Development and Validation of Innovative Food Processing Interventions

Goals / Objectives
1: Further studies on the ARS-patented use of RFP for shell eggs through the development of pilot plant and commercial prototypes of continuous RFP equipment for multiple eggs. 2: Further studies on the use of innovative technologies to reduce microorganisms on fresh produce, and minimally preserved, brined, and fresh-cut refrigerated vegetables. 3: Evaluate the use of biochars to reduce pathogens in manures, compost, and soils used for the production of fresh (both conventional and organic) produce.

Project Methods
A pilot plant-scale radio frequency pasteurization (RFP) unit will be developed, capable of continuously processing multiple shell eggs. Initial efforts will use a 60 MHz RFP unit similar to the unit used to write the ARS patent. The single-egg RFP unit is capable of pasteurizing shell eggs with significantly better quality than industry eggs (currently pasteurized using hot water immersion). RFP operating parameters will be optimized, while experimental factors to be investigated will include cooling water flow rate, cooling water conductivity, cooling water temperature, and amount and duration of RF power applied. Equally important for reducing pasteurization operating costs is reducing equipment costs. To this end, we will study egg roller minimum rotation speed, and feasibility of adjusting frequency to 40.68 MHz, which is within the radio band internationally reserved for industrial, scientific and medical purposes. Optimized RF operating and equipment costs will be estimated. Quality and functionality characteristics of RFP eggs will be evaluated. The RFP process will be scaled up by developing RF power supplies, matching networks, and power distribution schemes to evenly heat hundreds of egg simultaneously. Finally, a continuous RFP pilot plant unit will be designed and assembled, which will convey eggs through the unit. To reduce microorganisms on fresh and fresh-cut vegetables, several innovative technologies will be researched. The ability of novel washes, developed during the previous project cycle, to remove pathogenic biofilms will be investigated. Bacterial cell surface charges will be determined using hydrophobic and electrostatic interaction chromatography. Also, the occurrence of sublethal injury to pathogens, following treatment with the produce wash, will be determined. The previously-developed antimicrobial wash will be improved with additional ingredients and pH adjustment. Wet steam technology has been successfully applied to cantaloupes, and will be extended to other produce. Finally, pilot plant scale testing of the produce intervention technologies will be conducted and costs of applying them estimated. In order to evaluate the use of biochars to reduce pathogens in manures, compost, and soils, non-pathogenic bacteria will be validated as surrogates for pathogenic bacteria in soil and manure survival studies with biochar. Antimicrobial efficacy of biochar will be optimized by adjusting production time and temperature as well as by comparing various biofeedstocks. The optimized biochar will be evaluated to determine its potential to inactivate surrogate bacteria in compost, in lab and greenhouse settings as well as in scaled-up field experiments. Cost estimates for applying lethal doses of the optimized biochar to compost and fields will be determined.

Progress 02/10/16 to 02/09/21

Outputs
PROGRESS REPORT Objectives (from AD-416): 1: Further studies on the ARS-patented use of RFP for shell eggs through the development of pilot plant and commercial prototypes of continuous RFP equipment for multiple eggs. 2: Further studies on the use of innovative technologies to reduce microorganisms on fresh produce, and minimally preserved, brined, and fresh-cut refrigerated vegetables. 3: Evaluate the use of biochars to reduce pathogens in manures, compost, and soils used for the production of fresh (both conventional and organic) produce. Approach (from AD-416): A pilot plant-scale radio frequency pasteurization (RFP) unit will be developed, capable of continuously processing multiple shell eggs. Initial efforts will use a 60 MHz RFP unit similar to the unit used to write the ARS patent. The single-egg RFP unit is capable of pasteurizing shell eggs with significantly better quality than industry eggs (currently pasteurized using hot water immersion). RFP operating parameters will be optimized, while experimental factors to be investigated will include cooling water flow rate, cooling water conductivity, cooling water temperature, and amount and duration of RF power applied. Equally important for reducing pasteurization operating costs is reducing equipment costs. To this end, we will study egg roller minimum rotation speed, and feasibility of adjusting frequency to 40.68 MHz, which is within the radio band internationally reserved for industrial, scientific and medical purposes. Optimized RF operating and equipment costs will be estimated. Quality and functionality characteristics of RFP eggs will be evaluated. The RFP process will be scaled up by developing RF power supplies, matching networks, and power distribution schemes to evenly heat hundreds of egg simultaneously. Finally, a continuous RFP pilot plant unit will be designed and assembled, which will convey eggs through the unit. To reduce microorganisms on fresh and fresh-cut vegetables, several innovative technologies will be researched. The ability of novel washes, developed during the previous project cycle, to remove pathogenic biofilms will be investigated. Bacterial cell surface charges will be determined using hydrophobic and electrostatic interaction chromatography. Also, the occurrence of sublethal injury to pathogens, following treatment with the produce wash, will be determined. The previously-developed antimicrobial wash will be improved with additional ingredients and pH adjustment. Wet steam technology has been successfully applied to cantaloupes, and will be extended to other produce. Finally, pilot plant scale testing of the produce intervention technologies will be conducted and costs of applying them estimated. In order to evaluate the use of biochars to reduce pathogens in manures, compost, and soils, non-pathogenic bacteria will be validated as surrogates for pathogenic bacteria in soil and manure survival studies with biochar. Antimicrobial efficacy of biochar will be optimized by adjusting production time and temperature as well as by comparing various biofeedstocks. The optimized biochar will be evaluated to determine its potential to inactivate surrogate bacteria in compost, in lab and greenhouse settings as well as in scaled-up field experiments. Cost estimates for applying lethal doses of the optimized biochar to compost and fields will be determined. In produce-related research, a new solution aimed at reducing the browning of fresh-cut apple pieces and, at the same time, reducing microbial populations was developed by combining specific short-chain organic acids, generally regarded as safe (GRAS). A hurdle technology using the above solution and combining it with non-thermal intervention technologies to lower or kill pathogenic bacteria after treatment was investigated. The new antibrowning-antimicrobial solution led to 8 log inactivation of Salmonella, E. coli O157:H7, and Listeria monocytogenes in vivo and a 3.8 log inactivation of attached bacteria on produce surfaces. Combination treatment with UV-light and 0.5 kGy of irradiation led to a 5 log reduction, suggesting that these treatments would reduce bacterial populations on produce, minimizing the incidence of foodborne illness, saving the produce industry costly recalls, and improving consumer confidence. Regarding research on biochar soil amendments for the safer production of produce, studies were conducted with a newly-constructed lab-scale muffle furnace pyrolyzer to generate biochar to have antimicrobial properties. Pyrolysis conditions consisted of the following: First, finely ground mixed hardwood chips were pyrolyzed for 30 min. at temperatures of 350, 400, 450, 500, 550, and 600°C. The resulting biochar was added at a 10% concentration (wt.:wt.) into cultivable soils inoculated with ca. 7 log CFU/g of a four-strain cocktail of attenuated E. coli O157:H7 and held for four weeks. Populations over time did not differ from the no-biochar control samples. Next, finely ground switchgrass was pyrolyzed at 500°C for times of 15, 30, 45, 60, 75, and 90 min. The biochar was then challenged in soil with E. coli O157:H7, as described above. The results were the same, with no difference in populations over time compared to no- biochar control samples. A major limitation with this means of biochar production is that once the target pyrolysis time and temperature is reached and the muffle furnace is turned off, the system must cool to room temperature overnight before the chamber can be opened for safety purposes. During this cooling period, the temperature remains high for a period of time; hence, the biochar may be over-processed, and the antimicrobial properties of the biochar may dissipate. Efforts are underway to secure a biochar reactor that can eject biochar samples at the target pyrolysis time and temperature which does not require extended time or extended for cooling down. Record of Any Impact of Maximized Teleworking Requirement: Because the Eastern Regional Research Center facility is on maximal telework, no lab experiments could be performed for the majority of the reporting period. ACCOMPLISHMENTS 01 Antimicrobial treatment for fresh and fresh-cut fruits and vegetables. Risks associated with human pathogens on fresh fruit and vegetables remain a concern for consumers. ARS researchers at Wyndmoor, Pennsylvania, have developed a combination of organic acids and safe additives that kills pathogens such as Listeria monocytogenes, Salmonella, and E. coli on a variety of fruits and vegetables. These newly created antimicrobial solutions provide alternatives for safe decontamination of fresh produce.

