Progress report for LNE20-412R
Problem, Novel Approach and Justification: The foodborne pathogens Salmonella, Escherichia coli O157:H7 and Listeria monocytogenes cause over 2 million annual illnesses in the U.S, largely due to consumption of contaminated fresh produce and poultry products. Current decontamination strategies using quaternary ammonium compounds or chlorine wash are not completely effective in reducing pathogen load on food products. Additionally, these chemicals pose significant health hazards for farmers that include the risk of cancer. This project aims to develop novel strategies for improved produce and egg decontamination. Our approach combines the antimicrobial efficacy of Generally Recognized as Safe status antimicrobial gas Ozone with novel ultra-fine bubble technology to develop safe and rapid-acting sanitizers for egg and produce washing.
Hypothesis and Research plan: We hypothesize that ultra-fine ozone (UFO) bubble water would significantly reduce Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes on eggs and fresh produce. The efficacy of UFO bubble water will be more than currently used antimicrobials (e.g. chlorine and peracetic acid). Our research plan consists of three stages. In the first stage, UFO bubble water will be generated using a dual-machine system consisting of an ozone generator and bubble mixer followed by characterizing the quality and stability of UFO bubbles. The PI has successfully standardized this stage. In the second stage, the efficacy of UFO bubble water in rapidly killing Salmonella on eggs and Salmonella, Escherichia coli O157:H7 and Listeria monocytogenes on produce (lettuce, cantaloupes) will be investigated. Preliminary research indicates that washing with UFO bubble water is effective in killing Salmonella on eggs and cantaloupes as early as 5 minutes at room temperature. No change in the color/appearance of eggs and cantaloupes was observed. In the third stage, the effect of organic load on the antimicrobial efficacy of most effective UFO bubble treatment-time combinations will be tested.
Outreach plan: As part of our extension/outreach objective, Dr. Shuresh Ghimire and Dr. Indu Upadhyaya will conduct workshops and technology demonstrations for extension personnel (train-the-trainer) in the Northeast to update stakeholders on outcomes of the project and to receive their feedback. They will also interact directly with organic and non-organic vegetable growers, egg-producers in CT and Northeast to understand their perceptions towards ultra-fine bubble technology-based wash solutions.
Project Objective: Our goal is to develop a novel washing method using ultra-fine ozone (UFO) bubble water to improve the microbiological safety of eggs and produce in the Northeast. The first objective will investigate the efficacy of UFO bubble water washing in killing Salmonella, Escherichia coli O157:H7 and Listeria monocytogenes on eggs and produce. The second objective will implement an extension/outreach program to disseminate our findings to stakeholders and extension personnel for field adoption of the technology.
Our goal is to develop a novel washing method using ultra-fine ozone (UFO) bubble water to improve the microbiological safety of eggs and produce in the Northeast. The first objective will investigate the efficacy of UFOB water washing in killing Salmonella, Escherichia coli O157 and Listeria monocytogenes on eggs and produce. The second objective will implement an extension/outreach program to disseminate our findings to stakeholders and extension personnel for field adoption of the technology.
Year 1 progress:
The PI hired a graduate student to work on the project starting September 2020. During the reporting period, the PI and graduate student have standardized the production of ozone micro bubbles under various temperature and time combinations. In addition, the effect of ozone in inactivating Listeria monocytogenes on cantaloupe rind and in produce wash water was investigated. Specific details on the experiments and results are provided below.
Objective 1: Investigating the solubility and stability of ozone in water maintained at different temperatures.
Rationale: Since ozone is generated on-site, it is critical to study the impact of type of machine, liquid temperature, and bubble time on ozone solubility. Also, since ozone naturally disintegrates to oxygen, understanding the disintegration kinetics of ozone in liquid medium and the various factors that impact the rate of disintegration is important for designing appropriate food safety experiments and develop recommendations for farmers.
Method: The solubility and stability of ozone in water maintained at different temperatures (25, 4℃) was measured at different bubbling time (0, 5, 10, and 15 min). The ozone was generated using an ozone generator (Ivation; ozone generation rate of 600 mg/h) and bubbled in 500 ml of DI water using a micro bubble diffuser. The concentration of ozone was measured post-bubble time at 0, 5, 10, 15, 20, 40 and 60 min using the Vacu-vials kit (SAM, OZONE, CHEMetrics, I-2019). Next, the generation of ozone was investigated using VMUS-4 ozone generator with or without an oxygen concentrator. VMUS-4 produces ozone at the rate of 4 g/h from dry air and 10 g/h from oxygen. This ozone generation rate is significantly higher than Ivation generator due to double corona discharge tubes present in the VMUS-4.
