SURVIVAL STRATEGIES OF FOODBORNE PATHOGENS AND COMMODITY CONTAMINATION IN PRODUCTION FIELDS AND RETAIL OUTLETS.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1007407
• Project No.: NJ10170
• Project Start Date: Oct 1, 2015
• Project End Date: Sep 30, 2020

Project Director
Matthews, KA.

Recipient Organization
RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY
3 RUTGERS PLZA
NEW BRUNSWICK,NJ 08901-8559

Performing Department
Food Science

Non Technical Summary
Most of the major supermarkets in the United States crisp and mist fresh vegetables or at minimum mist vegetables in display cases. Crisping generally entails soaking of a commodity in water to rehydrate and misting is done on commodities while in display cabinets to limit wilting and improve appearance. During crisping product may become contaminated through exposure to contaminated equipment, workers, and other product. Misting of product may have unintended consequences if for example the lines delivering water develop biofilms harboring human pathogens. In the present study, formation of biofilms in mist water systems and its effect on microbial load of misted/crisped product is investigated. Experiments are also conducted to evaluate the efficacy of various sanitizers to reduce or eliminate cross-contamination during crisping. The shelf-life of crisped and misted product in retail and home setting is determined. Collectively, crisping and misting may contribute to improving product quality and shelf-life, but may also increase the likelihood of product and equipment contamination. Potential also exist for increase in harmful chemical by-products. The research will support the development of science based "best practices" to ensure that crisping, misting, or both do not adversely impact the microbial and chemical safety of fresh produce.Bacteria have the ability to adapt to varying environments which has facilitated their existence for millions/billions of years. From a practical food safety and shelf-life position recognizing and understanding how bacteria survive is essential. Studies conducted in the open-environment and in greenhouses suggests that enteric bacterial population of ≤104 CFU survive for short periods; not detectable on crops by culture or molecular methods. Companies conducting monitoring and test and hold programs indicate that enrichment of samples (water, soil, commodity) is required to detect pathogens of interest. Thus, it would seem that consumers suffer cases of foodborne illness from consuming produce that is apparently contaminated with extremely low levels of the causative agent. This may or may not be the case. Enteric foodborne pathogens may be at much greater levels but in dormant states that impede detection and facilitate contamination. In the present project, experiments that focus on ability of bacteria to undergo desiccation and entry in to a viable but-nonculturable state (VBNC) and subsequent contamination of or recovery on leafy greens is investigated. E. coli O157:H7 is exposed to water, soil, and chicken manure pellets and desiccated or VBNC state determined. Assessment of crop contamination and resuscitation on crops is investigated.

Animal Health Component

0%

Research Effort Categories

Basic

40%

Applied

40%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	1430	1100	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
1430 - Greens and leafy vegetables;

Field Of Science
1100 - Bacteriology;

Keywords

foodborne pathogens

viable but nonculturable

crisping

misting

e. coli o157:h7

Goals / Objectives
Objectives: Establish conditions that influence entry of E. coli O157:H7 into a VBNC state prior to interaction with a crop.Assess whether VBNC E. coli O157:H7 recover or survive on leafy greens.Assess change in microbial population of crisped and misted products and shelf-life (retail/in-home).Define cross-contamination during crisping focusing on E. coli O157:H7, Salmonella and Listeria monocytogenes and utility of various sanitation processes in preventing cross-contamination.