Impacts
(N/A)

Publications

Yang, Y., Geveke, D.J. 2019. Shell egg pasteurization using radio frequency in combination with hot air or hot water. Food Microbiology. https://doi.org/10.1016/j.fm.2019.103281.

Progress 10/01/19 to 09/30/20

Outputs
Progress Report Objectives (from AD-416): 1: Further studies on the ARS-patented use of RFP for shell eggs through the development of pilot plant and commercial prototypes of continuous RFP equipment for multiple eggs. 2: Further studies on the use of innovative technologies to reduce microorganisms on fresh produce, and minimally preserved, brined, and fresh-cut refrigerated vegetables. 3: Evaluate the use of biochars to reduce pathogens in manures, compost, and soils used for the production of fresh (both conventional and organic) produce. Approach (from AD-416): A pilot plant-scale radio frequency pasteurization (RFP) unit will be developed, capable of continuously processing multiple shell eggs. Initial efforts will use a 60 MHz RFP unit similar to the unit used to write the ARS patent. The single-egg RFP unit is capable of pasteurizing shell eggs with significantly better quality than industry eggs (currently pasteurized using hot water immersion). RFP operating parameters will be optimized, while experimental factors to be investigated will include cooling water flow rate, cooling water conductivity, cooling water temperature, and amount and duration of RF power applied. Equally important for reducing pasteurization operating costs is reducing equipment costs. To this end, we will study egg roller minimum rotation speed, and feasibility of adjusting frequency to 40.68 MHz, which is within the radio band internationally reserved for industrial, scientific and medical purposes. Optimized RF operating and equipment costs will be estimated. Quality and functionality characteristics of RFP eggs will be evaluated. The RFP process will be scaled up by developing RF power supplies, matching networks, and power distribution schemes to evenly heat hundreds of egg simultaneously. Finally, a continuous RFP pilot plant unit will be designed and assembled, which will convey eggs through the unit. To reduce microorganisms on fresh and fresh-cut vegetables, several innovative technologies will be researched. The ability of novel washes, developed during the previous project cycle, to remove pathogenic biofilms will be investigated. Bacterial cell surface charges will be determined using hydrophobic and electrostatic interaction chromatography. Also, the occurrence of sublethal injury to pathogens, following treatment with the produce wash, will be determined. The previously-developed antimicrobial wash will be improved with additional ingredients and pH adjustment. Wet steam technology has been successfully applied to cantaloupes, and will be extended to other produce. Finally, pilot plant scale testing of the produce intervention technologies will be conducted and costs of applying them estimated. In order to evaluate the use of biochars to reduce pathogens in manures, compost, and soils, non-pathogenic bacteria will be validated as surrogates for pathogenic bacteria in soil and manure survival studies with biochar. Antimicrobial efficacy of biochar will be optimized by adjusting production time and temperature as well as by comparing various biofeedstocks. The optimized biochar will be evaluated to determine its potential to inactivate surrogate bacteria in compost, in lab and greenhouse settings as well as in scaled-up field experiments. Cost estimates for applying lethal doses of the optimized biochar to compost and fields will be determined. In produce-related research, a new solution aimed at reducing browning of fresh-cut apples pieces and, at the same time, reducing microbial populations was developed by combining specific short chain organic acids, generally regarded as safe (GRAS). A hurdle technology using the above solution and combining it with non-thermal intervention technologies to lower or kill pathogenic bacteria after treatment was investigated. The new antibrowning-antimicrobial solution led to 8 log inactivation of Salmonella, E. coli O157:H7 and Listeria monocytogenes in vivo and a 3.8 log inactivation of attached bacteria on produce surfaces. A combination treatment with UV-light and 0.5 kGy of irradiation led to a 5 log reduction, suggesting that these treatments would reduce bacterial populations on produce, minimizing incidence of foodborne illness, saving the produce industry costly recalls, and improving consumer confidence. Risks associated with human pathogens on fresh fruit and vegetables remain a concern for consumers. We developed a patent-pending combination of organic acids and GRAS additives that effectively inactivates human pathogens on a variety of fruits and vegetables. Isopropyl citrate (IC) (0.16% acidulant) plus 0.05% total concentration of two GRAS surfactants (0.025% sodium-2-ethyl-hexyl sulfate (EHS) and 0.025% sodium dodecylbenzene-sulfonate (SDBS)) inactivated L. monocytogenes in suspension by up to 7.0 log CFU/mL within 2 min, while 0.27% IC plus surfactants inactivated Salmonella and E. coli O157:H7 by up to 6.54 log CFU/ml (99.9999+%). On grape tomatoes, 0.54% IC plus 0.05% total concentration of EHS + SDBS inactivated L. monocytogenes, E. coli O157:H7 and Salmonella by 4.2 (99.994%), 4.9 (99.998%), and 5.5 log CFU/g (99. 999+), respectively. A patent pending combination of 0.35% lactic or citric acids plus 0.05% total concentration of two surfactants (0.025% EHS and 0.025% SDBS) inactivated up to 7.0 log CFU/ml (99.99999+%)of L. monocytogenes, Salmonella and E. coli O157:H7. On grape tomatoes, 0.61% lactic or citric acids plus 0.05% total concentration of EHS + SDBS inactivated L. monocytogenes, E. coli O157:H7 and Salmonella by up to 4.0 (99.99%), 4.4 (99.996%), and 4.9 log CFU/g (99.998%), respectively. Newly created antimicrobial solutions may provide alternatives for the decontamination of fresh produce. Regarding research on biochar soil amendments for the safer production of produce, studies were conducted with a newly-constructed lab-scale, muffle furnace pyrolyzer to generate biochar with the goal of having antimicrobial properties. Pyrolysis conditions consisted of the following: First, finely ground mixed hardwood chips were pyrolyzed for 30 min. at temperatures of 350, 400, 450, 500, 550, and 600°C. The resulting biochar was added at a 10% concentration (wt.:wt.) into cultivable soils inoculated with ca. 7 log CFU/g of four-strain cocktail of attenuated E. coli O157:H7 and held for four weeks. Populations over time did not differ from the no-biochar control samples. Next, finely ground switchgrass was pyrolyzed at 500°C for times of 15, 30, 45, 60, 75 and 90 min. The biochar was then challenged in soil with E. coli O157:H7, as described above and the results were the same, with no difference in populations over time in comparison to no-biochar control samples. A major limitation with this means of biochar production is that once the target pyrolysis time and temperature is reached, and the muffle furnace is turned off, the system must cool to room temperature overnight before the chamber can be opened, for safety purposes. During this cooling period, the temperature remains high for a period of time; hence, the biochar may be over processed, and the antimicrobial properties of the biochar may dissipate. Efforts are underway to secure a biochar reactor that can eject biochar samples at the target pyrolysis time and temp, which does not require extended time periods for cooling down. In other work, a patent application was filed for a novel combination of surfactants and acidulants for the inactivation of foodborne pathogens in solution or on fresh produce. Accomplishments 01 New antimicrobial anti-browning solution for fresh-cut fruits. Human pathogens can survive on fresh fruit, creating a safety hazard for consumers. ARS researchers at Wyndmoor, Pennsylvania, developed and filed for a patent for a new antimicrobial solution, which also stops browning in cut fruits. The antimicrobial and anti-browning solution can kill Listeria, Salmonella, or E. coli bacteria on treated freshly- cut fruits. The treated fruit has low microbial counts, are safe, and will help to reduce costly recalls of the fresh-cut fruits by produce industries.

