Umass Food Science EXTENSION RESEARCH

SANITATION AND VALIDATION OF PRODUCE RINSE WATER TO ENHANCE FOOD SAFETY

Monitoring Equipment Chemical Test Kits Chemical Test Strips Portable ORP and pH Meter Considerations for Farms Accuracy Sensitivity Time and Patience of handler www.thermoscientific.com www.sensafe.com Google image

Research Objectives Evaluate the use of portable Oxidation Reduction Potential (ORP) as an indicator of antimicrobial efficacy and as a rapid test method for sanitation management. Assess Free Chlorine test strips as a rapid test method for postharvest sanitation. Assess Free Chlorine efficacy on reduction of pathogenic Escherichia coli O157:H7 ATCC 43895 suspended in various turbid wash solutions MA Produce: Cucumbers Next is the Methods…And …Materials Google image

Methods and Materials

Postharvest Process Water Preparation Synthetic postharvest wash process water was designed to reproduce and simulate small commercial MA farm conditions. Organic matter: Soil and Cucumber Suspended Solids were diluted with ultrapure water to certain turbidities Five Autoclaved Process Water solutions: Clear water: Control Soil in water Cucumber Suspended Solids “SS” in water Soil and Cucumber SS in water Organic Challenge water Magnone, Natick Labs 2012 0 ntu 25 ntu 50ntu CW Portable HACH 2100Q Turbidimeter

Inoculum and Sanitizer Efficacy Procedure Autoclaved Wash Solutions 100 mL Clean H2O (0 NTU) Soil + H2O (25, 50 NTU) Cucumber + H2O (25, 50 NTU) Cucumber + Soil (25, 50 NTU) Organic Challenge Water 2.5 g/L TOC Tannic acid & 2.5 g/L TDS Sodium chloride (5 NTU) (Exposed for 2 minutes) Sanitizer Chlorine Treatments 0 ppm NaClO 50 ppm NaClO 200ppm NaClO Validate Free Chlorine Levels E. Coli O157:H7 Inoculation 106-107 CFU/mL (Exposed for 2 minutes) Validate Free Chlorine Levels Add 100 μl Sodium thiosulfate (1N Na2S2O3) for neutralization Validate Neutralization Serial Dilution and Spread Plate onto TSA Incubation for 24 hours at 37°C Enumeration

Physicochemical Analysis All physicochemical analysis was conducted according to standard protocols and within manufacturer’s instructions Organic Challenge Water 2.5 g/L TOC Tannic acid & 2.5 g/L TDS Sodium chloride (5 NTU) Clean H2O (0 NTU) Soil + H2O (25, 50 NTU) Cucumber + H2O (25, 50 NTU) Cucumber + Soil (25, 50 NTU) Sanitizer Chlorine Treatments 0 ppm NaClO 50 ppm NaClO 200ppm NaClO Record initial ORP (mV) after adding Bleach. Record ORP (mV) every 30 sec for 8 mins ORP Analysis ORP#1 ORP#2 Sample solutions were prepared in parallel for microbial and physicochemical analysis. Varying types of simulated wash solutions were prepared at varying organic loads to evaluate the QA tools ag water interferences. Figure 2. Eutech Instruments OAKTON Handheld ORPTestr 10 Figure 1. Thermo Scientific Orion Star A221 Portable ORP Meter Free Chlorine Analysis

Physicochemical Analysis ORP Analysis ORP#1 ORP#2 Perform Free Chlorine test strip analysis immediately after ORP. Within 2 mins. for max Chlorine exposure of 10 mins. Free Chlorine Analysis Standard: Free Chlorine compared to HACH DPD colorimetric measurements “Fr1” WaterWorks Free Chlorine Check Ultra High: #480024 Range: 0, 25, 50, 100, 200, 300, 400, 500, 750 ppm “Fr2” HF Scientific Free Chlorine MicroCheck: # 09940 Range: 0, 0.05, 0.2, 0.4, 0.6, 0.8, 1.2, 1.5, 2.0, 2.6, 4.0, 6.0,10 ppm “Fr3” LaMotte Chlorine Test Papers: #4250-BJ Range: 0, 10, 50, 100, 200 ppm “Fr1” “Fr2” “Fr3”

Results

Comparison of ORP Measurements with Reduction of E.coli O157:H7 by Chlorine Bleach in Different Turbid Organic Matter Wash Solutions Wash Solutions Turbidity (NTU) NaClO added (ppm) Residual Free Cl (HACH ppm) ORP# 1 ORP# 2 E.coli O157:H7 Target: 650-800 mV Clean H2O Control: 0 NTU 444.6 455 + 0 NTU 50 680.7 701 - Soil + H2O Control: 50 NTU 475.3 457 25 NTU 25 693.0 700 50 NTU 718.8 717 Cucumber + H2O Control:50 NTU 350.0 303 11 834.9 830 1.2 584.3 560 Cucumber + Soil 381.0 370 13 761.6 774 10 833.1 816 Organic Challenge H2O Control:2.5g/L TOC/TDS 220.1 223 2.5 g/L TOC/TDS 209.9 224 200 211.8 210 Results – Type of simulated wash water impacts the organic matter (influences the residual free chlorine) when residual free chlorine is present, its an affective sanitizer Handheld ORP readings of at least 560mV will inactivate E.coli.