Project Methods
Objective 1.VBNC: Induction in water.A cocktail of four E. coli O157:H7 strains composed of clinical and produce associated strains is used in all experiments Bacteria are exposed to conditions that will induce VBNC state and then leafy greens inoculated with the cells and behavior on growing crops or harvested commodities evaluated. Irrigation water (collected from farm well, pond, etc) is inoculated with E. coli O157:H7 cocktail to achieve 108 CFU/ml. Water is sampled at predetermined intervals (e.g., every 3d) and change in culturable cell concentration determined by plating. When plating of 10 ml (concentrated to 100 µl) of sample results in no colony formation then differential staining is used to determine bacterial viability (Li et al., 2003).Control: E. coli O157:H7 suspended in TSB is subjected to same treatment.Bacterial viability and recovery of VBNC cells. Differential staining (Live/Dead Baclight Bacterial viability assay) is used to determine population of cells that are VBNC. To determine whether VBNC cells can recover samples are plated on media for recovery of VBNC cells according to Kolling and Matthews (2001). Alternative, methods for detection of VBNC cells are used if required (Dreux et al., 2007).Objective 2.Bacterial viability and recovery of VBNC are determined using several methods. See objective 1.Lettuce and Spinach Plants: Seeds are purchased and plants grown. Age of plants in this study refers to time after emergence.Bacterial challenge of plants: Reminder: Bacteria cultured under ideal conditions will serve as "controls" and are used in all experiments. This will provide guidance on persistence and survival of E. coli O157:H7 and a comparison for VBNC E. coli O157:H7.Microbiological analysis. Weighed plant samples are placed in a sterile stomacher bag with 0.1% peptone water (1:10, w/v), macerated by hands, and stomached for 3 min. The bacterial populations calculated and reported as log CFU per g fresh weight of plants/head.Enumeration of VBNC cells. Samples from plants treated with VBNC cells are plated on media for recovery of VBNC cells according to Kolling and Matthews (2001).Statistical Analysis: The effects of factors are tested by ANOVA and the differences in frequencies of positive samples by a Chi-square test levelofsignificance set at P<0.05, using the SPSS software version 19.0 (SPSS Inc, Chicago, IL).Post-harvest recoveryPlants inoculated for the above pre-harvest experiments are used in these experiments. The harvested leafy greens are placed in open top plastic containers, clamshell containers, and film bags. They are placed in refrigerator set to 3oC and 95% humidity. Temperature and humidity are monitored. Product is sampled at 1, 3, 5, 7, 10, 15, and 20 d of storage. The 20d point is to represent product typically considered worst case scenario as product is at spoilage point.A set of experiments are conducted in which product is harvested, cut, washed in 30 ppm chlorine water and air dried. The packaged products are placed in refrigerator set to 3oC and 95% humidity. Temperature and humidity are monitored. Product is sampled at 1, 3, 5, 7, 10, 15, and 20 d of storage. The 20d point is to represent product typically considered past use by date or worst case scenario.Microbiological analysis. A composite 25 g sample is processed for microbiological analysis and reported as log CFU per g fresh weight of plants. Enumeration of VBNC cells. Samples from plants treated with VBNC cells are plated on media for recovery of VBNC cells according to Kolling and Matthews (2001). Homogenates of plant samples treated with VBNC are processed for differential staining.Objective 3: Product contaminated with VBNC E. coli O157:H7 as outlined above will also be used in these experiments. Methods for detection are as described above. As such specific reference to VBNC cells will not be indicated under this objective.Crisping: A thin slice of the butt-end of the product (e.g., lettuce) is removed. Product (2.5 Kg) is placed into a stainless steel sink filled with 76 L of water (control) or water plus chlorine (50 ppm). An additional sanitizer (e.g., PAA) may be evaluated if time and resources permit. The product is immersed for 5 min, removed from the water, placed on crisping trays, and then onto crisping tower. So, Treatments include: control (water only) and chlorine (50 ppm). Samples are collected for microbiological analysis of leafy greens to provide baseline bacterial populations for misting experiments. Microbiology of Chill Cabinets: A pre-determined list of chill cabinet sampling sites is used to ensure consistency between experiments.Microbiology of Misted Produce: A commercial chill display cabinet fitted with a commercial misting system will be used for all experiments. Experiments are designed to determine change in microbial load on selected commodities based on days of storage in chill cabinets and home refrigerator. Product is held in a chill cabinet for up to 2 d and then analyzed for microbial load and chlorine by-products or placed into a refrigerator (simulating consumer storage conditions) and held for 5d. Microbial analysis of home refrigerated stored commodity is sampled at 1, 3 and 5 d of storage.Treatments: Sanitizer - chlorine; Water alone (control). In both instances water temperature is controlled so that water temperature does not become a variable.Sample Processing: A minimum of three units (e.g., three heads of lettuce) at each sampling time are processed for microbiological analysis.Aerobic Mesophilic and Psychrophilic Total aerobic bacteria counts based on (BAM, Chapter 3; Maturin & Peeler, 2001): For the mesophilic plate counts, plates are incubated at 37°C for 24 h and for the psychrophilic plate counts, plates are stored at 7°C for 5 d and the number of colonies recordedYeast and Mold Count (BAM, Chapter 18; Tournas, Stack, Mislivec, Koch, & Bandler, 2001): Following the methods described in the BAM for determination of yeasts and molds; the sample processed as above. Enumeration of Coliforms and E. coli : Total coliforms and total E. coli are enumerated using 3M Petrifilm Coliform Count Plates based on manufacturers procedures.Chlorine Residues on Product: Product is analyzed for chlorite residues according to the method of Tsai et al. (2001) or by Rutgers Analytical services, a fee for services facility.Statistical Analysis: Allstatistical analyses are conducted using the SPSS software version 19.0 (SPSS Inc, Chicago, IL).Objective 4: Commodities: Romaine lettuce, Red leaf lettuce, and cilantro.Sanitizers: Commercial products: Based on collaborator input. Chlorine is included in addition to other products. In total two sanitizers are evaluated, one being chlorine based.Bacteria: Cocktails of four strains of Listeria monocytogenes or Salmonella are used. E. coli O157:H7 as described under objective 1 are used. VBNC E. coli O157:H7 induced in water are included.Inoculation of Product: Product is inoculated through immersion or spot methods to achieve approximately 105 CFU/g.Crisping: The protocol for crisping is as described above, but product is processed for microbiology prior to placing in the cooler or chill cabinet.A single experiment will include: no-crisping, water alone, 50 ppm chlorine, and sanitizer X. It is necessary to include all variables in a single experiment since natural microbiotia of the commodity can vary by 1 to 3 log CFU/g and this can substantially influence outcome.Crisping Water Testing: Water (six 100 ml samples) are collected and processed for microbiological analysis.Microbiological Testing of product: A 25 g sample of product is prepared as described under previous objectives.Statistical Analysis: Allstatistical analyses will be conducted using the SPSS software version 19.0 (SPSS Inc, Chicago, IL).