Impacts
(N/A)

Publications

Gurtler, J. 2020. Two generally recognized as safe surfactants plus acidulants inactivate Salmonella, Escherichia coli 0157:H7, and Listeria monocytogenes in suspension or on dip-inoculated grape tomatoes. Journal of Food Protection. 83:637-643.
Rodriguez, A.B., Olanya, O.M., Ukuku, D.O., Niemira, B.A., Orellana, L.E., Mukhopadhyay, S., Cassidy, J.M., Boyd, G. 2019. Reduction of Listeria monocytogenes on post-harvest carrot and tomato by radiation, santizer and biocontrol treatments and their combinations. LWT - Food Science and Technology. 118:1-8.
Leng, J., Mukhopadhyay, S., Sokorai, K., Ukuku, D.O., Fan, X., Olanya, O.M. , Juneja, V.K. 2019. Inactivation of Salmonella in cherry tomato stems cars and quality preservation by pulsed light treatment and antimicrobial wash. Food Control. 110:107005.
Gurtler, J., Mullen, C.A., Boateng, A.A., Masek, O., Camp, M.J. 2020. Biocidal activity of fast pyroloysis biochar against E.coli 0157:H7 in soil varies based on production temperature or age of biochar. Journal of Food Protection. 83:1020-1029.
Gurtler, J., Juneja, V.K., Jones, D.R., Pruohit, A. 2019. Thermal inactivation kinetics of three heat-resistant Salmonella in whole liquid egg. Journal of Food Protection. 82(9):1465-1471.
Fan, X., Sokorai, K., Gurtler, J. 2019. Advanced oxidation process for the inactivation of Salmonella Typhimurium on tomatoes by combination of gaseous ozone and aerosolized hydrogen peroxide. International Journal of Food Microbiology. 312.
Yu, Y., Jin, Z.T., Fan, X., Gurtler, J. 2019. Effects of carvacrol wash and ally isothiocyanate vapor treatment to extend the shelf life of blackberries. Jacobs Journal of Food and Nutrition. 6(4):46-57.
Wang, L., Fan, X., Gurtler, J., Wang, W. 2019. Interaction of gaseous chlorine dioxide and mild heat on the inactivation of Salmonella on almonds. Journal of Food Protection. 82(10):1729⿿1735.
Berrios-Rodriguez, A., Ukuku, D.O., Olanya, O.M., Cassidy, J.M., Orellana, L.E., Mukhopadhyay, S., Niemira, B.A. 2019. Nisin based organic acids inactivation of Salmonella on Grape tomatoes: Efficacy of treatment using bioluminescences ATP Assay. Journal of Food Protection. 83:68-74.
Mukhopadhyay, S., Sokorai, K.J., Ukuku, D.O., Fan, X., Olanya, O.M., Juneja, V.K. 2019. Effects of pulsed light and sanitizer wash combination on inactivation of Escherichia coli 0157:H7, microbial loads and apparent quality of spinach leaves. Food Microbiology. 82:127-134.
Olanya, O.M., Hoshide, A.K., Oluwatosin, I., Ukuku, D.O., Mukhopadhyay, S., Niemira, B.A., Ayeni, O. 2019. Cost estimation of listeriosis (Listeria monocytogenes) occurrence in South Africa in 2017-2018 and its food safety implications. Food Control.
Gurtler, J., Keller, S.E., Fan, X., Olanya, O.M., Jin, Z.T. 2020. Survival of desiccation-resistant salmonella on apple slices following antimicrobial immersion treatments and dehydration. Journal of Food Protection. 83:902-909.

