The presence of Vibrio parahaemolyticus (Vp) at various stages of seafood production has adversely affected public health and threatened the sustainability of the industry. Driven by the advancement of next-generation-sequencing technologies and public health data sharing initiative, an increasing volume of public Vp genomic data with metadata has become available, which serve as the foundation for building learning models to accurately differentiate isolation sources and further uncover gene-level determinants of pathogenic potential. The primary goal of this study was to develop and validate machine learning (ML) and deep learning (DL) algorithms to differentiate Vp strains from different isolation sources (clinical vs. environmental isolates) using pangenome assemblies and achieved robust and precise pathogenic potential prediction of Vp. The secondary goal of this study was to obtain critical biological insights revealing pathogenic potential-associated genes contributing to the isolation source difference from these established learning models. Based on the results, the developed learning models demonstrated strong performance, achieving an AUC greater than 0.95 in distinguishing clinical and environmental isolates using pangenome signals. Besides, the gene feature weight analysis from RF revealed the importance of specific accessory genes during Vp evolution including but not limited to functional unknown cloud genes, vspR, sctC5, and tdh1, which provides biological insights as potential future research directions. These findings essentially highlight critical importance of accessory and cloud genes in differentiating clinical and environmental isolates, and provide new insights into how recently acquired genes may contribute to pathogenic evolution of Vp. Additionally, the framework demonstrated in this study provides a cost-effective intelligent strategy by leveraging large public genomic datasets to support surveillance and risk assessment of seafood-associated pathogens.

Chard, collard greens, and kale juices as representative dark leafy green vegetable juices (DLGVJs) have gained increasing attention due to their rich nutritional profiles and health-promoting properties. Conventional dark leafy green vegetables (DLGVs) represent the majority of the DLGV market; however, the microbial changes in conventional DLGVJs during refrigerated storage remain poorly understood. This study aimed to characterize the microbial profiles of fresh and spoiled conventional DLGVJs using both culture-dependent and culture-independent methods, alongside monitoring physicochemical changes (pH and color) over 21 days of refrigerated storage. Results showed a general decrease in pH and a shift toward more saturated color in all DLGVJs. Initial aerobic plate counts (APC) in chard, collard greens, and kale juices were 7.71 ± 0.03, 7.57 ± 0.14, and 7.61 ± 0.09 Log CFU/mL, respectively, while fungal populations were 5.85 ± 0.03, 6.01 ± 0.15, and 6.15 ± 0.03 Log CFU/mL. APC showed an overall decrease while fungal populations exhibited an initial increase followed by a decline during refrigerated storage. High-throughput sequencing revealed Pseudomonas, Leuconostoc, and Periweissella as core spoilage-associated bacterial genera, while Sporobolomyces, Alternaria, and Symmetrospora were predominant fungal genera in spoiled conventional DLGVJs. Although microbial compositions showed similarities between conventional and organic DLGVJs, distinct microbial communities were observed, with some taxa uniquely associated with a specific cropping system. The dominant lactic acid bacteria isolated from DLGVJs were identified as Enterococcus faecium and Leuconostoc mesenteroides. These findings advance our understanding of microbial ecology in DLGVJs and provide a scientific basis for developing targeted interventions to ensure microbial quality and safety, supporting the growth of the DLGVJ market.

Postharvest cooling is a critical pillar of fresh produce preservation. However, the ways in which different cooling methods shape the microbial communities associated with fresh produce remain largely unexplored. This opinion paper synthesizes the mechanisms by which postharvest cooling technologies influence the microbial quality and safety of fresh produce and proposes a framework to guide food safety risk assessment of different cooling options. Each cooling technology creates distinct temperature, pressure, moisture, and water-contact conditions that interact with produce physiology and microbial behavior. The effects of these conditions on microbial survival and growth, cross-contamination, and pathogen internalization provide the foundation for understanding how cooling technologies influence postharvest microbial risk. Finally, the identify key priorities for future applied research will support the development of cooling strategies that better balance cooling efficiency with microbial safety while preserving product quality, extending shelf life, and reducing food loss.

