Reduction of Pathogen Surrogate Bacteria using Biosafe on Diced Fruits and Vegetables

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Summary

This study was conducted to determine the antimicrobial efficacy of Biosafe intervention applied to frozen and fresh vegetables. To determine results relating to microbial surface reduction, random samples of the vegetables were selected as cell attachment samples (before intervention). For intervention application, after attachment, 10 samples will be placed into a commercial spray cabinet and treated according to the manufacturer’s specifications. Also, samples Microbiota (not inoculated) samples were collected to determine natural microflora on the vegetables.

Methodology

Preparation of Pathogen Cocktail

  1. Five non-pathogenic ATCC surrogate strains of Escherichia coli (BAA 1427, 1428, 1429, 1430, and 1431), previously shown to mimic Salmonella and E. coli O157:H7 behavior (Niebuhr, 2008 & Cabrera-Diaz, 2009).
  2. The surrogate strains will be independently propagated in a free pathogen BSL-I laboratory on the Texas Tech University campus in room 232 in the Animal and Food Sciences Building.
  3. Cocktail inoculums for the in-plant challenge trial to target an estimated 5 log10 CFU/g on the vegetable’s surface.
  4. Individual ATCC surrogate frozen isolates will be independently inoculated into 5 ml tubes of sterile Buffered Peptone Water (BPW), for incubation for 18-24 hours at 37°C.
  5. Culture tubes will be transferred into sterile 45 mL conical tubes of BPW for cocktail preparation.  
  6. Each tube will be screened for Salmonella, E. coli O157:H7, and Listeria monocytogenes using real-time PCR before use in the plant. 
  7. The containers will be properly labeled as biological hazards and a spilling kit will be transported in a Texas Tech Vehicle by University graduate students who will drive directly to the facility in Omaha, Nebraska on the day of analysis

Vegetables Preparation & Inoculation

  1. For each vegetable tested, a total of twenty, 25g samples will be sprayed using a plastic spray bottle (710 mL) with the E. coli surrogate cocktail. Treatments were distributed as follows:
  1. Microbiota: 10 samples total without inoculation nor intervention will be collected for natural microbiota analysis. 
  2. Before intervention: 20 samples total of vegetables before treatment were collected as cocktail attachment samples.
  3. After intervention: 20 samples total of inoculated vegetables were collected after Biosafe treatment spray.
  1. 20 – 30 minutes were allowed for cell attachment while resting at ambient temperature on plastic plates inside a plastic bag to contain the spray.
  2. The 25g samples will be aseptically placed into a whirl pack bag and stored in a Styrofoam cooler with cooler packs (before intervention).
  3. After attachment, samples were placed into a commercial spray cabinet and treated according to the manufacturer’s specifications. Immediately after the intervention, samples were held for 1-2 minutes on a mesh bowl to drip the excess water and then placed into a whirl pack bag (after intervention).
  4. Bags will be disinfected with the bleach solution before disposal and the bag will be swabbed to ensure the destruction of the culture.
  5. Mesh bowls, equipment used, and the remaining cocktail culture were treated with a bleach solution to inactivate the culture.
  6. Samples were shipped back to Texas Tech for microbial analysis. Samples that were placed inside whirl pack bags were sealed and then placed inside a leak-proof container. This secondary containment was sealed inside additional leak-proof packaging to meet the triple containment requirements for shipping as Category B.

 

Microbiological Enumeration

  1. Microbiological enumeration was conducted in the Experimental Sciences Building at Texas Tech University. Samples received at the lab were added 225 mL of BPW and homogenized for 1-minute at 230 RPM and serially diluted if needed. Total aerobic counts (APC) and E. coli counts (EC) will be conducted using the TEMPO® AC (aerobic colony count), and TEMPO® EC (E. coli Count) methods, according to the manufacturer’s instructions. Results will be log-transformed before statistical analysis. 
  2. The total number of samples consisted of 20 samples/vegetable × 5 vegetables (broccoli, blueberries, sliced carrots (carrots and peas), lettuce, and spinach) × 2 sampling points (attachment and after intervention), resulting in 200 samples. Microbiota samples consisted of 10 samples/vegetable × 5 vegetables resulting in 50 samples. A total of 250 samples including all sampling points

Results

Table 1. Microbial counts of pathogen surrogate bacteria using BioSafe in diced fruits and vegetables on natural microbiota (n= 10 e /o), attachment and after intervention treatments for Blueberries, Lettuce, Spinach (n= 20 samples) as for Broccoli and Carrots (n=30 samples).

 

Vegetable/

Fruit

 Natural Microflora

(Log CFU/g ± SE)

 Sampling Location Average Reduction

(Log CFU/g ± SE)

 P-value
Attachment

(Log CFU/g ± SE)

 After Intervention

(Log CFU/g ± SE)
Blueberries 2.66 ± 0.46 6.55 ± 0.09a 5.23 ± 0.14b 1.32 <0.001
Broccoli 3.33 ± 0.46 6.71 ± 0.04a 6.76 ± 0.08a -0.05 0.590
Carrots 2.12 ± 0.18 7.27 ± 0.09a 7.22 ± 0.05a 0.05 0.710
Lettuce 3.16 ± 0.29 7.38 ± 0.05a 5.30 ± 0.13b 2.08 <0.001
Spinach 5.58 ± 0.30   7.52 ± 0.05a 5.94 ± 0.20b 1.58 <0.001

 

Figure 1. Escherichia coli counts (Log CFU/g) before and after treatment application (n= 20 samples per sampling point, n= 30 samples after treatment Broccoli and Carrots). Boxes with different letters are significantly different according to T-test analysis. The points represent the actual points.

Summary of Results 

Aerobic counts were analyzed to obtain the amount of natural microbiota present previously inoculating each vegetable. Escherichia coli counts (Log CFU/g) were significantly reduced (P < 0.001) between attachment and after BioSafe treatment on average by 1.32, 2.08, and 1.58 Log CFU/g on Blueberries, Lettuce, and Spinach respectively. Escherichia coli counts (Log CFU/g) on Broccoli and Carrots were not significantly reduced comparing attachment and after treatment intervention. In these 2 vegetables, 3 repetitions are reflected in the results and all of them showed no reduction between attachment and after-treatment intervention analyzed independently and altogether. Based on the data, it is reasonable to conclude that the BioSafe spray treatment is effective in reducing Escherichia coli in Blueberries, Lettuce and Spinach.

Reference

Niebuhr SE, Laury A, Acuff GR, Dickson JS. Evaluation of nonpathogenic surrogate bacteria as process validation indicators for Salmonella enterica for selected antimicrobial treatments, cold storage, and fermentation in meat. J Food Prot. 2008;71(4):714-718. doi:10.4315/0362-028x-71.4.714.

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