Summary of Awards to Date

Evidence for the industrial application of bacteriophages to control Listeria monocytogenes in leafy greens


Jan. 1, 2023 - Dec. 31, 2023

Amount Awarded



Pilar Truchado, Ph.D.
CEBAS-CSIC Campus de Espinardo, Spain


Maria I. Gil, Ph.D., Ana Allende, Ph.D.


The control of Lm occurrence in processing environments and end products is an important challenge for the fresh-cut produce industry and the public health sector. The use of bacteriophages has provided a promising option for the control of Lm in fresh produce at the lab scale. However, there is a lack of knowledge on the effectiveness of this phage biocontrol on fresh products in real industrial settings.

The proposed research plan is aimed at (a) validating the application of bacteriophages as a post-treatment in baby spinach, providing the industrial-scale operating procedure; (b) demonstrating the benefits of the use of this treatment despite the variability of the raw material by seasonality and abusive storage conditions (c) cost-benefit analysis of the industrial application. Achieving this goal will be possible thanks to the collaboration of the CEBAS-CSIC team with one major fresh produce company, where the validation in their commercial facility will be performed every two weeks for 10 months to confirm the effectiveness of bacteriophages by the absence of Lm/Listeria spp compared with control while it preserves quality. This ‘Proof of Concept’ will provide solid evidence from an independent team on the cost-benefit application of bacteriophages at large-scale operations.

Technical Abstract

Providing safe products is still a great challenge for the fresh-cut industry regarding microbial safety, especially in the context of Listeria monocytogenes. Due to the ability of survival and proliferation in preservation conditions, this pathogenic bacteria can be transmitted to fresh produce before harvest or during processing, increasing the likelihood of finished product contamination, recalls, and outbreaks. Apart from prevention, effective strategies to control, reduce, or eliminate Lm/Listeria spp. are necessary. Among them, the use of bacteriophages is a promising post-process treatment to control/reduce the presence of Lm in fresh produce. In the previous CPS Project led by the CEBAS-CSIC team (CPS2020-CPS01), PhageGuard Listex P100 (Micreos Food Safety, The Netherlands), a Listeria specific bacteriophage, has shown to be a promising post-process treatment, achieving 2 log reductions on artificially inoculated Lm. Industrial validation showed that the concentration of bacteriophages recommended by the supplier (106-107 cfu/g) is easy to apply in the industry during processing and does not cause any detrimental impact on the sensory quality of the selected commodities or shorten their shelf life. However, before processors can adopt this practice on an industrial scale, conclusive evidence is needed for its technical implementation. Details on how to perform the industrial operating procedure, demonstrating the benefits over different seasons, and the positive cost balance are needed. The current proposal aims at the industrial validation of the application of bacteriophages as a prevention tool to control Lm growth in leafy greens applied in a large-scale industrial operation when the quality of raw materials fluctuates by seasonality and abusive storage conditions. Three objectives have been identified. The first objective will verify in an industrial setting the effectiveness of bacteriophages to control Lm growth while preserving the quality of leafy greens as affected by seasonality. In the second objective, the beneficial impact of bacteriophages controlling Lm growth at abusive temperatures will confirm and the interaction with the natural microbiota will be assessed. The third objective will estimate the cost-benefits for the industrial application of bacteriophages as a post-process treatment after washing and drying just before packaging to control Lm. As a result, we expect to confirm to the industry whether the application of bacteriophages is an affordable tool for the benefit of controlling Lm growth in leafy greens to be implemented in their facilities.

The current project aims to impact the fresh produce industry in several ways including:

i) Developing a standardization protocol for the application of this treatment as a food safety measure,

ii) Understanding the effect of bacteriophages on product quality and shelf-life,

iii) Knowing the interaction of bacteriophages, microbiota and Lm through abuse storage temperatures,

iv) Knowing if processors will have a net positive cost with this treatment.