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A commercial phage biocontrol for reducing Listeria monocytogenes has been described as an effective tool for improving fresh produce safety. Critical challenges in the phage application must be overcome for the industrial application. The validation studies were performed in two processing lines of two industry collaborators in Spain and Denmark, using shredded iceberg lettuce as the ready-to-eat (RTE), high process volume product. The biocontrol treatment optimized in lab-scale trials for the application of PhageGuard ListexTM was confirmed in industrial settings by four tests, two in Spain and two in Denmark. Results showed that the method of application that included the device and the processing operation step was appropriate for the proper application. The proper dose of Phage Guard ListexTM was reached in shredded iceberg lettuce and the surface was adequately covered for the successful application of phages. There was no impact on the headspace gas composition (CO2 and O2 levels), nor on the color when untreated and treated samples were compared. The post-process treatment with PhageGuard ListexTM did not cause any detrimental impact on the sensory quality, including flavor, texture, browning, spoilage, and visual appearance over the shelf-life as the phage solution was applied as a fine, mist solution.

Haloacetic acids (HAAs) are one of the most important chlorinated disinfection by-products generated during water disinfection in the fresh-cut industry, and they can remain in the product, resulting in a consumer health risk. In this study, ultra-high-pressure liquid chromatography-tandem multiple reaction monitoring mass spectrometry (UHPLC-MRM) analysis used for drinking water was optimized and applied for the quantification of nine HAAs (HAA9) in fresh-cut lettuce and process water samples, with the complex matrix interferences for separation, and quantification problems. The method showed good selectivity, specificity and linearity, satisfactory values for trueness (recoveries of 80–116 %), precision (<22 %), and uncertainty (<55 %). Quantification limits varied from 1 to 5 µg L−1 or µg kg−1. The matrix effect for tribromoacetic, bromochloroacetic and chlorodibromoacetic acid was corrected by matrix-matched calibration and standard addition. After storage at -20 °C, only monobromoacetic acid was the HAA which loss happened after 7 days. The application of the methodology in lettuce and process water samples from the industry was successfully implemented. Therefore, this method could be employed for the quality control and regulatory analysis of HAAs in fresh products and process water from the fruit and vegetable industry

Since 2020, there is a new European Regulation (EU, 2020/741) on minimum requirements for water reuse, where routine and validation monitoring requirements (log reductions of indicator microorganisms and reference pathogens) have been established. Many reclamation facilities that are already in operation might have difficulties to comply with these performance targets. Existing disinfection systems must be expanded and upgraded. In the case of UV disinfection systems, fluence requirements must be determined to properly design with a focus on the safety and economic-environmental viability of reclaimed water. This study can be used as a reference to develop fluence-response curves for Clostridium perfringens spores, Escherichia coli, and total and F-specific coliphages, indicator microorganisms referred to in the new European Regulation. Eight UV-LED collimated beam tests were performed. Samples were obtained from filtered effluent of secondary treatment from two wastewater treatment plants (WWTPs) which ranged between 30 and 54%. Results showed UV sensitivity of 33.46 mJ/cm2 log I for C. perfringens spores and 2.86 mJ/cm2 log I for E. coli, both from environmental origin. Coliphages were inactivated below the limit of quantitation. The non-dominance of MS2 phages in environmental F-specific coliphages was observed

