Progress 09/01/20 to 08/31/23
Outputs
Target Audience:The research from this Phase II SBIR project is meant to serve all growers and processors of apples (and fresh produce and proteins more broadly) who seek to improve current product and equipment sanitization practices to improve food safety outcomes. Changes/Problems:Objectives 2 and 3 were not fully completed as contemplated in the original goals and milestones. Initial efforts toward each of these objectives were described in earlier reports (including experiments to evaluate the effectiveness of ozone nanobubbles at pathogen destruction on the surface of apples and at neutralizing certain mold species that contribute to the shortening of apple shelf-life), but Objectives 2 and 3 were not pursued to completion because we prioritized efforts and resources to focus on biofilms (Objective 1) instead. Both customers and industry experts contend that the overwhelming impacts ofL. monocytogenesbiofilms warrant particular focus on developing new solutions for their prevention and eradication. Biofilms have been identified as a vector for foodborne illness outbreaks and, further, the issue of cross contamination on food contact surfaces such as brushes results in the highest impact to public health versus the ability to clean individual apples/products. Given the promising results of our preliminary testing of ozone nanobubbles on biofilms (described in earlier reports), encouraging similar outcomes documented in recent literature, and the overwhelming commercial importance of biofilm prevention and destruction, we deemed it most beneficial to aggressively target biofilms (Objective 1) for the remainder of our research. 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 did occur for a graduate student at Washington State University (subaward to the project). The graduate student, Zi Hua, was mentored by Dr. Meijun Zhu, subaward key person, who validated the experimental design and performed the microbial analytical work as part of her doctoral studies.? How have the results been disseminated to communities of interest?To better communicate the benefits of our technology, we joined the Western Growers Association's Innovation Center Food Safety Initiative, a program that helps fresh produce farmers discover and adopt new technology. This unique networking opportunity has provided access to almost a thousand companies growing fresh produce on the West Coast. We have also participated in various industry speaking engagements, including the Produce Marketing Association's special webinar on nanobubbles for food safety (where registration included over 500 industry professionals), the 2022 annual meeting of the Western Growers Association in Las Vegas, and the AgTech podcast called Voices of the Valley. The results of the Washington State University studies regarding the efficacy of ozone nanobubbles against biofilms will be presented at the annual meeting of the International Association for Food Protection in 2024 and submitted to a peer-reviewed journal for publication. 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 Phase II research contributed to the development of ascientifically validated, commercially ready, cost-competitive nanobubble ozonation system capable of enhancing the productivity, quality, and safety of apple crops. This technology allows for cleaner foods without the use of chemicals or harmful by-products. It generates the cleaning solution from oxygen in the atmosphere, water, and electricity, which eliminates supply chain concerns.Our technology can improve many aspects of current apple facility operations, including providing labor and water savings, removal of variable and rising chemical costs, decreased dependency on chemical availability and the necessity of chemical storage, mitigation of extensive and broad-reaching liability from food safety events, and the advancement of company sustainability goals.The research conducted here, however, has implications beyond apples. Data regarding the performance of nanobubbles in pressurized spray conditions will inform application of the ozone nanobubble solution across a wide variety of food safety and sanitization functions, from washing other varieties of fresh produce to cleaning proteins and food processing equipment. Further, the impressive laboratory results validating the effectiveness of ozone