Microbiological Evaluation of Mechanically- Harvested Citrus Fruit

Microbiological Evaluation of Mechanically- Harvested Citrus Fruit L.M. Friedrich, R.M. Goodrich-Schneider, R.C. Ebel, T.M. Spann, and M.D. Danyluk April 2 nd, 2012 mddanyluk@ufl.edu

Why are we worried about fruit and juice microflora? Sensitivity to food safety risks exists due to: Salmonella outbreaks associated with fresh orange juice in the mid-1990s Recent outbreaks and recalls associated with produce and produce related items (leafy greens, tomatoes, peanut butter) Increased scrutiny, legal actions and media coverage during produce related outbreaks FSMA MH systems that may place fruit in contact with the grove floor need to be explored to quell food safety fears

Objectives Understand the impact of mechanical harvesting on: 1. Fruit surface microflora 2. Corresponding juice microflora Influence of CMNP on citrus microflora during post harvest storage

Objectives Different harvesting systems: OXBO Pick-Up Machine OXBO 3210 pull-behind harvester OXBO 3220 self-propelled continuous canopy shaker with catch frame CMNP Application (OXBO 3210 pull-behind harvester)

Study Objectives Fruit collected: OXBO Pick-Up Machine Hand, ground, Pick-Up (PU) OXBO 3210 pull-behind harvester Hand, ground OXBO 3220 self-propelled continuous canopy shaker Hand, ground, catch frame (CF) CMNP Application Hand, ground; CMNP, No CMNP

Study Objectives Microbiological tests Total Plate Count enumerates most microoranisms Acidophilic Plate count enumerates microorganisms capable of growth in Acid conditions Generic Escherichia coli enrichment identifies if generic E. coli a possible fecal indicator is present Salmonella enrichment identifies if Salmonella is present Alicyclobacillus count enumerates Alicyclobacillus, a juice spoilage organism

Log CFU/fruit Pick-Up Machine Total Plate Count - Fruit 7.0 6.0 5.0 4.0 3.0 2.0 Tree Ground PU 1.0 0.0 05/06-1 05/06-2 05/06-3 05-06 - 4 06/07-1 06/07-2 06/07-3 06/07-4 07-08 - 1 07/08-2 Sample Year and Number Take away message: Differences in location, fruit and weather may be more impactful than harvest method

Pick-Up Machine E. coli - Fruit Trial Tree Ground PU E. coli enrichments 05/06 - all 0/20 0/20 0/20 06/07-1 0 z 3 3 06/07-2 0 0 0 06/07-3 0 1 0 06/07 4 0 0 0 07/08-1 0 0 0 07/08-2 0 0 5 z number of positive tests out of 5 enrichments Take away message: Fecal indicators isolated from ground and PU fruit. These data indicate a potential for cross contamination during use of Pick-Up machine.

Pick-Up Machine Salmonella - Fruit Trial Tree Ground PU Salmonella enrichments 05/06 - all 0/20 0/20 0/20 06/07-1 0 z 0 0 06/07-2 0 1 0 06/07-3 0 0 0 06/07 4 0 0 0 07/08-1 0 0 0 07/08-2 0 0 0 z number of positive tests out of 5 enrichments Take away message: Pathogens and fecal indicators isolated, but not in the same trial, all from fruit that has touched the ground

Log CFU/fruit Oxbo 3220 Catch Frame Total Plate Count - Fruit 7.0 6.0 5.0 4.0 3.0 2.0 Tree Ground CF 1.0 0.0 07/08-1 07/08-2 08/09-1 08/09-2 09/10-1 09/10-2 Sample Year and Number Take away message: Differences in location, fruit and weather may be more impactful than harvest method

Oxbo 3220 Catch Frame E. coli - Fruit Trial Tree Ground CF E. coli enrichments 07/08-1 0 z 1 4 07/08-2 0 0 0 08/09-1 0 0 4 08/09-2 0 0 5 09/10 1 0 0 4 09-10 - 2 0 0 5 z number of positive tests out of 5 enrichments Take away message: Fecal indicators isolated from ground and CF fruit. These data indicate a potential for cross contamination during use a catch frame.