Progress 10/01/15 to 09/30/20

Outputs
Target Audience:Retail processors of fresh fruits and vegetables including supermarkets and farm stands would benefit from the water antimicrobial research. Research directed at photosynthesizers curcumin would have greatest value to meat and poultry processors. The research will likely be of interest to agencies developing guidance on the non-commercial washing and crisping of vegetables. Changes/Problems:As indicated earlier, the bulk of research was devoted to Objectives 3 and 4. Research associated with these objectives garnered the greatest interest from stakeholders and is why they became the primary focus. Experiments were added to address questions presented by stakeholders. The net result was a very robust project that provided significant benefit to stakeholders. What opportunities for training and professional development has the project provided?During the full project duration, 11 undergraduate students, five graduate students and two visiting scientists contributed directly or indirectly to the project. Development of communication, public speaking, and leadership skills were promoted. Growth in development of critical thinking skills was achieved by all. All researchers involved participating in writing peer-reviewed papers, grant proposals, and other materials designed to disseminate results of the research. How have the results been disseminated to communities of interest?Results have been presented both orally and by poster at greater than 20 national and international meetings over the full duration. Research papers and abstracts have been published. Meetings with industry representatives were set-up to convey detailed information about results. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During the final year of the project, two main areas had reportable output: Photodynamic treatment is a nonthermal disinfection technology, which uses photosensitizers under specific light conditions to inactivate microorganisms. Curcumin, a component of turmeric and with many medicinal properties, has been used as food additive in many countries. In the study, curcumin was used as a photosensitizer. The results showed that photodynamic inactivation effects relied on curcumin concentration, co-incubation time and illumination time. Curcumin photodynamic technology is expected to show great potential application for the inactivation of microbials on the surface of food equipment and fresh food products. Fresh produce shall be thoroughly washed at the retail level prior to serving to the consumer with potable water. Foodborne pathogens if present on a product may transfer to the wash water and cross-contaminate other products immersed in the water. Typically, an entire carton of lettuce (24 heads) is washed together increasing the likelihood of cross-contamination due to the close contact between each head. This study aimed to compare the effects of two wash batch volumes - "low" (8 heads) and "high" (24 heads) on the efficacy of two commercial water antimicrobials and cross-contamination. Red leaf lettuce was spot-inoculated (~5.0 log CFU/g) with Shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes. In the first batch of washing, inoculated lettuce was washed with non-inoculated lettuce, followed by reuse of the water/antimicrobials twice washing only non-inoculated lettuce. Samples of inoculated and non-inoculated lettuce were collected to determine aerobic plate count (APC) as well as the populations of STEC and L. monocytogenes. Microbiological analysis of the wash water was also conducted. Wash volume (8 versus 24 lettuce heads) had limited effects on the antimicrobial activities of the interventions evaluated. Instead, high-volume wash increased the rate of cross-contamination between non-inoculated and inoculated lettuce, and cross-contamination of non-inoculated lettuce through wash water reuse. The conclusion is that retailers should consider volume of product processed per batch, reuse of wash water, and use of an appropriate water antimicrobial in consideration of mitigating potential product cross-contamination. Over the full project period, the project focused most on addressing objectives 3 and 4. The research was deemed very applicable and feedback from stakeholders led to additional studies of value to the retail produce industry. Moreover, research on alternatives to well used water antimicrobials including peracetic acid and chlorine was aggressively pursued. Ultimately, the project resulted in several peer-reviewed publications and research data used in proposals submitted to USDA NIFA grant programs. Projects were not funded, but reviewers' comments led to new avenues of research. The research on photosensitizers was initially conducted with application toward fresh fruits and vegetables. Through discussion with researchers in the field, change in commodity focus from fresh fruits and vegetables to meat and poultry was made. Significant progress has been made in development of a light system couple with a photosensitizer that can be evaluated in poultry processing. Collaboration with the University of Alabama was initiated since the research group there has access to a poultry pilot plant processing facility. The intent is to develop technology that can be used by poultry and beef processors to control spoilage and foodborne pathogens.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Gao, J., H. Jang, L. Huang, and K. R. Matthews. 2020. Influence of product volume on water antimicrobial efficacy and cross-contamination during retail batch washing of lettuce. International Journal of Food Microbiology. https://doi.org/10.1016/j.ijfoodmicro.2020.108593
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Li, T., Y. Zhao, K. Matthews, J. Gao, J. Hao, S. Wang, J. Han, Y. Ji 2020. Antibacterial activity against Staphylococcus aureus of curcumin-loaded chitosan spray coupled with photodynamic treatment. LWT  Food Science and Technology. https://doi.org/10.1016/j.lwt.2020.110073