nanobubbles againstListeriabiofilms on food contact surfaces underscores the potential for the technology to drastically improve current sanitization practices in fresh food processing. Although our initial market focus has been on fresh produce processing and packaging, the technology has broad applicability in additional areas like proteins, irrigation, and food distribution channels. Objectives.Objective 1: Quantify the effectiveness of ozone nanobubbles at destroyingListeriabiofilms on major food-contact surfaces. In satisfaction of Objective 1, a comprehensive analysis of the impact of ozone nanobubbles onL. monocytogenesbiofilms on major food contact surfaces was completed in cooperation with Washington State University (WSU). We first evaluated the efficacy of ozone nanobubbles against 2-day-oldListeria monocytogenesbiofilms at different concentrations (2 or 4 ppm), at different contact times (1 min or 10 min), and on commonly used food contact surfaces in apple packing facilities, including stainless steel (SS) and polyester (PET). We then evaluated the effectiveness of ozone nanobubble treatments against 7-day-oldL. monocytogenesbiofilms on these surfaces. Water and oxygen nanobubble treatments were included as controls. A 1-min contact with 2 ppm ozone nanobubble solution resulted in 1.1 and 0.7 log CFU/coupon reductions ofL. monocytogenesin the 2-day-old biofilm on SS and PET surfaces, respectively. Increasing the ozone concentration and contact time of the ozone nanobubble solution enhanced its antimicrobial activity. After a 1-min treatment with 4 ppm ozone nanobubbles, reductions of 1.4 and 1.3 log CFU/coupon ofL. monocytogenesin 2-day-old biofilms on SS and PET surfaces were observed, respectively. Furthermore, after a 10-min treatment, reductions of 2.2 and 1.6 log reductions were observed on SS and PET surfaces, respectively. Of note, the efficacy of ozone nanobubbles was independent of the age of biofilm. Next, we investigated the impact of different surface-to-volume ratios and methods of application ozone nanobubble solution on its efficacy againstL. monocytogenesbiofilms. Our data revealed that increasing the volume of ozone nanobubble solution only marginally impacts efficacy on reducing biofilms on SS coupons. However, the method of exposure influenced the efficacy of ozone nanobubbles in removingListeriabiofilms. Dynamic exposure to ozone nanobubble solution at 4.0 ppm using a flow rate of 1 L/min for 1 min showed significantly improved results compared to a 1-min static immersion, with 3.24vs.1.44 log CFU/coupon reduction on SS; however, there was no significant difference in the efficacy of 4.0 ppm ozone nanobubble solution when used at 1 L/min or 6 L/ min. We also observed a reduction in biofilmfrom exposure to dynamic exposure of flow of oxygen nanobubbles, discussed further below. Our results indicate that ozone nanobubble solutions are highly effective againstL. monocytogenesbiofilms. The data support an additive effect of both a physical and a chemical cleaning of these biofilms as the dynamic flow experiments reveals approximately 1.5 log reduction from ozone in a static immersion and an additional 1.5 log reduction from oxygen nanobubbles with a minimum flow rate; these effects combine to produce a greater than 3 log reduction when used in combination. The capability to reduce 7-day-old biofilms by 3 log represents a significant new capability for food processing facilities. These results have implications for the application of ozone nanobubbles in real-world operations, where secondary benefits to overall facility cleanliness can be realized by simply introducing an ozone nanobubble solution to pipes and drains at low flows and nominal pressure as part of a primary operations orcleaning applications. Objective 2:Quantify the effectiveness of ozone nanobubbles at pathogen destruction on the surface of apples.Objective 2 was not fully completed as contemplated in the original goals and milestones. Initial efforts toward the objective were described in earlier reports, but Objective 2 was not pursued to completion because we prioritized efforts and resources to focus on Objective 1 (more detail in the Changes/Problems section). Objective 3: Quantify the effectiveness of ozone nanobubbles at extending the shelf-life of apples.Objective 3 was not fully completed as contemplated in the original goals