08/09-1 08/09-2 08/09-3 08/09-4 09/10-1 09/10-2 09/10-3 09/10-4 08/09 - CMNP 1 08/09 - CMNP 2 09/10 - CMNP 1 09/10 - CMNP 2 Log CFU/g Oxbo 3210 with and without CMNP Total Plate Count - Fruit 7 6 5 4 3 2 1 Tree Ground 0 Sample Year and Number Take away message: Differences in location, fruit and weather may be more impactful than harvest method

Oxbo 3210 with and without CMNP E. coli - Fruit Trial Tree Ground E. coli enrichments 08/09 1 0 z 0 08/09 2 0 0 08/09 3 0 5 08/09 4 0 0 08/09 CMNP 1 1 1 08/09 CMNP 2 0 0 z number of positive tests out of 5 enrichments Take away message: Rain during 08/09-3 may have lead to high ground E. coli levels

Other counts APC fruit Acidophilic counts showed similar results to Total plate counts No trends could be related to harvest system Alicyclobacillus fruit Isolated from: 2 OXBO 3210 ground fruit samples Both samples were collected following rain Juice TPC and APC Both total and acidophilic counts in juice were very low Harvesting system does not impact juice microbial load

E. coli and Salmonella Results Juice No E. coli or Salmonella enriched from any juice sample

Study Objectives Evaluate microbiology of fruit after CMNP application Aerobic Plate Count enumerates most microoranisms Acidophilic Plate count enumerates microorganisms capable of growth in Acid conditions Alicyclobacillus enumeration enumerates Alicyclobacillus, a juice spoilage organism Evaluate juice quality of fruit after CMNP application Brix Acid Color Three harvest dates; one Hamlin and two Valencia

Methods & Materials Sampling 5 non-defective fruit randomly selected from each of the trees from each of the sample groups described on days 0, 3, 5, and 7 Fruit brought to lab for microbiological analysis 30 ml buffer; shake/rub/shake to remove microorganisms from fruit surface Plate on Plate Count Agar (for total plate count) and Orange Serum Agar (for total acidophilic plate count) Alicyclobacillus count Samples heat shocked 75 C for 15 min, and plated onto Ali agar Results reported in log CFU/orange or CFU/orange (Ali)

Total Plate Count - Hamlin Take away message: 1. Counts are not significantly different 2. Population after 7 days with CMNP application at 30 C is slightly higher

Total Plate Count Valencia 1 Take away message: 1. No significant differences 1. Treatment or temperature

Total Plate Count Valencia 2 Take away message: 1. No significant differences 1. Treatment or temperature

Alicyclobacillus spp. No Alicyclobacillus spp. detected in any samples

Juice Quality Hamlin Control CMNP Temp ( C) Time (day) Ratio Color Ratio Color 0 15.5 37.0 15.1 37.2 10 3 13.6 37.2 15.1 37.1 5 14.5 37.2 15.1 37.4 7 15.5 36.8 14.8 37.5 20 3 14.1 37.2 14.8 37.1 5 14.3 37.0 14.3 37.2 7 15.4 37.1 14.4 37.4 30 3 16.6 36.7 16.8 37.1 5 16.1 36.8 16.4 37.5 7 16.6 37.6 16.9 38.2 Ambient 3 15.2 36.8 15.6 36.6 5 14.7 36.2 15.6 36.7 7 15.1 36.8 15.4 37.4

Summary No indication that fruit in contact with ground is consistently and significantly higher in microbial surface contamination During PU and CF trials multiple samples positive for E. coli indicate the potential for cross contamination during MH Cross contamination from harvesting equipment E. coli isolation from one tree orange following CMNP application indicate a need for good quality water use. Potential contamination during spray application Independent of CMNP presence

Summary Salmonella was only isolated in one sample in 2006 2007 Further classified as Salmonella Munchen Alicyclobacillus only isolated during one trial and only from ground samples Weather may play a role in contamination levels Weather conditions and grove floor maintenance during the season may contribute to these results Harvesting following rain and irrigation may lead to contamination

Summary No significant differences in fruit surface microflora CMNP application followed by storage at different temperatures does not appear to impact total microflora levels Alicyclobacillus not isolated from any samples CMNP followed by storage does not seem to play a role in contamination levels In general, no differences are seen in juice quality parameters CMNP/storage studies currently in year 2.

Acknowledgements Technical assistance Lorrie Friedrich Gwen Lundy OXBO International Corp. Grower cooperators State of Florida Citrus Industry Initiative funding

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