Produce surface treatments based on bacteriophages and bacteriocin-producing cultures to consistently reduce 2-log of Listeria monocytogenes on leafy greens and pre-cut fruit and vegetables

The large number of outbreaks and recalls associated with Listeria monocytogenes (Lm) evidences that control of Lm in fresh produce requires a multiple hurdle approach including the implementation of effective innovative strategies to reduce, control and/or eliminate the risk of listeriosis. This proposal focuses on the search of commercially available post-process treatments able to provide a consistent 2-log reduction of Lm on the surface of leafy greens and pre-cut fruit and vegetables. Considering that growth kinetics of Lm in fresh produce is strongly commodity-dependent, a risk categorization will be performed to provide the produce industry available information about which commodities are more susceptible, making necessary the application of a post-process treatment. Promising results against Listeria spp. and Lm have been already reported for several commercial treatments tested under lab-scale experiments. However, experience shows that results obtained in the laboratory are difficult to extrapolate to the industry. After a preliminary evaluation under controlled conditions for the selection of the most promising treatments, this project focuses on the validation and verification of control strategies in commercial processing plants with the help of the industry cooperators. The impact of the selected post-process treatments on the quality and sensory aspects will be evaluated during shelf-life.

Produce decontamination practices represent a useful tool in further reducing the number of pathogenic microorganisms when used as part of a fully integrated control program. Nowadays, the fresh produce industry is still searching for effective control strategies to reduce, control and/or eliminate the risk of listeriosis in produce grown, packed and manufactured/processed. The aim of the present proposal is to evaluate the implementation of commercial post-process treatments to control Listeria monocytogenes (Lm) growth in leafy greens and fresh-cut fruits and vegetables. Several commercial post-process treatments based on bacteriophages and bacteriocin-producing cultures have shown promising results under lab-scale conditions, but there is a lack of information about their performance under commercial processing conditions. In this project, commercial brands including ListShield (Intralytix, US), PhageGuard Listex (PhageGuard, US), SafePro® (CHR Hansen, DK) and HOLDBAC® Listeria (DowDuPont, US) will be tested as control strategies against Lm. In the case that new market releases are identified, they will be included in the study. The first objective will focus on a risk prioritization analysis of the most relevant commodities, considering that the growth kinetics of Lm in fresh produce is strongly commodity-dependent. Available data on the growth kinetics of Lm and the new data generated will be analyzed to provide the produce industry with relevant information about which commodities are more susceptible, making the application of a post-process treatment necessary as an additionally control point. The three/five top commodities identified in the risk categorization will be selected for further studies. The second objective consist on the evaluation of the efficacy of commercially available post-process treatments against Listeria spp. and Lm under controlled conditions to determine the most effective treatments and optimized conditions. The impact of the selected treatments and operational standards on the quality and sensory aspects of the product will be evaluated in the third objective of the project. The proof of concept on the influence of these commercial treatments in reducing Lm loads on different fresh produce, mode of application, doses, and storage temperature must be validated under commercial conditions. Thus, the fourth objective of the project will focus on the validation of selected postprocess treatments and establishment of operational standards for the application of control strategies in commercial fresh produce facilities. The validation and verification activities associated with Listeria spp. and Lm preventive controls will be possible thanks to the collaboration with two industry cooperators, (Flensted (http://www.flensted.dk/) from Denmark and Florette (http://www.florette.es/) from Spain. Validation of the selected treatments will include monitorization of commercial processing lines to fine-tune critical parameters previously identified under controlled conditions including modes of application, selected doses and storage conditions. Operational standards will be established for each specific commodity and processing line design to facilitate the implementation of the new produce surface treatments by the fresh produce industry.

1. Risk prioritization analysis of critical fresh produce commodities based on the ability of Listeria monocytogenes (Lm) to grow at different temperatures in different fresh commodities, and search for commercially available produce surface treatments that meet regulatory requirements for use on food. 

2. Establishment of the efficacy of commercially available post-process treatments against Lm and Listeria spp. by lab-scale trials mimicking commercial conditions. 

3. Evaluation of the impact of the selected post-process treatments on the organoleptic quality and shelf-life of selected fresh produce. 

4. Validation of selected post-process treatments in commercial fresh produce facilities and establishment of operational standards.

• Leafy greens support the growth of Lm better than other vegetables, such as cucumber and asparagus, as well as some fruits. A screening to determine the capacity of different commodities to support the growth of Lm before a post-process treatment was implemented. 

• For leafy greens, predictions made by the Gamma Concept were in agreement with the observations obtained in the challenge tests. Therefore, this model can be used to predict the Lm behavior in different types of leafy greens, avoiding the need to perform more demanding trials for these commodities. 

• Listex™ was the treatment capable of consistently reducing about Lm by 2 log during the product shelf-life when compared to the untreated product. Therefore, Listex™ was the treatment selected for further analyses. 

• The selected post-process treatment, Listex™, can be considered as a processing aid, as in most of the cases no further reductions than the ones observed initially after 24 h were observed during storage. 

• The post-process treatment Listex™ does not cause any detrimental impact on the sensory quality of the selected commodities or reduce shelf-life. Therefore, this treatment can be recommended to be applied in commercial facilities to control Lm growth in fresh produce. 

• The validation trials demonstrated that it is possible to reach the optimal concentration of Listex™ needed to consistently reduce the load of Lm by 2 log in iceberg lettuce. The data obtained in lab-scale trials for the application of the Listex™ treatment was validated in industrial settings.

• The selected treatment, Listex™, did not cause any detrimental effect on the quality of iceberg lettuce when applied in an industrial setting. Listex™ can be safely applied in commercial processing plants without causing any quality impact on the product.