The new European regulation on minimum quality requirements (MQR) for water reuse (EU, 2020/741) was launched in May 2020 and describes the directives for the use of reclaimed water for agricultural irrigation. This Regulation will be directly applicable in all Member States from 26 June 2023. Since its publication in 2020, concerns have raised about potential non-compliance situations in water reuse systems. The present study represents a case study where three different water reuse systems have been monitored to establish their compliance with the MQR. Each water reuse system includes a wastewater treatment plant (WWTP), a distribution/storage system and an end-user point, where water is used for irrigation of leafy greens. The selected water reuse systems allowed us to compare the efficacy of water treatments implemented in two WWTPs as well as the impact of three different irrigation systems (drip, furrow and overhead irrigation). The presence and concentration of indicator microorganisms (Escherichia coli and C. perfringens spores) as well as pathogenic bacteria (Shiga toxin-producing, E. coli (STEC), E. coli O157:H7, and Salmonella spp.) were monitored in different sampling points (influent and effluent of the WWTPs, water reservoirs located at the distribution system and the end-user point at the irrigation system as well as in the leafy greens during their growing cycle. Average levels of E. coli (0.73 ± 1.20 log cfu E. coli/100 mL) obtained at the point where the WWTP operator delivers reclaimed water to the next actor in the chain, defined in the European regulation as the ‘point of compliance’, were within the established MQR (<1 log cfu/100 mL) (EU, 2020/741). On the other hand, average levels of E. coli at the end-user point (1.0 ± 1.2 log cfu/100 mL) were below the recommended threshold (2 log cfu E. coli/100 mL) for irrigation water based on the guidance document on microbiological risks in fresh fruits and vegetables at primary production (EC, 2017/C

Growth of L. monocytogenes is among the most important factors affecting the risk of human listeriosis. In ready to eat leafy greens, the use of anti-Listeria treatments represents a good alternative to inhibit growth during storage. Several commercially available antimicrobial agents have been suggested as effective intervention strategies. Among them, phage preparations and bacteriocin-producing strains have shown promising results against L. monocytogenes. In this study, we investigate the efficacy of two commercially available surface treatments, the bacteriophage formulation PhageGuard Listex (Micreos Food Safety B.V., NL) and the bacteriocin-producing culture SafePro® (CHR Hansen, DK) to inactivate L. monocytogenes in fresh-cut curly endive after processing and during storage. Fresh-cut endive was inoculated with a cold-adapted L. monocytogenes cocktail of 6 strains (4.4 ± 0.0 log cfu/g) and treated with the anti-Listeria treatments. The treatments were applied using a spray system at two different places within the processing line, on the conveyor belt and in the centrifuge. A total of 5 different treatments were applied: i) Untreated (CT); ii) PhageGuard Listex on the conveyor belt (Listex_Conveyor); iii) PhageGuard Listex during centrifugation (Listex_Centrifuge); iv) SafePro on the conveyor belt (SafePro_Conveyor); and v) SafePro during centrifugation (SafePro_Centrifuge). Samples were stored 3 days at 5 °C plus 5 days at 8 °C. PhageGuard Listex treatment reduced L. monocytogenes in fresh-cut endive by 2.5 logs, regardless of the place of treatment application (conveyor belt or centrifuge). On the other hand, SafePro only reduced L. monocytogenes by 0.2 and 0.4 logs, at the conveyor belt and centrifuge, respectively. Maximum L. monocytogenes reductions of about 3.5 log units were observed in fresh-cut endive treated with PhageGuard Listex after 3 days of storage. At the end of the shelf life (8 days), the initial trends were maintained and the fresh-cut cu

Postharvest research must enable us to translate fundamental knowledge into innovative and sustainable solutions to reduce postharvest losses. These include genetic improvements by selecting cultivars with good flavour and texture and long postharvest life, sensor improvements to monitor and control the ripeness and the optimal maturity for harvest, and improvements in quality control and storage conditions. New packaging technologies using biodegradable and recycled materials are promising solutions for helping with environmental sustainability. However, they should also be adequate for fresh produce quality maintenance. The innovative actions should combine appropriate optimal temperature and relative humidity during storage, transport, and distribution to create the desired conditions around the commodity for freshness preservation. To achieve these goals, collaborative actions that integrate all the aspects related to postharvest losses are needed starting from the agricultural production activities to processing, marketing, distribution, and even consumers.

Lettuce is one of the most commonly consumed leafy vegetables worldwide andis available throughout the entire year. Lettuce is also a significant source ofnatural phytochemicals. These compounds, including glycosylated flavonoids,phenolic acids, carotenoids, the vitamin B groups, ascorbic acid, tocopherols,and sesquiterpene lactones, are essential nutritional bioactive compounds. Thisreview aims to provide a comprehensive understanding of the composition ofhealth-promoting compounds in different types of lettuce, the potential healthbenefits of lettuce in reducing the risks of chronic diseases, and the effect ofpreharvest and postharvest practices on the biosynthesis and accumulation ofhealth-promoting compounds in lettuce.

