Progress report for GNE19-213
The overall objective of the proposed study is to determine the antimicrobial efficacy of select lactic cultures for controlling L. monocytogenes at high (5 log CFU/fruit) and low inoculation (3 log CFU/fruit) levels on apples. A high inoculum level (5 log CFU/fruit) will be used to enable measurement of several log reductions in pathogen counts during the study (Beuchat et al., 2002). Additionally, this study will incorporate a low level of inoculum (3 log CFU/fruit) in order to simulate low levels of pathogen contamination that are likely to occur under normal postharvest handling and storage conditions (Behrsing et al., 2003). 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.
Over the last decade, the inclusion of fresh produce in the American diet has been steadily increasing due to heightened consumer awareness of the associated health benefits. This increase in fresh produce consumption has been associated with a concomitant increase in foodborne outbreaks. More specifically, the recent recall of several popular apple varieties including Honey Crisp and Red Delicious due to potential Listeria monocytogenes contamination, indicates that there is a critical need for antimicrobial strategies, which are effective, and sustainable along the production continuum. In this regard, lactic acid bacteria (LAB) can serve as excellent candidates for reducing pathogen levels on fresh and minimally-processed produce, like apples.
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 brain heart infusion 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) to an absorbance of 0.2%. The approximate bacterial count in each culture was determined spectrophotometrically. 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. In the first round of experiments, apple plugs and apple halves were used in place of whole apples. Fruits were spot inoculated with the bacterial cocktail (6 log CFU/plug or apple half) by placing 50 µl of a five-strain mix around the stem end of the apple/ on the plug. 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 (8 log CFU/ml). 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 and 22°C for 14 days. Surviving L. monocytogenes and LAB population on the apples were enumerated at different times over the 14 day period (days 0, 1, 3, 5, 7, 14). The apples/plugs were individually transferred to sterile stomacher bags containing 100 ml of BPW, each fruit hand rubbed for 2 min, and the BPW was analyzed for surviving Listeria and LAB population.
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.
The present study investigated the application of LAB namely, Lactococcus lactis B-23802 (LL1), L. lactis B-23804 (LL2), Lactobacillus rhamnosus B-442 (LR) and Lactobacillus plantarum B-4496 (LP) for improving the microbial safety of apples. Specifically, the study evaluated spray application of LAB strains to control Listeria on apples during storage at the packing-house (4°C) and retail/ home (22°C) conditions. Spray application of LAB significantly (P <0.05) reduced Listeria populations on apple plugs/apples by ~ 1 – 2 logs, on storage at ambient and refrigeration temperatures. However, ~ 6 log CFU of L. monocytogenes was still recovered from the control plugs/ apples. With regards to the LAB, ~ 5 log CFU of LAB was recovered from the samples by 14 days of storage.