Progress 10/01/18 to 09/30/19

Outputs
Progress Report Objectives (from AD-416): 1: Further studies on the ARS-patented use of RFP for shell eggs through the development of pilot plant and commercial prototypes of continuous RFP equipment for multiple eggs. 2: Further studies on the use of innovative technologies to reduce microorganisms on fresh produce, and minimally preserved, brined, and fresh-cut refrigerated vegetables. 3: Evaluate the use of biochars to reduce pathogens in manures, compost, and soils used for the production of fresh (both conventional and organic) produce. Approach (from AD-416): A pilot plant-scale radio frequency pasteurization (RFP) unit will be developed, capable of continuously processing multiple shell eggs. Initial efforts will use a 60 MHz RFP unit similar to the unit used to write the ARS patent. The single-egg RFP unit is capable of pasteurizing shell eggs with significantly better quality than industry eggs (currently pasteurized using hot water immersion). RFP operating parameters will be optimized, while experimental factors to be investigated will include cooling water flow rate, cooling water conductivity, cooling water temperature, and amount and duration of RF power applied. Equally important for reducing pasteurization operating costs is reducing equipment costs. To this end, we will study egg roller minimum rotation speed, and feasibility of adjusting frequency to 40.68 MHz, which is within the radio band internationally reserved for industrial, scientific and medical purposes. Optimized RF operating and equipment costs will be estimated. Quality and functionality characteristics of RFP eggs will be evaluated. The RFP process will be scaled up by developing RF power supplies, matching networks, and power distribution schemes to evenly heat hundreds of egg simultaneously. Finally, a continuous RFP pilot plant unit will be designed and assembled, which will convey eggs through the unit. To reduce microorganisms on fresh and fresh-cut vegetables, several innovative technologies will be researched. The ability of novel washes, developed during the previous project cycle, to remove pathogenic biofilms will be investigated. Bacterial cell surface charges will be determined using hydrophobic and electrostatic interaction chromatography. Also, the occurrence of sublethal injury to pathogens, following treatment with the produce wash, will be determined. The previously-developed antimicrobial wash will be improved with additional ingredients and pH adjustment. Wet steam technology has been successfully applied to cantaloupes, and will be extended to other produce. Finally, pilot plant scale testing of the produce intervention technologies will be conducted and costs of applying them estimated. In order to evaluate the use of biochars to reduce pathogens in manures, compost, and soils, non-pathogenic bacteria will be validated as surrogates for pathogenic bacteria in soil and manure survival studies with biochar. Antimicrobial efficacy of biochar will be optimized by adjusting production time and temperature as well as by comparing various biofeedstocks. The optimized biochar will be evaluated to determine its potential to inactivate surrogate bacteria in compost, in lab and greenhouse settings as well as in scaled-up field experiments. Cost estimates for applying lethal doses of the optimized biochar to compost and fields will be determined. Scaling-up of the radio frequency pasteurization (RFP) process for shell eggs continued. A 54-egg RFP unit is being assembled in collaboration with a CRADA industry partner. A branch line splitter, which distributes the power from one RF generator to many eggs, was assembled from inexpensive parts and tested. It was compared to a commercial 90-degree hybrid splitter, which divides the power between eggs exactly 50:50 but is prohibitively expensive. The branch line splitter divides the power 56:44, which is acceptable, at a fraction of the cost of the commercial splitter. The cost savings of using branch line splitters in a large- scale RFP unit is estimated to be $100K. In other RFP research, the efficacy of RF in combination with hot water spraying (HWS) on the inactivation of Salmonella in shell eggs was compared to that of the current method which uses hot water immersion (HWI). HWS is of interest because eggs are already sprayed with warm water during RF processing. The results indicated that the pasteurization times were comparable for RF/HWI and RF/HWS (19.5 and 24.5 min, respectively). In addition, the quality of the eggs from both treatments were essentially the same. These results showed that HWS, which simplifies the RF pasteurization process, is an attractive alternative to HWI. In produce-related research, the surface charge and hydrophobicity of strains of Salmonella, Escherichia coli and Listeria monocytogenes were determined and the initial bacterial attachment on produce was highest for individual strains of E. coli and lowest for L. monocytogenes, but Salmonella exhibited the strongest attachment during storage. A new solution aimed at reducing browning of fresh-cut apples pieces and, at the same time, reducing microbial populations was developed by combining specific short chain organic acids generally regarded as safe (GRAS). Also, a bioluminescent ATP assay that achieves results within minutes was compared to conventional microbial determination that requires 2-3 days incubation before results are acquired. The results were very comparable to plate count methods but not on antimicrobial washed fruits due to low bacterial count after treatment. The new antibrowning-antimicrobial solution also disrupted biofilms on the surfaces of treated fruits. An invention disclosure for this novel antibrowning-antimicrobial solution has been filed. This treatment and determination would save produce industry time and money in investigating microbial load on produce. Regarding research on biochar soil amendments for the safer production of produce, studies were conducted determining the minimum concentrations of biochar capable of inactivating attenuated E. coli O157:H7 in soil as well as if biochar was capable of reducing the pathogen in dairy manure compost. The rationale for using biochar as a soil amendment is to mitigate the potential for foodborne pathogen contamination in cultivable soils to prevent transfer from soil to fresh produce commodities, which could subsequently infect humans. Soil was amended with fast-pyrolysis biochar generated at 600°C in a newly-constructed bioreactor. Pathogen populations after only one week of storage were (biochar concentrations in parentheses) 6.93 log CFU/g (0%), 5.89 log (1.0%), 3.80 log (1.5%), 3. 78 log (2.0%), 3.25 log (2.5%), 1.46 (3.0%), and 0.00 (3.5%). When fresh dairy compost was supplemented with 10% walnut cyclone biochar from a collaborator, no growth was detected throughout 7 weeks of storage which was attributed to high pH in compost (10.76). However, when compost was supplemented with 10% high-temp walnut biochar (pH 8.71 in compost) from the collaborator, populations remained as high as 9.05 log CFU/g up to week 4, compared with the no-biochar control, which was 9.34 log. Further, when 3.5% of the same biochar was inoculated in soil, populations were reduced to 4.42 log as compared with the no-biochar control, which was 6.46 log, indicating biochar may have a greater antimicrobial efficacy in soil as compared with fresh dairy compost. Results should provide guidance on the application of biochar added to compost or soil to inactivate foodborne pathogens. Accomplishments 01 Novel antimicrobial antibrowning solution for fresh-cut produce. The ARS- developed antimicrobial-antibrowning solution was able to kill viable populations of Listeria monocytogenes, Salmonella or Escherichia coli O157:H7 bacteria and reduced their populations to below detectable levels on treated fresh-cut fruits. This antimicrobial-antibrowning solution will reduce foodborne outbreaks and produce recalls by the industries. Invention disclosure has been filed and work is in progress with an industry partner for possible licensing and patent application. 02 Inactivation of E. coli O157:H7 in manure by supplementing with biochar. ARS researchers in Wyndmoor, Pennsylvania have demonstrated that biochar is antimicrobial to foodborne pathogens in crop soil. Fresh dairy compost was supplemented with 10% walnut cyclone biochar from a collaborator. The 10% biochar supplement inactivated 7.95 log of the pathogen within 7 days. No E. coli was detected in the biochar- supplemented compost throughout 7 weeks of storage which was attributed to high pH in compost (10.76). This result provides guidance on the application of biochar to compost or soil to inactivate foodborne pathogens.