and milestones. Initial efforts toward the objective were described in earlier reports, but Objective 3 was not pursued to completion because we prioritized efforts and resources to focus on Objective 1 (more detail in the Changes/Problems section). Objective 4: Construct an experimental testing chamber and spray system to evaluate the stability of ozone nanobubbles when subjected to pressures relevant for spray bar applications. Objective 4 wascompleted during the performance period of this Phase II research. Procedures and outcomes related to Objective 4 were described in detail in earlier reports. Objective 5: Quantify the effect of organic load on the ability of ozone nanobubbles to neutralize pathogens. In satisfaction of Objective 5, we further quantified the ability of ozone nanobubbles to reduce microbial populations in real-world, high organic-load environments. Testing over the last 6 months confirmed the effectiveness of ozone nanobubbles as a replacement for peracetic acid (PAA) in brush cleaning applications, but failed to support ozone nanobubbles as a viable water recycling solution without significant investment in additional filtering systems. On the roller brush beds, ozone nanobubbles reduced microbial counts by an additional log 1.6 when compared to PAA. On the apple surfaces, the ozone nanobubbles performed similarly to PAA, with log reductions of 1.5 with ozone nanobubbles and 1.3 with PAA. Ongoing full-scale pilot activities at the apple cider processing plant have confirmed the commercial-readiness of our automated nanobubble ozonation technology. The installed ozonation unit has successfully operated for over 650 hours and provided over 3,000 gallons of ozone nanobubble solution at that facility. The switch to ozone nanobubbles has saved the facility thousands of dollars in expensive chemical costs and is helping to eliminate high wastewater processing fees associated with water disposal. Objective 6:Identify the maximum concentration of ozone nanobubbles that can be applied to apples without incurring damage to the integrity of the apple. Objective 6 wascompleted during the performance period of this Phase II research. Procedures and outcomes related to Objective 6 were described in detail in earlier reports.
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Progress 09/01/21 to 08/31/22
Outputs
Target Audience:
Nothing Reported
Changes/Problems:A major ongoing problem encountered by En Solución throughout this award has beenthe impact of the COVID-19 pandemic on access to facilities at Washington State University (WSU), the subawardee to this award.Due to staff and facility limitations at WSU, we were unable to commence the WSU work in the first performance period as originally scheduled. Although we now have our equipment installed in their facilities and have commenced some preliminary experiments, we continue to see delays due to backlog of work across the lab. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?No formal presentation results have been disseminated as of the date of this report; however, many informal conversations have been had with subject matter experts in industry, government, and academia as we continue to engage relevant stakeholders. What do you plan to do during the next reporting period to accomplish the goals?We will complete Objectives 1, 2, 3, and 5 during the final NCE reporting period. Objectives 1, 2 and 3 will be accomplishedin collaboration with Washington State University (WSU), a subawardee to this proposal. Work on Objectives 1 and 2 will build upon the experiments already commenced in the previous performance period, with experimental protocols and variables adjusted as needed based on prior results. Objective 3 will be conducted as originally proposed in the USDA approved Phase II application. We will complete Objective 5 in connection with pilot testing at a Washington apple processing facility. We anticipate installing our nanobubble ozonation unit to replace their current traditional aqueous ozone system, utilizing the existing overhead spray nozzles for application on processed apples and to explore the potential to replace the chlorine in the primary dump tank. We also will conduct a series of dump tank trials, exploring the possibility of replacing chlorine with ozone nanobubbles in this immersion step.
Impacts
What was accomplished under these goals?