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

Outputs
Target Audience:The research has been particularly informative for retail food establishments. The manufacturers of antimicrobials used to treat water in retail environments are also following the research. The research is being used by the Conference on Food Protection to address issues associated with the use of antimicrobials in wash water. The research has been presented at international professional scientific meetings. A new research area has emerged in the area of photosensitizers that have potential application on fruits and vegetables and poultry. This opens the target audience beyond the retail level and into commercial processing. Changes/Problems:The specific direction of the research has not changed. A new component has been added, investigating the use of a novel photosensitizer to control microbial populations on fruits and vegetables. What opportunities for training and professional development has the project provided?The project has facilitated the training of two undergraduate students, three graduate students and two visiting scientists. Moreover, the undergraduate students used the project as a base to receive an Aresty Undergradaute Research Fellowship through Rutgers University. The research was also presented at local and international research conferences including the International Association of Food Protection conference. How have the results been disseminated to communities of interest?Results of the research have been used in academic courses taught by the PI, in continuing education courses taught by the PI, and presented at national and international professional meetings. The research has also been published in peer reveived journals. What do you plan to do during the next reporting period to accomplish the goals?Several of the research studies are nearing conclusion. The final sets of data will be analyzed and decisions made whether any of the studies (experiments) require repeating. Manuscripts will be written and submitted for publication in peer-reviewed journals. Students working toward MS and Ph.D. degrees will complete writing of thesis and dissertation.

Impacts
What was accomplished under these goals? Completed research clearly demonstrated the importance of using a water antimicrobial during processing of fresh fruits and vegetables under retail or small process operation (e.g., farm stand). Interestingly, the act of misting actually had a negative influence on the survial of Listeria monocytogenes. However, misting has no impact on survival and gorwth of commensal bacteria. Another project demonstrated that processing of a large batch of a commodity (cantaloupe, lettuce, etc) in a small vessel (e.g., sink) increases the likelihood of cross-contamination even in the presence of a water antimicrobial. A new direction of this research is invesigating novel photosensitizer compounds to prevent cross--contamination. Although poultry is seldom consumed raw, it carries a high safety risk as it provides optimum conditions for bacterial growth: high water activity, near neutral pH, and abundant nutrients. These conditions increase the survival and growth of bacteria, perhaps exacerbating cross-contamination. Chlorine and acids are the two most common chemical interventions to decontaminate the surfaces of poultry carcasses. However, high concentrations of chlorine and acids may result in off-flavor, discoloration, equipment corrosion, and other problems. A recent collaborative study demonstrated that water-soluble photosensitizer curcumin (PSC) inactivated L. monocytogenes and Salmonella in liquid media and on chicken skin. Under the experimental conditions in this study, incubation time and light dose did not influence the antimicrobial activity of PSC, suggesting that photoinactivation can be achieved in a short time.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Gao, J., and K.R. Matthews. 2019. Effects of the Photosensitizer Curcumin in Inactivating Foodborne Pathogens on Chicken Skin. Food Control. 109:https://doi.org/10.1016/j.foodcont.2019.106959.