Impacts
(N/A)

Publications

Ukuku, D.O., Niemira, B.A., Uknalis, J. 2019. Nisin-based antimicrobial combination with cold plasma treatment inactivate Listeria monocytogenes on granny smith apples. LWT - Food Science and Technology. 104:120-127.
Yang, Y., Geveke, D.J., Brunkhorst, C.D., Sites, J.E., Geveke, N., Tilman, E.D. 2019. Optimization of the radio frequency power, time and cooling water temperature for pasteurization of Salmonella typhimurium in shell eggs. Journal of Food Engineering. 247:130-135.
Ukuku, D.O., Mukhopadhyay, S., Olanya, O.M. 2018. Survival and growth of transferred salmonella and aerobic mesophilic bacteria from melon rinds to fresh juices during juices preparation. Frontiers in Sustainable Food Systems.
Gurtler, J., Fan, X., Jin, Z.T., Niemira, B.A. 2019. Effects of antimicrobials on the thermal sensitivity of foodborne pathogens: A review. Journal of Food Protection. 82(4):628-644.
Singh, A., Geveke, D.J., Jones, D.R., Tilman, E.D. 2019. Can acceptable quality angel food cakes be made using pasteurized shell eggs? The effects of mixing factors on functional properties of angel food cakes. Food Science and Nutrition. 7: 987-996.
Guo, M., Jin, Z.T., Gurtler, J., Fan, X., Yadav, M.P. 2018. Inactivation of E.coli O157:H7 and Salmonella on fresh strawberries by antimicrobial washing and coating. Journal of Food Protection. 81(8):1227-1235.