Impact. This Phase II research contributes to the development of acommercially ready, cost-competitive nanobubble ozonation system capable of enhancing the productivity, quality, and safety of apple crops.Our technology will provide additional and needed protections for the vast economic investments made during the decade-long process of growing trees to maturity for high-profitability, innovative apple varietals. Due to the increasing specialization of the apple industry, devastating brand damage can result from negative consumer events such as a foodborne illness, recalls, decreased quality, and premature decay and spoilage. This research will ultimately lead to the production of novel ozone nanobubble technology that can dramatically improve food safety systems and extend product shelf-life of postharvest-washed apple crops.The research conducted here, however, has implications beyond apples. Data regarding the performance of nanobubbles in pressurized spray conditions will inform application of the ozone nanobubble solution across a wide variety of food safety and sanitization functions, from washing other varieties of fresh produce to cleaning proteins and food processing equipment. These efforts will ultimately lead to the production of innovative technology with the potential to make food safer in a way that is more effective, cost-competitive, and better for workers and the environment. Objectives. Objectives 4 and 6 were completed in prior performance periods. Objectives 1, 2, and 3 will be completed in collaboration with Washington State University (WSU), a subawardee to this proposal, during the final NCE performance period. Objective 5 will be completed via pilot testing at an apple processing facility in Washington. In the last performance period, progress was made on Objectives 1, 2, and 5 as follows: In support of Objective 1,we collaborated with WSU to conduct a series of microbial experiments to quantify the effectiveness of ozone nanobubbles at destroyingL. monocytogenesbiofilms on major food-contact surfaces. These tests indicatethat ozone nanobubbles can provide up to a 2 log reduction of pathogen levels on stainless steel and a 1.5 log reduction on rubber surfaces.These preliminary results indicate that ozone nanobubbles show promise in providing superior neutralization againstListeriabiofilms on food-contact surfaces. Further work to quantify the impact of ozone nanobubbles onListeriabiofilms will be conducted in cooperation with WSU during the final NCE performance period. In support of Objective 2,we collaborated with WSU to conduct initial microbial experiments to evaluate the effectiveness of ozone nanobubbles at destroyingListeria innocuaon the surface of apples. These tests showed some reduction in pathogen levels on the surface of the apples, but significant anomalies in the collected data necessitate additional procedural review before the experiments are replicated. Further work to quantify the impact of ozone nanobubbles onListerialevels on the surface of apples will be conducted in cooperation with WSU during the final NCE performance period. In connection with Objective 5, we completed the preliminary administrative and engineering requirements to commence a full-scale pilot at an apple processing facility in WA.
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Progress 09/01/20 to 08/31/21
Outputs
Target Audience:Through participation in the USDA Food Loss and Waste Innovation Fair on May 26, 2021, we engaged with a wide audience of business and industry professionals interested in reducing food loss and waste in the US food system. We also engaged with service providers from Pace International and Fruit Growers Supply, a subsidiary of Sunkist, during the course of our research efforts this reporting period. Changes/Problems:A major problem encountered by En Solución during the period leading to this mid-term report was the impact of the COVID-19 pandemic on access to facilities at Washington State University (WSU), the subawardee to this award. Due to staff and facility limitations at WSU, we were unable to commence the WSU work in the first half of the performance period as originally scheduled. We have entered into the necessary contractual arrangements with WSU, have installed our equipment in their laboratory, and have had initial conferences with the students and staff to permit the commencement of the WSU work in October 2021. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?No formal presentation results have been disseminated as of the date of this mid-term report; however, many informal conversations have been had with subject matter experts in industry, government, and academia as we continue to engage relevant stakeholders. What do you plan to do during the next reporting period to accomplish the goals?We will complete Objectives 1, 2, 3, and 5 during the next reporting period. Objectives 1, 2 and 3 will be accomplished in collaboration with Washington State University (WSU), a subawardee to this proposal, and will be conducted as originally proposed in the USDA approved Phase II application. These objectives could not be accomplished in the first reporting period due to restrictions at WSU related to the COVID 19 pandemic. We will complete Objective 5 in connection with pilot testing at the Domex Superfresh Growers' apple processing facility. For Objective 5, we will account for the impact of high organic load on ozone by exploring the application of ozone nanobubbles via pressurized spray bar, roller brush, or in secondary flume water. Further, we will evaluate the effect of organic load on higher concentrations of ozone, up to the maximum achievable by our latest prototype unit. Finally, we will investigate the impact of pre-filtering the charging water to get optimal chemistry and minimal organic loading prior to generating ozone nanobubbles to further enhance performance.
Impacts
What was accomplished under these goals?
