from various gases (i.e., CO2, air, O2, O3, and N2) could effectively inactivate E. coli O157:H7. For each of the air, O2, CO2, and N2 nanobubble wash waters, the results showed a reduction of less than 0.5 log of E. coli when compared to the positive control. The nanobubbles of ozone gas (with concentration of 1.4 ppm) had the highest ORP value (1,207 mV) when compared to the nanobubbles formed from other gases, suggesting that they have strong oxidizing capabilities and can successfully break down contaminants. As a result, ES modified the scope of Objective 1 to focus on testing ozone exclusively, since the other gases showed little potential for pathogen neutralization. Testing showed a low concentration of ozone nanobubbles can produce a potent antimicrobial effect against all of five-strain pathogen cocktails of E. coli O157:H7, listeria monocytogenes, and salmonella ranging from log reduction of -4 to -7 CFU/mL. Objective 2: In support of Objective 2, ES designed a protocol for leafy green inoculation consistent with academic literature and industry testing. The leaf pathogen inactivation experiment was performed using ozone nanobubbles over six trials. A target ozone concentration of 0.65 ppm was achieved with resultant ORP more than 1100 mV. The pretreatment leaves were inoculated to log 7.93+/-0.32 CFU/g. Post-treatment with ozone nanobubbles, the leaves were measured to have an average log change of -1.36 +/- 0.19 CFU/g. By incorporating the results of the control studies, we can attribute log 0.9 CFU/g of this reduction to water washing from DI water and log -0.5 CFU/g of this reduction to ozone nanobubbles. This nanobubble leaf surface inactivation result matches the dissolved ozone result, indicating that the gas inside of nanobubbles is not "locked-away" and remains available for interactions within the liquid solution. Objective 3: In support of Objective 3, ES followed the protocol for leafy green inoculation created for Objective 2 and included testing for shedding by sampling the wash water for pathogen concentration 60 seconds after the removal of the inoculated product. The antimicrobial effect of 0.65 ppm ozone nanobubble wash water was tested against five-strain cocktails of E. coli on spinach leaves in a 5 L wash water bath. Results showed that ozonated nanobubbles in a wash water bath produced a total transfer rate from the inoculated leaves of log -3.85 +/- 0.42 CFU/mL. Using DI water as a control study, the inactivation rate in the water by ozone nanobubbles was observed to be approximately log -3. Objective 4: In support of Objective 4, ES has conducted comparative control studies using both chlorine and peracetic acid (PAA). The results of the comparative studies show chlorine (concentration of 50 ppm) can produce a 5-6 log pathogen reduction with a contact time of 60 seconds, and PAA (concentration of 80 ppm) can produce up to a 6 log pathogen reduction. These results are consistent with industry expectations and practice and validate the methodology used to test nanobubbles. As compared to industry standard sanitizers, the low-worker-hazard and environmentally friendly ozone nanobubbles are as effective at inoculation of pathogens. Objective 5: In support of Objective 5, several shelf life studies were conducted to compare the antimicrobial efficacy of PAA, ozone nanobubbles, and PAA in combination with ozone nanobubbles in their capacity to impact shelf-life of fresh produce. We continued our study with leafy greens and used spinach leaves for one of the washed products. Additionally, we added zucchini noodles as a more complex and processed product. The shelf-life studies followed the immersion treatment protocol described above, except that the spinach and zucchini noodle samples were not inoculated prior to treatment. The results of the shelf-life studies indicate that treatment of both a leafy green and a processed fresh squash with both PAA and ozone nanobubbles are effective at removing and suppressing the long term growth of microbial populations, providing a positive impact upon shelf-life considerations. Significant reductions of yeast, mold, lactic acid, and aerobic bacteria occurred in all products, independent of sanitizer applied. The ultimate cause of food spoilage is an excess of microbial populations, and all treatments were effective at keeping APC and LAB counts low over the course of a week. The ozone nanobubble were observed to have a greater impact upon mold and yeast when examining recovery rates of spore populations. Leafy green spoilage is dominated by general bacteria, but commodities such as tree fruits have shelf-life and spoilage rates dominated by mold. If similar results are observed outside of the laboratory, the addition of an ozone nanobubble treatment to tree fruits would have a positive impact upon shelf-life considerations (in addition to improving food safety via inactivation of harmful pathogens). ES expects that ozone nanobubbles alone or in conjunction with traditional sanitizers will improve food safety and extend the shelf-life and overall quality of produce.
Publications
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