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

Outputs
Target Audience:The research has been particularly informative for retail food establishments. The manufacturers of antimicrobials used to treat water in retail environments are also closely following the research Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A team of three undergraduates worked on projects related to completion of objectives 3 and 4. They were supervised by two graduate students while in the laboratory. The graduate students attended both local and international meetings in which they presented the research. This also provided an opportunity to interact with other students and professionals in the field of microbial food safety. How have the results been disseminated to communities of interest?The research was used by the Conference on Food Protection to address issues associated with the use of water antimicrobials in water for the washing and handling of fresh and fresh-cut produce. The research has been presented at international professional scientific meetings and at a food safety and food security course held at Central University of Haryana, India. The research has been presented in oral and poster format at two local and three international scientific meetings. Two peer reviewed papers have been published. The research was also used in the teaching of a short-course at the Central University of Haryana. What do you plan to do during the next reporting period to accomplish the goals?Additional studies will be conducted to evaluate efficacy of various water antimicrobials to impact safety and shelf life of leafy greens and other commodities including apples. Experiments will be developed to address small scale processing as practiced at farm stands, retail outlets, and in the home.

Impacts
What was accomplished under these goals? For this period, research was centered on addressing objectives 3 and 4. The efficacy of various water antimicrobials in reducing MS2 bacteriophage (serving as a surrogate for human norovirus) on the surface of strawberries was investigated. The water antimicrobial served two purposes, the reduction of MS2 on strawberries and the control of cross-contamination. The study was also conducted with a cocktail of Shiga-toxin producing Escherichia coli. The shelf-life of fresh produce was evaluated following exposure through immersion to various water antimicrobials. In short, commensal bacteria populations quickly recovered and several days post-exposure populations were similar to those on produce receiving no treatment or treatment with water. Evaluation of several "home" or "kitchen" antimicrobials was completed. The studies demonstrated that sodium bicarbonate and sodium chloride have minimal effect on reducing population of commensal bacteria. Foodborne pathogens cannot be effectively removed from fruits and vegetables through washing. Understanding the behavior of foodborne pathogens on fresh fruits and vegetables will aid in developing strategies to identify and inactivate pathogens of concern when present. Research on bacteria that enter a survival state (viable but non-cultural; VBNC) builds on studies previously completed by the PI demonstrating long-term survival of pathogens in water. For instance, VBNC foodborne pathogens on leafy greens may not be detected using biochemical or molecular typing methods. Therefore, testing using those methods may under estimate the risk associated with a particular lot of lettuce. VBNC bacteria may also be less susceptible to the action of antimicrobial agents.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Jang, H. and K.R. Matthews. 2018. Survival and interaction of Escherichia coli O104:H4 on Arabidopsis thaliana and lettuce (Lactuca sativa) in comparison to E. coli O157:H7: Influence of plant defense response and bacterial capsular polysaccharide. Food Research International. 108:35-41.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Huang, Licheng & Luo, Xin & Gao, Jingwen & R. Matthews, Karl. (2018). Influence of water antimicrobials and storage conditions on inactivating MS2 bacteriophage on strawberries. International Journal of Food Microbiology. 10.1016/j.ijfoodmicro.2018.11.009.

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

Outputs
Target Audience:The research has been particularly informative for retail food establishments. The manufactureres of antimicrobials used to treat water in retail environments are also following the research.The research is being used by the Conference on Food Protection to address issues associated with the use of sanitizer in wash water. The research has been presented at international professional scientific meetings and at a food safety course held at Shanghai Jiao Tung University. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students particpiated in the project fulltime. A visiting scientist form China participated for four months. Two undergraduate students participated for six months. The project provided leadership training, utilization of interpersonal communication skills, and critical thinking skills. How have the results been disseminated to communities of interest?We had multiple peer reviewed publications and researchwas reported at several international meetings and used in "short courses" presented in China and the US. What do you plan to do during the next reporting period to accomplish the goals?Studies will be initiaed addressing virus the cross-contamination of commodities during soak washing andcrisping. Studies will also be conducted to determine diferences in stability of viruses on frozen, refrigerated, and room temperature strawberries.