Impact. This Phase II research contributes to the development of a commercially ready, cost-competitive nanobubble ozonation system capable of enhancing the productivity, quality, and safety of apple crops. Our technology will provide additional and needed protections for the vast economic investments made during the decade-long process of growing trees to maturity for high-profitability, innovative apple varietals. Due to the increasing specialization of the apple industry, devastating brand damage can result from negative consumer events such as a foodborne illness, recalls, decreased quality, and premature decay and spoilage. This research will ultimately lead to the production of novel ozone nanobubble technology that can dramatically improve food safety systems and extend product shelf-life of postharvest-washed apple crops. The research conducted here, however, has implications beyond apples. Data regarding the performance of nanobubbles in pressurized spray conditions will inform application of the ozone nanobubble solution across a wide variety of food safety and sanitization functions, from washing other varieties of fresh produce to cleaning proteins and food processing equipment. These efforts will ultimately lead to the production of innovative technology with the potential to make food safer in a way that is more effective, cost-competitive, and better for workers and the environment. Objectives. Objectives 1, 2, and 3 will be completed in collaboration with Washington State University (WSU), a subawardee to this proposal, during the second half of the performance period. To date, progress has been made on Objectives 3, 4, 5, and 6 as follows: In support of Objective 3, we conducted a series of preliminary microbial experiments to quantify the effectiveness of ozone nanobubbles at extending the shelf-life of apples. These tests indicate that low concentrations of ozone nanobubbles (1 ppm) are more effective than high concentrations of chlorine (200 pm) at neutralizing shelf-life reducing mold spores over short contact periods and equally effective over longer contact periods. These preliminary results indicate that ozone nanobubbles show promise in effectively eradicating mold spores that lead to the decay of fresh apples. Further work to quantify the impact of ozone nanobubbles on apple shelf-life will be conducted in cooperation with WSU during the second performance period. In satisfaction of Objective 4, we constructed an experimental testing chamber and spray system to evaluate the stability of ozone nanobubbles when dispensed at pressures consistent with industry-standard spray bar applications. The experiments ultimately aimed to determine whether a high-concentration ozone nanobubble sanitizer could be applied as a spray to product surfaces without any OSHA atmospheric ozone safety concerns. Nozzles were selected for the types and brands commonly used in industry for overhead spray application on apples washed on roller brush-beds. The nozzles spanned a full range of hole sizes, producing "mist" to "flood" type effects. In general, the conversation efficiency was relatively insensitive to pressure and increased with nozzle size (i.e. the largest nozzle, TK 10.0, had a 18%-22% conversation rate and the smallest nozzle, TK 1.0, had 2.5%-5% conversation rate of dissolved ozone). We determined that nanobubbles retain their unique physical, chemical, and physiochemical properties when flowing through nozzles of at least 1.3 gpm at 30 psi. The mid-range nozzle, TK 7.5 represents the optimal sized nozzle in terms of minimizing off-gassing while maintaining the highest aqueous ozone nanobubble conversation rate. This nozzle will be the specified nozzle for applications of En Solución's product going forward. In connection with Objective 5, we performed a series of preliminary organic load tests at Pace International's research and manufacturing facility in Union Gap, WA, to begin quantifying the ability of ozone nanobubbles to neutralize pathogens in high load environments. For these trials, ozone nanobubble solution (at 3 ppm) was added to apple rot water (the highest organic load water in an apple processing facility) in a 50/50 ratio in a 5L container. Ozone levels crashed immediately to 0 ppm with the addition of the rot water. We determined that further study of the effects of organic load on competitor sanitizers (like peracetic acid and chlorine) was not needed due to the overwhelming impact of heavy organic load on ozone levels These tests indicated that it is simply unrealistic to achieve and maintain the ozone concentrations required to provide sanitizing effects in a large dump tank with high levels of organic load. In satisfaction of Objective 6, we determined that the maximum concentration of ozone nanobubbles achievable by En Solución equipment, 10 ppm, can be applied to apples without incurring damage to the integrity of the apple. Testing was conducted on twelve organic, unwaxed Honeycrisp apples. Results indicated that exposure to ozone nanobubbles in concentration-times of up to 40 ppm-min had no negative impact on the physical appearance, weight, or overall product quality. Brix measurements for each apple indicate that dissolved sugar content also was unaffected by ozone exposure. These results confirm that ozone nanobubbles can be applied at the highest concentrations achievable to maximize pathogen neutralization without concern for impacts on product integrity.
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