One solution to current water scarcity is the reuse of treated wastewater. Water reuse systems have to be examined as a whole, including the efficacy of water-reclamation treatments and the operation steps from the wastewater inlet into the WWTP to the irrigation endpoint, including the irrigated crop. In this study, the monitoring of human enteric viruses and coliphages were assessed in two water reused systems. The presence of hepatitis A virus (HAV) and human noroviruses genogroups I and II (GI and GII) were analyzed by real-time RT-PCR (RT-qPCR) in water (n = 475) and leafy green samples (n = 95). Total coliphages were analyzed by the double-layer agar plaque technique. The prevalence of HAV in water samples was very low (c.a. 2%), mostly linked to raw sewage, while for leafy green samples, none was positive for HAV. In leafy greens, prevalence of norovirus was low (less than 5–6%). The highest reductions for norovirus were observed in samples taken from the water reservoirs used by the growers near the growing field. The virus die-off during water storage due to solar radiation could be considered as an additional improvement. Reclamation treatments significantly reduced the prevalence and the counts of noroviruses GI and GII and coliphages in reclaimed water. However, the coliphage reductions (c.a. 5 log) do not comply with the specifications included in the new European regulation on reclaimed water (≥6.0 log). Correlations between noroviruses GI and GII and coliphages were found only in positive samples with high concentrations (>4.5 log PFU/100 mL). A high percentage of samples (20–25%) negative for total coliphages showed moderate norovirus counts (1–3 logs), indicating that coliphages are not the most suitable indicator for the possible presence of human enteric viruses.

A case study of 15 wastewater treatment plants (WWTPs) at a full-scale was assessed for the risks of disinfection byproduct (DBP) formation, mainly the regulated trihalomethanes (THMs) and haloacetic acids (HAAs) and chlorate as an inorganic byproduct regulated recently in the EU. Raw wastewater from large, medium/small urban areas were treated with single or combined disinfection processes (i.e., chlorine, peracetic acid (PAA) and ultraviolet (UV) radiation). Sampling was executed once a month over seven months for the medium/small WWTPs and twice a month for the large ones. Due to the potential risk of SARS-CoV-2 contaminated wastewater, several inactivation methods were examined before the DBP analysis. Due to the inactivation step, the stability of THM4 and HAA9 suffered reductions, monitoring their presence only in the effluents after the disinfection treatments. In contrast, chlorate levels remained unchanged after the inactivation treatment; thus both raw wastewater and effluents were examined for their occurrence before disinfection treatments. Results showed that chlorate residues in the raw wastewater varied greatly from undetected levels to as high as 42.2 mg L−1. As the continuous monitoring of DBPs was performed, a positive correlation with chlorine or chlorine/UV was found. Changes in the physicochemical parameters indicated that the quality of the raw wastewater varied considerably depending on the WWTPs, and it influenced byproduct formation. In all WWTPs, chlorine alone or combined with UV significantly increased the presence of THMs, HAAs, and chlorate levels in the treated effluents. When the same WWTPs changed to PAA or PAA/UV, DBPs were diminished completely. This study highlights the risk of chlorate residues in raw wastewater during the pandemic. It also showed how the chemical risks of DBP formation could be reduced by changing the chlorinated disinfection technologies to PAA or PAA/UV, particularly if reclaimed water is intended for agricult

