Use of Lactic Acid Bacteria to Control L. monocytogenes on Apples under Simulated Commercial Conditions

Progress report for GNE19-213

Project Type: Graduate Student
Funds awarded in 2019: $15,000.00
Projected End Date: 08/31/2022
Grant Recipient: University of Connecticut
Region: Northeast
State: Connecticut
Graduate Student:
Faculty Advisor:
Mary Anne Amalaradjou
University of Connecticut
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Project Information

Project Objectives:

The overall objective of the proposed study is to determine the antimicrobial efficacy of select lactic acid bacteria for controlling L. monocytogenes at high (5 log CFU/apple) and low inoculation (3 log CFU/apple) levels on whole apples. A high inoculum level (5 log CFU/apple) will be used to enable measurement of several log reductions in pathogen counts during the study. Additionally, this study will incorporate a low level of inoculum (3 log CFU/apple) in order to simulate low levels of pathogen contamination that are likely to occur under normal postharvest handling and storage conditions. In order to simulate commercial conditions, Listeria control on apples will be specifically evaluated following: 1. Spray application of LAB and fruit storage under conditions simulating cold storage at the packing house. 2. Application of LAB containing fruit finish and apple storage under conditions simulating storage at the packing house, retail outlet and home. 3. Application of LAB containing fruit finish and effect on subsequent pathogen transfer while cutting.

Introduction:

To date, there are two multistate foodborne outbreaks linked to Listeria monocytogenes with the consumption of apples, indicating that contaminated whole apples may serve as a vehicle for foodborne listeriosis. Apples can get contaminated pre-harvest in the field, post-harvest in the processing plants and during storage and shipping. Listeria monocytogenes can survive on apples under refrigeration and ambient storage conditions. Therefore, there is a critical need for effective antimicrobial strategies to control pathogens on apples. Lactic acid bacteria (LAB) can serve as excellent candidates to reduce pathogen levels on apples.

 

Research

Materials and methods:

A five-strain cocktail of L. monocytogenes consisting of produce isolates (580-072, 583-060, LM1, LM2, LM3 – apple isolates) was used for the study. Each strain was cultured separately in 10 ml of sterile brain heart infusion broth (BHI) and NA (50 µg/ml) at 37 °C for 24 h with agitation (100 rpm). Cultures were then transferred for two consecutive 24-h periods onto modified Oxford media agar plates containing NA to produce a bacterial lawn. To prepare the inoculum, growth from the bacterial lawn was transferred to 0.1% buffered peptone water (BPW). Equal portions from each of the five strains were then combined to make the pathogen cocktail.  The bacterial population in the five-strain mixture was determined by plating 0.1-ml portions of appropriate dilutions on modified Oxford media with NA followed by incubation at 37°C for 48 h. Appropriate dilutions of the five-strain mixture in BPW was used to obtain the desired level of inoculum. Whole apples were spot inoculated with the bacterial cocktail by placing 100 µl of a five-strain mix around the stem end of the apple to get the desired inoculum (5 or 3 log CFU/apple). After inoculation, fruits were held for 2 h at room temperature in a biosafety hood for the inoculum to dry. For the antimicrobial treatment, appropriate dilutions of the overnight LAB cultures (LL1/LL2/LP/LR) were washed and suspended in BPW to obtain the desired inoculum level (7 log CFU/apple). Each LAB treatment was sprayed on to the inoculated apples using the air-nozzle sprayer and held in the biosafety cabinet for an additional hour to allow for drying. They were then placed in sterile containers and stored at 4 °C for 90 days. Surviving L. monocytogenes and LAB population on the apples were enumerated at different times over the 90-day period (day 0, 1, 7, 21, 30, 60 and 90). The apples were individually transferred to sterile stomacher bags containing 100 ml of BPW, each fruit hand rubbed for 2 min and analyzed for surviving Listeria and LAB populations by serial dilution and plating.

Research conclusions:

The purpose of this project is to develop a sustainable, natural and safe biocontrol strategy to improve the microbial safety of apples thereby promoting the fresh produce industry.  Based on the results, we expect that LAB can be effectively applied to control Listeria monocytogenes on apples along the production process in handling and packaging facilities.

 

Participation Summary

Education & Outreach Activities and Participation Summary

Participation Summary:

Education/outreach description:
  1. Poster presented at International Association for Food Protection Annual meeting, Oct 2020 : Use of Lactic Acid Bacteria to Control Listeria monocytogenes on Apples during Simulated Storage Conditions (2020) Deepa Ashwarya Kuttappan, Mairui Gao & Mary Anne Amalaradjou

Project Outcomes

Project outcomes:

We have completed the half apple trials and the apple plug trials. Also, we standardized the protocol for the whole apple trial. We are in the process of repeating the whole apple trials at the high and low inoculum. In the current project period, experiments were performed to evaluate the antimicrobial effect of different LAB strains on Listeria on whole apples. Specifically, the present study investigated the application of Lactococcus lactis B-23802 (LL1), Lactococcus lactis B-23804 (LL2), Lactobacillus rhamnosus B-442 (LR) and Lactobacillus plantarum B-4496 (LP) for reducing Listeria monocytogenes populations on whole apples under simulated commercial storage conditions. Following Listeriainoculation and LAB application, apples were placed in sterile containers and held in cold storage at 90-95% humidity for 90 days. At specified times, apples were processed to enumerate surviving Listeria and LAB populations. Our results show that Listeria populations reduced from ~ 5 log CFU/apple at day 0 to ~ 2 log CFU/apple at day 7 in the control group. Spray application of LL1, LL2 and LP reduced Listeria populations by greater than 0.5 log when compared to the control. With regards to the LAB population, ~ 7 log CFU/apple was recovered from the apples by 7 days of storage. In the next project period, we will continue sampling of apples stored under simulated commercial storage conditions. Further, we will perform experiments using LAB containing fruit finish (Apple Lustr® 331) to evaluate its effect on Listeria population on apples under simulated retail and at home storage.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.