Progress 10/01/17 to 09/30/18

Outputs
Progress Report Objectives (from AD-416): 1: Further studies on the ARS-patented use of RFP for shell eggs through the development of pilot plant and commercial prototypes of continuous RFP equipment for multiple eggs. 2: Further studies on the use of innovative technologies to reduce microorganisms on fresh produce, and minimally preserved, brined, and fresh-cut refrigerated vegetables. 3: Evaluate the use of biochars to reduce pathogens in manures, compost, and soils used for the production of fresh (both conventional and organic) produce. Approach (from AD-416): A pilot plant-scale radio frequency pasteurization (RFP) unit will be developed, capable of continuously processing multiple shell eggs. Initial efforts will use a 60 MHz RFP unit similar to the unit used to write the ARS patent. The single-egg RFP unit is capable of pasteurizing shell eggs with significantly better quality than industry eggs (currently pasteurized using hot water immersion). RFP operating parameters will be optimized, while experimental factors to be investigated will include cooling water flow rate, cooling water conductivity, cooling water temperature, and amount and duration of RF power applied. Equally important for reducing pasteurization operating costs is reducing equipment costs. To this end, we will study egg roller minimum rotation speed, and feasibility of adjusting frequency to 40.68 MHz, which is within the radio band internationally reserved for industrial, scientific and medical purposes. Optimized RF operating and equipment costs will be estimated. Quality and functionality characteristics of RFP eggs will be evaluated. The RFP process will be scaled up by developing RF power supplies, matching networks, and power distribution schemes to evenly heat hundreds of egg simultaneously. Finally, a continuous RFP pilot plant unit will be designed and assembled, which will convey eggs through the unit. To reduce microorganisms on fresh and fresh-cut vegetables, several innovative technologies will be researched. The ability of novel washes, developed during the previous project cycle, to remove pathogenic biofilms will be investigated. Bacterial cell surface charges will be determined using hydrophobic and electrostatic interaction chromatography. Also, the occurrence of sublethal injury to pathogens, following treatment with the produce wash, will be determined. The previously-developed antimicrobial wash will be improved with additional ingredients and pH adjustment. Wet steam technology has been successfully applied to cantaloupes, and will be extended to other produce. Finally, pilot plant scale testing of the produce intervention technologies will be conducted and costs of applying them estimated. In order to evaluate the use of biochars to reduce pathogens in manures, compost, and soils, non-pathogenic bacteria will be validated as surrogates for pathogenic bacteria in soil and manure survival studies with biochar. Antimicrobial efficacy of biochar will be optimized by adjusting production time and temperature as well as by comparing various biofeedstocks. The optimized biochar will be evaluated to determine its potential to inactivate surrogate bacteria in compost, in lab and greenhouse settings as well as in scaled-up field experiments. Cost estimates for applying lethal doses of the optimized biochar to compost and fields will be determined. Progress on scaling up radio frequency pasteurization (RFP) of shell eggs continued. The ARS patented RFP technology kills 99.999% of Salmonella in shell eggs in 1/3 the time of the current commercial process. Assembly of a third generation RFP unit is nearing completion. The unit is currently capable of simultaneously processing 9 eggs. Work is ongoing to maximize processing uniformity of the eggs and to increase the number of eggs processed. A fourth generation, large-scale RFP unit is being assembled in collaboration with a CRADA industry partner. Two major advancements were made that will facilitate commercialization of RFP; (1) the equipment was modified to operate at a frequency of 40.68 MHz, which is an international frequency reserved for industrial, scientific and medical (ISM) purposes rather than the non-ISM (60 MHz) frequency previously used, and (2) matching was obtained using inexpensive ferrites instead of with a matching network costing $10-100K. A second patent (U.S. 9,883,682) was awarded for RFP. Also, the effect of RF power, treatment time and cooling water temperature on the inactivation of Salmonella Typhimurium was investigated. Five operating conditions (of various power, time, and cooling water temperature) achieved more than 99.999% kill without any observable quality change. The results showed, for the first time, that RFP is a flexible process. This study will help to determine the processing parameters of RF for the food industry. In produce-related research, fruits and vegetables were inoculated with Salmonella, Escherichia coli and Listeria monocytogenes. Bacterial attachment was highest for spinach and lettuce compared to cucumber, apples and tomatoes. Salmonella exhibited the strongest attachment. In another study, a solution with dual purpose (antibrowning/antimicrobial) for treatment of fresh-cut produce was developed and tested against E. coli O157:H7, Salmonella spp. and L. monocytogenes bacteria inoculated on melon, apple, tomato, cucumber, and pear surfaces. The antibrowning- antimicrobial solution was effective in reducing bacterial populations and inhibiting browning reaction. An invention disclosure on the novel antimicrobial-antibrowning treatment has been filed. Also, a hurdle technology using a combination of cold plasma and antimicrobial treatment was developed to inactivate bacteria on fruits surfaces. Regarding research on soil amendment for the safer production of produce, a study was conducted to evaluate survival of non-pathogenic surrogate strains of E. coli in soil with 10 percent biochar. The rationale for using biochar as a soil amendment is to mitigate the potential for foodborne pathogen contamination in cultivable soils to prevent transfer from soil to fresh produce commodities, which could subsequently infect humans. Five types of biofeedstock were used to produce the pyrolysis biochars including (1) switchgrass, (2) wheat straw, (3) miscanthus grass, (4) oil rapeseed straw, and (5) rice hulls. Results should provide guidance on biofeedstock type that go into producing biochar with the greatest biocidal activity to inactivate foodborne pathogens in the soil. Accomplishments 01 Improvements in radio frequency pasteurization of shell eggs. Raw shell eggs can be contaminated with Salmonella, causing illnesses and recalls. The ARS-patented radio frequency pasteurization (RFP) process produced safer eggs with exceptional quality in a small-scale prototype. ARS researchers at Wyndmoor, Pennsylvania, in collaboration with a CRADA industry partner, assembled and successfully tested a larger- scale RFP unit, thus paving the way for a commercial-scale RFP unit. In addition, two breakthroughs were achieved that will facilitate commercialization. The first was the modification of RFP to operate at 40.68 MHz, which is an international frequency reserved for industrial, scientific and medical purposes. The second was another modification of the RFP to enable inexpensive ferrites to provide a match between the eggs and equipment. This will save between $10 to $100K per RFP unit. This technology has the potential to address a significant, widespread source of foodborne illness and make shell eggs safer for the American consumer.

Impacts
(N/A)

Publications

Lacombe, A., Niemira, B.A., Gurtler, J., Kingsley, D.H., Li, X., Chen, H. 2018. Surfactant-enhanced disinfection of the human norovirus surrogate, Tulane virus, with organic acids and surfactant. Journal of Food Protection. 81:(2)279-283.
Yan, R., Yun, J., Gurtler, J., Fan, X. 2017. Radiochromic film dosimetry for UV-C treatments of apple fruit. Postharvest Biology and Technology. 127:14-20.
Yan, R., Liu, Y., Gurtler, J., Fan, X. 2017. Sensitivity of pathogenic and attenuated E. coli O157:H7 strains to ultraviolet-C light as assessed by conventional plating methods and ethidium monoazide-PCR. Journal of Food Safety. doi: 10.1111/jfs.12346.
Hu, M., Gurtler, J. 2017. Selection, validation and utility of pathogen surrogate bacteria used in food safety challenge studies: A Review. Journal of Food Protection. 80:1506-1536.
Gurtler, J. 2017. Pathogen decontamination in crop soil: A review. Journal of Food Protection. 80:1461-1470.
Geveke, D.J., Bigley, A., Brunkhorst, C., Jones, D.R., Tilman, E.D. 2018. An improved radio frequency method to pasteurize salmonella in shell eggs. International Journal of Food Science and Technology.
Olanya, O.M., Ukuku, D.O., Solaiman, D., Ashby, R.D., Niemira, B.A., Mukhopadhyay, S. 2018. Effects of temperature and storage time on inactivation of Listeria monocytogenes, Salmonella enterica, and Escherichia coli 0157:H7 populations by sophorolipid and sanitizer in- vitro and on tomato. International Journal of Food Science and Technology. 53:1303-1315.
Mukhopadhyay, S., Sokorai, K.J., Ukuku, D.O., Jin, Z.T., Fan, X., Olanya, O.M., Juneja, V.K. 2018. Inactivation of Salmonella in tomato stem scars by organic acid wash and chitosan-allyl isothiocyanate coating. International Journal of Food Microbiology. 266:234-240.
Fan, X., Gurtler, J., Sokorai, K.J. 2018. Type of tomatoes and water rinse affect efficacy of acid washes against salmonella enterica inoculated on stem scar areas of tomatoes and on product quality. International Journal of Food Microbiology. 280:57-65.