Impacts
What was accomplished under these goals? Progress remains focused on objectives 3) Assess change in microbial population of crisped and misted products and shelf-life (retail/in-home) and 4) Define cross-contamination during crisping focusing onE. coliO157:H7,SalmonellaandListeria monocytogenesand utility of various sanitation processes in preventing cross-contamination. Summary below relates to accomplishments for both objectives. The practices of washing and crisping have the potential to result in cross-contamination of commodity. A prudent practice would be the addition of a chemical antimicrobial agent in the water to control microbial populations in the water and thereby reduce the likelihood of cross-contamination. In this project, experiments were designed based on retail practices, and commercial equipment used. The water antimicrobial agents Produce Maxx (hypochlorous acid-based electrolyzed water, Chemstar/Sterilox), Suma Eden Antimicrobial Fruits & Vegetable Wash (lactic acid/phosphoric acid-based, Diversey) and Ecolab Antimicrobial Fruits & Vegetable Treatment (AFVT; Sodium dodecylbenzenesulfonate/lactic acid-based, Ecolab) were evaluated. Potable water served as a control. Commodities used in the study included whole head Romaine and red leaf lettuce, cantaloupes, and strawberries. Shiga toxin-producing E. coli (STEC) and L. monocytogenes associated with fresh produce outbreaks were used to inoculated product. The project was specifically designed to evaluate crisping of high volumes of raw agricultural commodities (heads of Romaine lettuce and red leaf lettuce) and soaking of fresh-cut commodities (strawberries and cantaloupe cubes). Parameters evaluated included microbial water quality, cross-contamination, change in microbial population of commodity, contact surface microbiology, and treatment water turbidity and pH. A process "burden" was created by "re-use" of the treatment water; sequentially processing two to three batches of a single commodity. Previous research demonstrated that cross-contamination can occur when a single contaminated head of lettuce is soaked with non-contaminated heads of lettuce. In small volume processing (eights of lettuce per sink) Produce Maxx effectively mitigated cross-contamination and inactivated microorganisms in the processing water. In this phase of the project, studies were conducted to evaluate high volume processing and cross-contamination; a scenario in which soak water was used three times and 24 heads of lettuce (one case) processed each time (soaking for 5 minutes). Produce Maxx was more effective in mitigating cross-contamination with Listeria monocytogenes and STEC of Romaine (2 of 48 heads) and Red leaf (0 of 48 heads) lettuce heads compared to water alone or other water antimicrobials evaluated. Foodborne pathogens (L. monocytogenes and STEC) and native microbiota were not detected in 100 ml samples collected after each consecutive usage of process water containing Produce Maxx. Importantly, the APC (CFU/100 ml treatment water) for the Produce Maxx treatment remained negative after three consecutive uses; the APC increased following each use for water alone and other chemical antimicrobials. Cantaloupes are typically washed prior to cutting and preparation of edible flesh (cutting into pieces or "cubes"). The prepared cubes are then washed and ready for sale to the consumer. In this study, two batches of cantaloupe were washed/soaked, the treatment water replaced and cubes of edible flesh treated. In brief, whole inoculated (three cantaloupes) and non-inoculated (nine cantaloupes) cantaloupes were soaked for 5 minutes in water alone or water with one of the indicated chemical antimicrobials. The soak water was then re-used to process a second batch of 12 non-inoculated cantaloupes. Compared to other treatments Produce Maxx was more effective in preventing cross-contamination of whole cantaloupes with STEC and L. monocytogenes. However, no chemical antimicrobial was effective in preventing cross-contamination of cantaloupe cubes soaked for 90 seconds in treated water. Since the water containing Produce Maxx was negative for L. monocytogenes and STEC, physical contact of cantaloupe cubes prepared from contaminated cantaloupe with cubes from non-contaminated cantaloupe prior to soaking was the likely route of cross-contamination. Strawberries are a high value commodity that when not handled and held under appropriate conditions will rapidly decline in quality. Studies were designed to evaluate benefit of using chemical antimicrobials in treatment water to control microbial load of the water, prevent cross-contamination, and influence product quality during refrigerated storage. Strawberries inoculated with L. monocytogenes or STEC were soaked for 60 seconds in water alone or one of the chemical antimicrobials indicated previously. The strawberries were removed from the soak water, transferred to aluminum trays, and held at 4oC for 3 days. Produce Maxx was more effective at inactivating microorganisms (i.e., product microbiota, STEC, and L. monocytogenes) in soaking water compared to other antimicrobials evaluated. Cross-contamination of L. monocytogenes did not occur, whereas cross-contamination with STEC was noted with water alone and with the product "Antimicrobial Fruits & Vegetable Treatment". Regardless of treatment, aerobic plate count, yeast and mold, and psychrotrophic bacteria counts for strawberries were not significantly different at days 0, 1, and 3 of storage at 4oC. Produce Maxx was more effective in controlling microbial populations on equipment surfaces compared to other treatments.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Jang, H. and K.R. Matthews. 2017. Influence of Surface Polysaccharides of Escherichia coli O157:H7 on Plant Defense Response and Survival of the Human Enteric Pathogen on Arabidopsis thaliana and Lettuce (Lactuca sativa). Food Microbiology. Submitted/under review.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Yangjin, J., H. Jang, M. Guo, J. Gao, and K. R. Matthews. 2017. Sanitizer efficacy in preventing cross-contamination of heads of lettuce during retail crisping. Food Microbiology.64:179-185.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Yangjin, J., J. Gao, H. Jang, M. Guo and K. R. Matthews. 2017. Efficacy of electrolyzed water in preventing cross-contamination during retail preparation of whole and fresh-cut cantaloupe. Food Control. 75:228-235. http://dx.doi.org/10.1016/j.foodcont.2016.12.009