The rapid growth of the plant-based meat analogues (PBMAs) market has sparked interest in its nutritional and environmental benefits, yet the microbial safety and spoilage dynamics of these products remain poorly characterized. In this study we isolated 60 lactic acid bacteria (LAB) from soy- (SBM) and pea-based meat (PBM) products stored at 4 °C or 22 °C until spoilage and performed whole-genome sequencing to characterize their phylogeny, metabolic capacity, and secondary-metabolite profiles. Phylogenomic analysis revealed that Latilactobacillus curvatus and Latilactobacillus sakei dominated SBM, while Weissella viridescens and W. cibaria were prevalent in PBM. Functional annotations indicated heterofermentative metabolism and diverse glycoside hydrolase repertoires in Latilactobacillus, particularly GH1, GH13, and GH32, facilitating degradation of plant-derived carbohydrates in SBM such as trehalose, fructans, and polyphenol glycosides. In contrast, Weissella uniquely encoded complete pathways for menaquinone and tetrahydrofolate biosynthesis, which may enhance redox balance and nucleotide/amino acid synthesis under vitamin-limited storage conditions, supporting persistence in PBM. Across both storage conditions, temperature exerted minimal selective pressure on dominant LAB identity. The heterofermentative physiology common to these LAB supports spoilage in PBMAs. In addition, their dominance likely reflects high initial loads in raw ingredients and genomic capacity for inhibitory metabolites (e.g., class II bacteriocins and, in some lineages, 2-deoxy-streptamine-like aminocyclitol pathways). Building on prior amplicon surveys, this study provides species-level resolution and foundational functional-genomic insights into dominant PBMA spoilage taxa, revealing potential targets for control to extend shelf life and improve the microbial safety of PBMAs.

ABSTRACT Oxytetracycline (OTC) is one of the few antibiotics approved by the U.S. Food and Drug Administration for catfish aquaculture. Unfortunately, OTC resistance has been frequently detected in production environments, with the fish gut identified as a potential hotspot for resistance selection. In aquaculture systems, water temperature is a critical factor influencing fish physiology, antibiotic pharmacokinetics, and water resistome development. However, its role in modulating OTC effects on the fish gut microbiome remains underexplored. This study examined temperature-dependent microbiome and resistome responses in channel catfish (Ictalurus punctatus) when treated with OTC at 20°C, 25°C, and 30°C. Gut contents collected at treatment completion and after withdrawal were analyzed via metagenomic sequencing. In untreated fish, temperature alone shaped microbial structure and function, with the Shannon diversity increasing with temperatures and the β-diversity differing significantly across temperature groups. After OTC exposure, microbial responses were markedly temperature dependent with few taxa affected at 20°C, whereas substantial shifts occurred at 25°C and 30°C, indicating reduced microbial resilience at higher temperatures. OTC elevated total antimicrobial resistance gene (ARG) abundance, enriching tetracycline and β-lactam resistant genes consistent with co-selection. ARG–host linkages were diffuse at 20°C but consolidated within Klebsiella, Enterococcus, Enterobacter, and Paraclostridium at 25°C and 30°C. Notably, OTC-induced dysbiosis persisted through the withdrawal period. These findings demonstrate that temperature modulates both the magnitude and persistence of OTC-driven microbiome disruption and resistome enrichment, underscoring the importance of temperature-aware antibiotic management to mitigate antimicrobial resistance risks and safeguard fish health and food safety in aquaculture.IMPORTANCEThis study reveals that water temperature critically shapes how antibiotics affect the gut microbiome and antimicrobial resistance in channel catfish. Metagenomic sequencing results showed that oxytetracycline (OTC) treatment caused minimal disruption of the microbiome at 20°C, but induced significant community shifts and enrichment of antimicrobial resistance genes (ARGs) at 25°C and 30°C. Higher temperatures reduced microbial resilience, consolidating ARGs within key bacterial genera such as Klebsiella and Enterococcus. Importantly, OTC-induced microbiome changes and resistance persisted through the withdrawal period. These findings highlight temperature as a major driver of antibiotic impact in aquaculture, emphasizing the prudent use of antibiotics at different disease breakout temperatures.