Progress 10/01/16 to 09/30/17

Outputs
Progress Report Objectives (from AD-416): 1: Further studies on the ARS-patented use of RFP for shell eggs through the development of pilot plant and commercial prototypes of continuous RFP equipment for multiple eggs. 2: Further studies on the use of innovative technologies to reduce microorganisms on fresh produce, and minimally preserved, brined, and fresh-cut refrigerated vegetables. 3: Evaluate the use of biochars to reduce pathogens in manures, compost, and soils used for the production of fresh (both conventional and organic) produce. Approach (from AD-416): A pilot plant-scale radio frequency pasteurization (RFP) unit will be developed, capable of continuously processing multiple shell eggs. Initial efforts will use a 60 MHz RFP unit similar to the unit used to write the ARS patent. The single-egg RFP unit is capable of pasteurizing shell eggs with significantly better quality than industry eggs (currently pasteurized using hot water immersion). RFP operating parameters will be optimized, while experimental factors to be investigated will include cooling water flow rate, cooling water conductivity, cooling water temperature, and amount and duration of RF power applied. Equally important for reducing pasteurization operating costs is reducing equipment costs. To this end, we will study egg roller minimum rotation speed, and feasibility of adjusting frequency to 40.68 MHz, which is within the radio band internationally reserved for industrial, scientific and medical purposes. Optimized RF operating and equipment costs will be estimated. Quality and functionality characteristics of RFP eggs will be evaluated. The RFP process will be scaled up by developing RF power supplies, matching networks, and power distribution schemes to evenly heat hundreds of egg simultaneously. Finally, a continuous RFP pilot plant unit will be designed and assembled, which will convey eggs through the unit. To reduce microorganisms on fresh and fresh-cut vegetables, several innovative technologies will be researched. The ability of novel washes, developed during the previous project cycle, to remove pathogenic biofilms will be investigated. Bacterial cell surface charges will be determined using hydrophobic and electrostatic interaction chromatography. Also, the occurrence of sublethal injury to pathogens, following treatment with the produce wash, will be determined. The previously-developed antimicrobial wash will be improved with additional ingredients and pH adjustment. Wet steam technology has been successfully applied to cantaloupes, and will be extended to other produce. Finally, pilot plant scale testing of the produce intervention technologies will be conducted and costs of applying them estimated. In order to evaluate the use of biochars to reduce pathogens in manures, compost, and soils, non-pathogenic bacteria will be validated as surrogates for pathogenic bacteria in soil and manure survival studies with biochar. Antimicrobial efficacy of biochar will be optimized by adjusting production time and temperature as well as by comparing various biofeedstocks. The optimized biochar will be evaluated to determine its potential to inactivate surrogate bacteria in compost, in lab and greenhouse settings as well as in scaled-up field experiments. Cost estimates for applying lethal doses of the optimized biochar to compost and fields will be determined. Progress was made on all objectives, all of which fall under National Program 108 � Food Safety, Component 1 � Foodborne Contaminants, Problem Statement 5, Intervention and Control Strategies. Progress on scaling up the radio frequency pasteurization (RFP) technology continued. The ARS patented RFP technology inactivates 99.999% of Salmonella in shell eggs in 1/3 the time of the current commercial process. Assembly of the 4-egg RFP unit was completed. Testing confirmed that the unit can uniformly pasteurize 4 eggs simultaneously. In addition, a 16-egg RFP unit is being assembled in collaboration with a CRADA industry partner. The rapidity of RFP results in eggs that retain their fresh-like appearance and significantly more functionality, especially of the heat sensitive egg whites. Angel food cakes, containing egg whites, made from RFP eggs had 40% more volume than those made from egg pasteurized using the commercially available process. In produce-related research, the surface charge and hydrophobicity of strains of Salmonella, Escherichia coli and Listeria monocytogenes were determined using electrostatic and hydrophobic interaction chromatography. Initial bacterial attachment was highest for individual strains of E. coli and lowest for L. monocytogenes, but Salmonella exhibited the strongest attachment during storage. A new solution aimed at reducing browning of fresh-cut apple pieces and, at the same time, reducing microbial populations was developed by combining specific short chain organic acids generally regarded as safe (GRAS). An invention disclosure for this novel antibrowning-antimicrobial solution has been filed. Also, flash steam technology was developed and used to inactivate bacteria on fruit surfaces. The flash steam treatments of whole cantaloupe surfaces before fresh-cut preparation reduced microbial populations on melon whole surface and transfer to fresh-cut pieces. Treatments also reduced biofilms on the surfaces of treated melons. The flash steam technology, as well as the RFP of shell eggs, were featured in AgResearch Magazine, as well as many other news outlets. Regarding research on soil amendment for the safer production of produce, a study was conducted to evaluate survival of 22 strains of avirulent E. coli and Salmonella in crop soil with 10% fast-pyrolysis switchgrass biochar. The goal was to validate effective surrogate strains of bacteria to use in future studies, as there is danger in conducting experiments with pathogenic (virulent) strains, which could lead to illness through dust aerosolization and infection of laboratory workers. Twenty-two soil samples were amended with 10% biochar and inoculated with 5 strains of non-toxigenic E. coli O157:H7, ten strains of non-pathogenic E. coli, and seven attenuated vaccine strains of Salmonella, respectively. Samples were held 9 weeks. By 7 days, two salmonellae and four E. coli were undetectable. Eight strains were undetectable by week 3. Twelve strains were undetectable by week 7, including all non-toxigenic O157:H7. At 9 weeks of storage, only seven of the twenty-two strains were still detectable. These results identified suitable surrogate bacteria, to be used in the place of virulent pathogenic strains, for use in future biochar-soil decontamination studies. This now provides researchers validated bacterial surrogates to use in biochar/soil studies which should significantly increase worker safety. Accomplishments 01 Pasteurizing multiple eggs using radio frequency. Raw shell eggs can be contaminated with Salmonella, posing an ongoing health risk. The ARS- patented radio frequency pasteurization (RFP) process produced safer eggs with exceptional quality, treating one egg at a time in the lab- scale prototype. ARS researchers at Wyndmoor, Pennsylvania, in collaboration with a CRADA industry partner, assembled and tested a RFP unit that can simultaneously pasteurize four eggs, thus paving the way for larger, commercial scale, RFP units. This technology has the potential to address a significant, widespread source of foodborne illness and make shell eggs safer for the American consumer to handle and consume. 02 New method kills food-poisoning bacteria. Cantaloupe rind surfaces are sometimes contaminated with Listeria monocytogenes, Salmonella or Escherichia coli O157:H7 bacteria. A novel antimicrobial solution was developed that kills these pathogenic bacteria and reduces their populations to below detectable levels on fresh-cut pieces of melons. ARS researchers at Wyndmoor, Pennsylvania licensed this antimicrobial solution to an industry partner and an invention disclosure was filed. Produce washed with the antimicrobial solution will be safer.