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

Outputs
Target Audience:Individuals in academia, government regulatory agencies, and industry may be interested in the research being conducted. The research is being used by the Conference on Food Protection to address issues associated with the use of sanitizer in wash water. The research has been presented at three professional scientific meeting: Institute of Food Technologists, International Association for Food Protection and The International Union of Food Science and Technology. Dissemination of the research to the international community facilities improving the outcome of the research. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Five graduate students (M.S. and Ph.D. candidates) have particpiated in the project. Four undergraduates have also been involved in the research. This provide leadership training for the graduate students and experitential learning opportunitiy for the undergraduate students. How have the results been disseminated to communities of interest?The research has been presented at three international professional scientifc meetings. What do you plan to do during the next reporting period to accomplish the goals?Crisping studies will be initiated testing additional sanitizing agents and commodities. Experiments focused on misting following crisping will be conducted with Salmonella and E. coli O157:H7. Several papers will be written and submitted for considered of publication in peer reviewed journals. The focus will be completion of objectives related to crisping and misting.

Impacts
What was accomplished under these goals? This first year of research has focused on addressing the goals: "Assess change in microbial population of crisped and misted products and shelf-life (retail/in-home)"; and "Define cross-contamination during crisping focusing on E. coli O157:H7, Salmonella and Listeria monocytogenes and utility of various sanitation processes in preventing cross-contamination." Regarding crisping and cross-contamination, the efficacy of commercially available electrolyzed water concentrate and acid-based sanitizers in preventing cross-contamination during crisping in a retail setting (e.g., supermarkets) was investigated. This study was conducted to provide information regarding mitigation of cross-contamination through the use of sanitizer during crisping at retail outlets. Seven non-inoculated heads and one inoculated head of lettuce were placed into commercial sink filled with tap water(TW), electrolyzed water (EW), lactic acid and phosphoric acid-based sanitizer (LPA), or citric acid-based sanitizer (CA) and soaked for 5 minutes. Two subsequent batches (eight non-inoculated heads per batch) were soaked in the same solution. Soaking with EW significantly reduced the population of Salmonella, E. coli O157:H7 and L. monocytogenes inoculated on Romaine lettuce compared to TW, LPA, and CA. On red leaf lettuce, EW significantly reduced Salmonella and E. coli O157:H7 populations, but not L. monocytogenes compared to other treatments. No significant difference was noted between TW, LPA, and CA in reducing foodborne pathogens or preventing cross-contamination. The impact of crisping and misting on survival and growth of foodborne pathogens has received minimal scientific attention. Review of the literature finds a single thesis project on the subject (Culbertson, 2014); no peer reviewed published papers. In the study conducted by the PIs laboratory the objective was to determine the influence of crisping and misting on growth of L. monocytogenes associated with Romaine lettuce. Inoculation and soaking (crisping) methods were similar to the study described above. Treatments included no soaking, soaking in water alone, soaking in electrolyzed water (50 ppm chlorine). Product was then processed and placed in chill cabinets and misted for 10 sec. every 10 min. with potable water for 24h or placed at 5C and 15C. Samples were processed to determine L. monocytogenes population. Reductions in the population of L. monocytogenes occurred on Romaine lettuce flowing soaking in water alone or electrolyzed water, respectively. The population of L. monocytogenes was approximately 1 log lower on to product held at 5C and 15C following soaking. All heads of Romaine lettuce were transferred to refrigeration units and held at 5C and 15C for 4 to 