Objective 2: Efficacy of ozone microbubbles in inactivating planktonic cells of Listeria monocytogenes (Scott A strain) in produce wash water.
Rationale: Contaminated wash water can act as a source of cross-contamination between produce and can act as a medium to spread foodborne pathogens. Therefore, it is critical to test the effectiveness of antimicrobial wash treatments in reducing pathogen survival in wash water.
Method: Ozone was bubbled in 500 ml of DI water maintained at 4℃ for 15 min. After bubbling, ozonated DI water was inoculated with L. monocytogenes (Scott A strain) at ~6 log CFU/ml. The wash water was sampled at 1, 5, 10, 15, 30 min and the surviving L. monocytogenes were enumerated by dilution and plating on oxford agar plates followed by incubation at 37℃ for 48 hours. Ozone concentration was simultaneously measured at the aforementioned timepoints. The concentration of ozone at 1 min was ~ 0.74 ppm. The ozone level reduced to ~0.4 ppm by 30 min.
Objective 3: Efficacy of ozone microbubbles in inactivating Listeria monocytogenes on cantaloupe rind plugs.
Rationale: Once the efficacy of ozone microbubbles in inactivating L. monocytogenes was established in wash water, the next step is to test the antimicrobial efficacy of ozone micro bubbles against L. monocytogenes present on food product surface.
Method: Ozone was bubbled in 500 ml of DI water maintained at 4℃ for 15 min. Cantaloupe rind plugs (~ 4 cm diameter) were inoculated with LM cocktail (Scott A, AT19115, LM1, LM2 and LM3) at 8 log CFU/plug. The inoculum was allowed to attach for 90 min. Inoculated rind plugs were treated with ozonated water for 1, 5, 10 min at 4℃ and the surviving L. monocytogenes were enumerated by dilution and plating on oxford agar plates followed by incubation at 37℃ for 48 h.
Year 2 progress:
Since the submission of last year’s progress report, the PI and graduate student custom designed ozone compatible nanobubble generator in collaboration with Aciniti LLC (Japan) that can generate ozone nanobubbles with size below 200 nm. Once the ozone nanobubble generator was received from Japan, the PI and graduate student standardized the nanobubble production (objective 1-progress report 2) followed by testing the efficacy of ozone nanobubble water in inactivating Listeria monocytogenes on lettuce (objective 2-progress report 2). All experiments were repeated at least three times with duplicate samples. Experiments investigating the potential of ozone nanobubble water in inactivating Salmonella spp. on eggs and Escherichia coli O157:H7 on lettuce, cantaloupes are currently underway.
Objective 1: Investigate the stability of ozone nanobubbles in water.
Experiment 1: Standardization of ozone nanobubble generation.
Method: Ozone nanobubbles were generated using the oxygen concentrator-ozone generator-nanobubble generator combination at various experiment settings (run time, temperature of water, oxygen pressure, oxygen flow, spin speed of nanobubble generator). Similarly, ozone was produced and dissolved in the water without the nanobubble generator to serve as control.
Experiment 2: Degradation kinetics of ozone in ozone nanobubbles in water.
Method: Ozone nanobubbles were generated using an oxygen concentrator-ozone generator-nanobubble generator combination. The run time was 15 min, oxygen pressure of 15 psi, oxygen flow of 3 liters per min, temperature of water was 25oC and spin speed of nanobubble generator was 27 Hz. For control, similar set up was used, except for the nanobubble generator. Dissolved ozone was measured immediately after generation (time zero) and at regular intervals till 24 h.
Objective 2: Investigate the efficacy of ozone nanobubble water in inactivating Listeria monocytogenes on lettuce.