The elaboration of guidelines for the industry to establish minimum concentration to prevent cross-contamination during washing practices based on operational limits is the core of the recommended criteria for the use of sanitizers. Several studies have evidenced that sanitizers reduced the levels of foodborne pathogens. However, they might lead to the progress into a viable but non-culturable (VBNC) state of the cells. This evidence has raised concerns regarding the effectiveness of the recommended washing practices for the inactivation of microbial cells present in the process wash water (PWW). The present study evaluated if the most commonly used sanitizers, including sodium hypochlorite (chlorine), peroxyacetic acid (PAA) and chlorine dioxide (ClO) at established operational limits induced the VBNC stage of Listeria monocytogenes and Escherichia coli O157:H7. Prevention of cross-contamination was examined in four different types of PWW from washing shredded lettuce and cabbage, diced onions, and baby spinach under simulated commercial conditions of high organic matter and 1 min contact time. The results obtained for chlorine showed that recommended operational limits (20–25 mg/L free chlorine) were effective in inactivating L. monocytogenes and E. coli O157:H7 in the different PWWs. However, the operational limits established for PAA (80 mg/L) and ClO (3 mg/L) reduced the levels of culturable pathogenic bacteria but induced the VBNC state of the remaining cells. Consequently, the operational limits for chlorine are satisfactory to inactivate foodborne pathogens present in PWW and prevent cross-contamination but higher concentrations or longer contact times should be needed for PAA and ClO to reduce the likelihood of the induction of VBNC bacteria cells, as it represents a hazard.

Sanitizers are an effective measure to maintain the microbiological safety of fresh produce process water. Proper water disinfection management is critical to avoid cross-contamination during the washing of fresh produce. Chlorine remains the most common sanitizer, and recent research deals, among other topics, with the comparison of different types of chlorine sources, optimization of doses for each product type and format, on-line monitoring systems, and mathematical modeling for process management. Peracetic acid (PAA) is the main industrial alternative but further research for the optimal dose and in-line monitoring and control systems is guaranteed. A plethora of other treatments are tested at a laboratory scale, but only a few are assessed under industrial conditions. Recent advances in the management of industrial fresh produce washing are presented and discussed.

An accurate sensor for peracetic acid (PAA) is needed to monitor and control the disinfection of fresh produce wash water. Inaccurate measurements can result in underdosing and unsafe produce or overdosing with huge cost implications. In this study, five methods were compared for measuring PAA in process water from washing apples, tomatoes, red sweet peppers, and lemons. A reversed phase HPLC-DAD method for the determination of PAA in water was adapted for process wash water and used as a reference method. The results showed that HPLC offered high selectivity for PAA and precise and reproducible measurement for its quantification. However, as HPLC is not suitable for in-line applications in processing plants and requires highly skilled technicians, installation and maintenance costs, other analytical methods were examined including a redox titration, a reflectometric method and two electrochemical methods (an amperometric in-line probe and a chronoamperometric method with disposable single-use electrode sensor). The drop titration kit overestimated PAA because of the interferences from other oxidizing agents such as hydrogen peroxide (HP) apart from it required substantial time to perform the measurements plus the extensive training of the technical personnel to ensure confidence in the results. The reflectometric method with disposable test strips, although fast and objective, overestimated PAA due to the wash water with a high content of suspended organic matter that needed the addition of high concentrations of PAA, coexisting with HP. From the two electrochemical methods studied, the in-line amperometric sensor underestimated the PAA concentration in lemon wash water as the PAA could not freely diffuse through the membrane through the active electrode because of the organic matter present in the wash water released from the lemon peel. In contrast, the chronoamperometric sensor showed good selectivity for PAA without interferences, with reliable results across a

The virological quality of process water (PW) used by the produce industry has received limited attention. As a first step to overcoming technical limitations in monitoring viruses in PW, the analytical performance of ultrafiltration was assessed to concentrate viral particles from 20 L of spiked PW. The selected method used for sample concentration of PW was carefully validated, thus enabling the accurate quantification and estimation of viral titers of human enteric viruses and phages. PW from the produce industry was collected periodically from the washing tanks of commercial facilities. The analysis of coliphages was performed by plaque assay, while the occurrence of enteric viruses and crAssphage was determined by molecular techniques. Significant differences in the physicochemical composition of PW, mostly due to the different nature of fresh produce types and differences in the sanitizer used in commercial operation, were observed. Accumulation of crAssphage and coliphages was observed in PW, but correlation with human enteric viruses was not possible due to the low prevalence of these pathogens in the PW analyzed. The obtained results showed that depending on the type of product washed, the product/water ratio and the residual concentrations of the sanitizers, the prevalence and concentration of bacteriophages changed significantly.