Comparison of Free Chlorine Test Strips in Turbid Organic Matter Wash Solutions Wash Solutions (50 NTU) Free Chlorine (ppm) Standard HACH Fr1 Fr2 Fr3 Clean H2O 0 ppm 50 ppm 50 > 10 Soil + H2O 25 ppm 25 Between 10-50 Cucumber + H2O 4 ppm 2.0 10 **10 ppm **< 25 **0.2 **50 Cucumber + Soil 5 ppm < 25 0.6 Organic Challenge H2O The presence of free chlorine will active as an effective sanitizer Presence of high levels of vegetable matter provides inconsistent free cl in some commercial test strips. Standard: Free Chlorine compared to HACH DPD colorimetric measurements “Fr1” WaterWorks Free Chlorine Check Ultra High: #480024 Range: 0, 25, 50, 100, 200, 300, 400, 500, 750 ppm “Fr2” HF Scientific Free Chlorine MicroCheck: # 09940 Range: 0, 0.05, 0.2, 0.4, 0.6, 0.8, 1.2, 1.5, 2.0, 2.6, 4.0, 6.0,10 ppm “Fr3” LaMotte Chlorine Test Papers: #4250-BJ Range: 0, 10, 50, 100, 200 ppm

Cucumber Matter Affects Free Chlorine Strips Readings Wash Solutions Standard HACH Fr1 Fr2 Fr3 Cucumber + H2O 0 ppm 4 ppm 50 2.0 10 **10 ppm **< 25 **0.2 **50 Fr1 Fr2 Fr3 Control 50ppm Free Cl2 Consistent **Fr1 **Fr2 **Fr3 Cucumber + H2O **10ppm Free Cl2 Inconsistent 10ppm <2 5ppm  10ppm ≠0.2ppm 10ppm ≠ 50ppm

Conclusions Presence of residual Free Chlorine reduced E.coli O157:H7 ATCC 43895 suspended in various turbid wash solutions. Vegetative organic matter can impact Free Chlorine Test strips with inconsistent readings. Portable ORP meters gave consistent results in controls and organic matter solutions. ORP may be a good indicator of sanitizer activity if kept within 650-800 mV range. However high turbid vegetation reduces ORP below threshold Free Chlorine Test strips are a cheap fast method of rapid method monitoring. However inconsistent readings will occur in high turbid vegetative process water. ORP correlates well with sanitizer efficacy, but challenging to use as it takes up to 8 minutes for a reading.

UMass Work In progress

Best Practices for Implementing an On-Farm Food Safety Plan Documentation Environmental monitoring River shed On-farm facilities Operators SOP Extension Programming

Microbial Counts from the CT River Average MPN and Average E.coli/ Coliform MPN of all water samples taken from the Connecticut River from June 2014 to October 2014.

Packing house Drain Average APC and Coliform Plate Counts for samples taken from drain grate closest to packing area door from June 2014 to October 2014.

Average APC and Coliform Plate Counts for samples taken from farm workers hands August 2014 to October 2014

Next Steps Develop and implement SOPs Establish training program Continue environmental monitoring Extension Activities On-line materials Workshops for best food safety practices

Validation of Commercial Produce Sanitizers Food Safety Validation Lab analysis Physiochemical Microbiological Organoleptic attributes Operational implementation Dosage calculations Cost per use Extension Dissemination

On Farm Research Needs Diagnostic tools that can support QA on-farm Must Account for Resource Limitations Ease of use: No/little sample preparation No/little technical skills Account for the farming conditions Limited electricity Affordable Rapid i.e. Agricultural water cannot be used unless the issue has been resolved

Natural Antimicrobials for Produce Safety Strategy: Natural Non-thermal highly effective antimicrobial Science Physiochemical emulsion stability Antimicrobial activity Product Development for application Figure 3: Food-grade antimicrobial emulsions can be formed using a simple spontaneous emulsification method food-grade antimicrobial emulsions as a non-thermal, potent, and safe means of combating pathogens associated with fresh and fresh-cut produce

Biosensors for Agricultural Water Figure 1: The bacteriophage will be used to recognize, infect and replicate within Salmonella spp. cells. Once replication is complete, the replicated bacteriophage will burst out of the bacteria. The increase in bacteriophage numbers can then be quantified with a fluorescent dipstick. Nugen, S. Bacteriophage Technology, CPS proposal, 2014.

Phage Amplification + = FDA approved key path