Impacts
(N/A)

Publications

Ukuku, D.O., Geveke, D.J., Chau, L.I., Bigley, A., Niemira, B.A. 2017. Appearance and overall acceptability of fresh-cut cantaloupe pieces from whole melon treated with wet steam process. LWT - Food Science and Technology. doi: 10.1016/j.lwt.2017.04.033.
Mukhopadhyay, S., Ukuku, D.O., Juneja, V.K., Ramaswamy, R. 2016. Impact of high-pressure processing on the microbial ecology of foods. In: de Souza Sant'Ana, A. (Ed). Quantitative Microbiology in Food Processing: Modeling the Microbial Ecology, First Edition. Chichester, West Sussex, UK. Wiley- Blackwell Publisher. p.194-216.
Mukhopadhyay, S., Sokorai, K.J., Ukuku, D.O., Fan, X., Juneja, V.K., Sites, J.E., Cassidy, J.M. 2016. Inactivation of Salmonella enterica and Listeria monocytogenes in cantaloupe puree by high hydrostatic pressure with/without added ascorbic acid. International Journal of Food Microbiology. 235:77-84. doi: 10.1016/j.ijfoodmicro.2016.07.007.
Jin, Z.T., Yu, Y., Gurtler, J. 2016. Effects of pulsed electrical field processing on microbial survival, quality change and nutritional characteristics of blueberries. LWT - Food Science and Technology. 77:517- 524. doi: 10.1016/j.lwt.2016.12.009.
Lacombe, A.C., Niemira, B.A., Gurtler, J., Sites, J.E., Boyd, G., Kingsley, D.H., Li, X., Chen, H. 2017. Nonthermal inactivation of norovirus surrogates on blueberries using atmospheric cold plasma. Food Microbiology. 63:1-5.
Lacombe, A.C., Beard, A., Hwang, C., Hill, D., Fan, X., Huang, L., Yoo, B. K., Niemira, B.A., Gurtler, J., Wu, V.C. 2016. Inactivation of Toxoplasma gondii on blueberries using low dose irradiation without affecting quality. Food Control. 73(2017):981-985.
Geveke, D.J., Bigley, A.B., Brunckhorst, C. 2017. Pasteurization of shell eggs using radio frequency heating. Journal of Food Engineering. 193:53-57.
Singh, A., Geveke, D.J., Yadav, M.P. 2016. Improvement of rheological, thermal and functional properties of tapioca starch using gum arabic. LWT - Food Science and Technology. 80:155-162.
Jiang, Y., Fan, X., Li, X., Gurtler, J., Mukhopadhyay, S., Jin, Z.T. 2016. Inactivation of Salmonella Typhimurium and quality preservation of cherry tomatoes by in-package aerosolization of antimicrobials. Food Control. doi: 10.1016/j.foodcont.2016.08.031.

Progress 10/01/15 to 09/30/16

Outputs
Progress Report Objectives (from AD-416): 1: Further studies on the ARS-patented use of RFP for shell eggs through the development of pilot plant and commercial prototypes of continuous RFP equipment for multiple eggs. 2: Further studies on the use of innovative technologies to reduce microorganisms on fresh produce, and minimally preserved, brined, and fresh-cut refrigerated vegetables. 3: Evaluate the use of biochars to reduce pathogens in manures, compost, and soils used for the production of fresh (both conventional and organic) produce. Approach (from AD-416): A pilot plant-scale radio frequency pasteurization (RFP) unit will be developed, capable of continuously processing multiple shell eggs. Initial efforts will use a 60 MHz RFP unit similar to the unit used to write the ARS patent. The single-egg RFP unit is capable of pasteurizing shell eggs with significantly better quality than industry eggs (currently pasteurized using hot water immersion). RFP operating parameters will be optimized, while experimental factors to be investigated will include cooling water flow rate, cooling water conductivity, cooling water temperature, and amount and duration of RF power applied. Equally important for reducing pasteurization operating costs is reducing equipment costs. To this end, we will study egg roller minimum rotation speed, and feasibility of adjusting frequency to 40.68 MHz, which is within the radio band internationally reserved for industrial, scientific and medical purposes. Optimized RF operating and equipment costs will be estimated. Quality and functionality characteristics of RFP eggs will be evaluated. The RFP process will be scaled up by developing RF power supplies, matching networks, and power distribution schemes to evenly heat hundreds of egg simultaneously. Finally, a continuous RFP pilot plant unit will be designed and assembled, which will convey eggs through the unit. To reduce microorganisms on fresh and fresh-cut vegetables, several innovative technologies will be researched. The ability of novel washes, developed during the previous project cycle, to remove pathogenic biofilms will be investigated. Bacterial cell surface charges will be determined using hydrophobic and electrostatic interaction chromatography. Also, the occurrence of sublethal injury to pathogens, following treatment with the produce wash, will be determined. The previously-developed antimicrobial wash will be improved with additional ingredients and pH adjustment. Wet steam technology has been successfully applied to cantaloupes, and will be extended to other produce. Finally, pilot plant scale testing of the produce intervention technologies will be conducted and costs of applying them estimated. In order to evaluate the use of biochars to reduce pathogens in manures, compost, and soils, non-pathogenic bacteria will be validated as surrogates for pathogenic bacteria in soil and manure survival studies with biochar. Antimicrobial efficacy of biochar will be optimized by adjusting production time and temperature as well as by comparing various biofeedstocks. The optimized biochar will be evaluated to determine its potential to inactivate surrogate bacteria in compost, in lab and greenhouse settings as well as in scaled-up field experiments. Cost estimates for applying lethal doses of the optimized biochar to compost and fields will be determined. This new Project Plan was recently certified through the ARS Office of Scientific Quality Review (OSQR). For further details on current work see the 2016 Annual Report for project 8072-41420-017-00D.

Impacts
(N/A)

Publications