7d. This process represents home storage under typical and abuse conditions. Romaine lettuce that was misted for 24h following crisping had significantly lower population of L. monocytogenes at day 4 of storage compared to non-misted product. By day 7, population of L. monocytogenes on misted and non-misted products were similar. Temperature abuse had no effect (P>0.05) on the growth of L. monocytogenes. A study was conducted involving the study of fresh-cut melon and cross-contamination. The objective of this study was to evaluate the efficacy of tap water (TW), commercial electrolyzed water (EW), and a commercial acid-based sanitizer (AS) in preventing cross-contamination of cantaloupe during processing in retail settings. A whole cantaloupe was dip-inoculated with a cocktail of Salmonella or L. monocytogenes. One inoculated and two non-inoculated whole cantaloupes were treated in TW, EW, or AS for 5 min. Subsequently, fresh-cut cantaloupe flesh from the inoculated and non-inoculated cantaloupes were soaked together in TW, EW, or AS for 90 seconds. EW treatment resulted in an approximately 1.5 log reduction in both Salmonella and L. monocytogenes on the rind of whole cantaloupe, which was significantly greater than with the TW treatment (P<0.05). Cross-contamination of non-inoculated whole cantaloupes occurred when washed with inoculated whole cantaloupe in TW or AS. Cross-contamination did not occur when whole cantaloupes were washed in EW. Additional washing of mixed fresh-cut cantaloupe flesh from the inoculated and non-inoculated cantaloupes prepared after washing of whole cantaloupes demonstrated that the EW treatment reduced the likelihood of cross-contamination compared with TW and AS. No viable Salmonella or L. monocytogenes were detected from 100 mL sample of EW processing water. The addition of a sanitizing agent to water used for the processing of whole and fresh-cut cantaloupe in a retail setting is recommended to prevent cross-contamination and reduce microbial load. Collectively, these experiments demonstrate the benefit of addition of a chemical sanitizer to water intended for the soaking, crisping, rehydrating of fresh and fresh-cut produce. The research also provides information on the impact that misting has on the population of L. monocytogenes. It is imperative to continue this research investigating fate of viruses in retail crisping solutions and efficacy of additional sanitizers to control cross-contamination.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Jung, Y., J. Gao, H. Jang, and K. R. Matthews. 2016. Sanitizer efficacy in preventing cross-contamination during retail preparation of whole and fresh-cut cantaloupe. IUFoST. Dublin, Ireland.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Jung, Y., M. Guo, J. Gao, K. R. Matthews. 2016. Preventing cross-contamination during retail crisping of leafy greens. IUFoST. Dublin, Ireland.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Jung, Y., J. Gao, H. Jang, and K. R. Matthews. 2016. Prevention of cross-contamination during retail preparation of whole and fresh-cut cantaloupe. In Program book. International Association for Food Protection. Annual meeting. St. Louis, MO.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Jung, Y., M. Guo, J. Gao, K. R. Matthews. 2016. Preventing cross-contamination of cilantro during retail crisping: efficacy of tap-water, commercial acid sanitizers and electrolyzed water. In Program book. International Association for Food Protection. Annual meeting. St. Louis, MO.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Jung, Y., H, Jang, M. Guo, and K. R. Matthews. 2016. Efficacy of electrolyzed water in preventing cross-contamination of lettuce during a crisping. In Program book. International Association for Food Protection. Annual meeting. St. Louis, MO.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Jang, H., and K. Matthews. 2016. Roles of extracellular polysaccharides of Escherichia coli O157:H7 in survival of the enteric pathogen on Arabidopsis and lettuce In Program book. International Association for Food Protection. Annual meeting. St. Louis, MO.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Guo, M., and K.R. Matthews. 2016. Survival and growth of Listeria monocytogenes on Romaine lettuce as influenced by misting and crisping. Institute of Food Technologists. Annual meeting, Chicago, IL.