Method: Circular lettuce samples were prepared using a sterile, steel corer. Five strains of L. monocytogenes (Scott A, AT19115, LM1, LM2 and LM3) was used for this experiment. For inoculum preparation, the individual 10 ml overnight culture was centrifuged at 7000 rpm for 15 minutes at 25℃. The bacterial pellet was washed three times and resuspended in 10 ml of sterile 1X PBS. Equal portion of the washed cultures were mixed and diluted appropriately to yield a final inoculum concentration of 6 log CFU/ml. The average inoculum on lettuce was ~5-5.5 log CFU/sample. Cocktail of L. monocytogenes was spot inoculated (200 µl volume with 20 spots of 10 µl each; ~5.5 log CFU/sample) on the prepared fresh produce samples followed by incubation for 2 h at 25℃ in the biosafety cabinet, to facilitate bacterial attachment. For control, sterile DI water was used for washing. Ozonated nanobubbles were generated by using Ozone generator (Oxidation technologies, USA) connected to nanobubble generator (Aciniti, Japan) by running the machine for 30 minutes with 15 Psi of oxygen pressure. The dissolved ozone was measured post-bubble using the Vacu-vials kit (SAM, OZONE, CHEMetrics, I-2019). For treatment, glass containers were filled with 500 ml of DI water (control) or 500 ml of ozonated nanobubble water (treatment). The inoculated lettuce was dipped in the treatment container and treated for 1, 3 or 5 minutes at 25℃. After treatment, the samples were transferred to Whirl-PakTM bag (Nasco, Fisher Scientific) containing 10 ml of Dey-Engley neutralizing broth. The samples were stomached for 1 min at 300 rpm followed by dilution and plating on Oxford agar plates. The plates were incubated at 37℃ for 24-48 hours, followed by bacterial enumeration.
Year 1 progress:
Results and Discussion (objective 1): The temperature of the liquid had a significant effect on Ozone solubility. For example, approximately 1 ppm of dissolved ozone was observed in the water maintained at 4℃ after 15 min of bubbling time. However, when the water was maintained at 25℃, the dissolved ozone level obtained after 15 min of bubbling time was ~ 0.5 ppm (~50% less solubility). The ozone disintegration time was also higher in water maintained at 25℃ than at 4℃. After 60 min post-bubble time, the ozone level reduced by ~90% in water maintained at 25℃ whereas the ozone level reduced by ~30% in water maintained at 4℃ suggesting that ozone has a higher stability at lower temperature. Use of oxygen concentrator facilitated in the production of ~5 ppm of ozone in 1500 ml of DI water maintained at 25℃ in 5 min. Without the oxygen concentrator, a maximum ozone concentration of ~1 ppm was observed in 5 min. These results suggest that in order to generate a higher ozone concentration in water, an ozone generator with a higher ozone output (g/h) is required. Moreover, an oxygen concentrator facilitates in generating higher level of dissolved ozone in solution.
Results and discussion (objective 2): Ozone micro bubbles (dissolved ozone concentration ~ 0.74 ppm) inactivated L. monocytogenes to below detection limits (~5 log CFU/ml reduction) as early as 1 min of treatment time indicating that dissolved ozone is very effective in inactivating L. monocytogenes in wash water.
Result and Discussion (objective 3): A reduction of ~ 1 log CFU/ml of L. monocytogenes was observed as early as 1 min of treatment time. Additional treatment times (5, 10 min) did not result in increased antimicrobial efficacy indicating that the maximum inactivation occurred in the first min of interaction between dissolved ozone and L. monocytogenes present on cantaloupe rind.
Year 2 progress:
Results and discussion (objective 1, experiment 1): We observed that a dissolved ozone level of ~ 3-4 ppm was obtained after 15 min of run time in both control (without nanobubble generator) and treatment (with nanobubble generator). When the water temperature was lowered to 4oC, a higher dissolved ozone level of ~ 9 ppm was obtained.
Results and discussion (objective 1, experiment 2): The figure (Fig. 1) below show the data obtained for experiment 2. We observed that a dissolved ozone level of ~ 3.5-3.8 ppm was obtained after 15 min of run time in both control (without nanobubble generator) and treatment (with nanobubble generator). After 1 h of storage at room temperature, the dissolved ozone level in ozonated nanobubble water (Blue line) was significantly higher as compared to normal ozone water (orange line) indicating that ozone nanobubbles might be facilitating higher dissolved ozone levels in the water.
Results and discussion (objective 2): The effect of ozone nanobubble water in inactivating L. monocytogenes on lettuce at 25oC is presented in Fig. 2. In case of control (washing lettuce with water), no reducing in pathogen load was observed as compared to baseline (lettuce inoculated with pathogen but not subjected to any wash treatment). Washing with water containing ozone nanobubbles for 1 min reduced pathogen load by ~ 2 log CFU/sample as compared to control (P<0.05). Increasing the wash time from 1 to 5 minutes did not further enhance the antimicrobial efficacy of ozone nanobubbles. Results suggest that ozone nanobubbles washing is effective in reducing L. monocytogenes on lettuce.
Education & Outreach Activities and Participation Summary
We are currently conducting survey to determine how they are currently managing product washing operations. In the coming months, survey data will be analyzed to determine common washing practices, their effectiveness and economic viability. Results will be used to identify areas for improvement and discussed with the project advisory committee to develop a comprehensive outreach program for the second and third year.