ABSTRACT Vibrio parahaemolyticus ( Vp ) is the major foodborne pathogen transmitted via shellfish products, which has posed significant threats to modern public health and resulted in significant economic damage to the seafood industry. Numerous studies have documented diverse aspects of Vp pathogenicity, among which thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH) are considered as the major virulence biomarkers of Vp . Despite their established roles as key biomarkers, a systematic understanding of the divergence of TDH and TRH across sequence, structure, and function remains limited. In this study, a multi-scale analysis of TDH and TRH was performed using publicly available (2131 and 99 records from NCBI and Uniprot database, respectively) amino acid sequence data combined with representation learning and structure prediction. Global alignment of curated TDH and TRH sequences revealed extensive, distributed mutations and clear separation between TDH and TRH at the amino acid level (percentage identity of between TDH and TRH ranging from 56.1–67.4%). In contrast, protein language model–derived embeddings showed high global functional similarity while preserving distinct clustering patterns, indicating conserved core functionality alongside nuanced divergence echoed with the structural inference by AlphaFold. Importantly, modeling of mutation trajectories demonstrated that the transition from TDH to TRH is driven by accumulated, genome-wide residue changes rather than a small set of key mutations. Together, these results suggested that TDH and TRH represent functionally conserved yet evolutionarily diverged toxins driven by accumulated sequence variation throughout the full-length amino acid sequence. These accumulated point mutations lead to major structural difference: TRH forms an α-helical tail that TDH lacks, which suggests that TDH and TRH disrupt host membranes by different mechanisms despite their conserved core function such as pore-forming and ion flux induction capability. These methods provided unprecedented detailed insights into the functional and structural properties of Vp haemolysin, offering critical information on how multi-dimensional variations in sequence might influence their role in Vp pathogenicity. Insights from this study reinforce the rationale for using Vp strains harboring tdh and trh genes in experimental design for environmental fitness investigation.

The consumption of collard greens (CG) and kale (KA) and different formats of their products, e.g. juices, has increased significantly. However, there have been limited studies focusing on the enhancement of the microbial quality and safety of dark leafy green vegetables (DLGVs), especially large DLGVs, despite the occurrence of several outbreaks. The larger and more rough leaf surface is one major characteristically difference between CG/KA and other DLGVs (e.g. spinach). This study evaluated the antimicrobial effectiveness of acid-based washing (vinegar, lemon juice, and peracetic acid [PAA]) in controlling the populations of native microorganisms and artificially inoculated bacteria (Enterococcus faecium) on CG and KA during washing and storage. The washing procedure included a 5 min soaking (w/v ration 1:50) and a 15 s rinsing. Results showed that PAA (80 ppm, pH ∼ 4.00) ed to the greatest reductions in native fungi (∼1.2 and 1.4 log reductions) and bacteria (∼1.0 and 1.2 log reductions on CG and KA respectively) compared to 35 % vinegar (pH ∼2.75) and 13 % lemon juice wash (pH ∼2.83). PAA-washed CG and lemon juice-washed KA also exhibited the greatest additional reductions in fungal and bacterial populations during storage. E. faecium, used as a pathogen surrogate, had the largest reductions on CG and KA washed by PAA, ∼1.5 and 2.3 log reductions respectively. Lemon juice-washed CG and PAA-washed KA showed the greatest additional reductions in E. faecium during storage. Acid washing showed promising potential in protecting the microbial safety and quality of large DLGVs; the tested washing steps can be used at retail and home settings.

Outbreaks and recalls of Listeria monocytogenes associated with commercial mushrooms have been reported in recent years. Unfortunately, knowledge about the survival of L. monocytogenes on different mushrooms remains limited. This study aims to characterize the survival of L. monocytogenes on growing and harvested Trumpet Royale (Pleurotus eryngii), Alba Clamshell (Hypsizygus tessellatus), and Brown Clamshell (Hypsizygus tessellatus) mushrooms. Mushrooms were spot-inoculated with rifampin-resistant cocktails of L. monocytogenes on the caps with an initial level of ca. 8 and 4 log CFU/mushroom and air-dried for 30 min until no visible inoculum was observed on the inoculated area. The survival of L. monocytogenes was monitored on growing and harvested mushrooms for up to 7 days after inoculation. L. monocytogenes inoculated on growing mushrooms dropped below the limit of detection after day 2 or day 3 regardless of the inoculation levels. L. monocytogenes persisted on harvested mushrooms for up to 7 days regardless of mushroom types and inoculation levels at 4 °C. By the end of 7-day storage, 6.1, 3.8, and 6.2 log CFU/mushroom of L. monocytogenes were recovered from Trumpet Royale, Alba Clamshell, and Brown Clamshell, respectively (inoculation level: ca. 8 log CFU/mushroom). At low inoculation levels (ca. 4 log CFU/mushroom), L. monocytogenes positive samples were found in all types of mushrooms after 7-day storage. The results of this study highlight one key important finding, that is the behavior of L. monocytogenes is significantly different between growing mushrooms and harvested mushrooms. Such information is critical for the development of food safety plans for the mushroom industry from pre-harvest to post-harvest stages.

To address the pressing challenges of economic loss and environmental concerns in the cold-chain sector due to temperature-related degradation and microbial contamination, we introduce reusable hydrogel cooling technology. “Jelly Ice Cubes” (JICs), bio-based hydrogel coolants offering a sustainable and efficient alternative to traditional cooling agents. This study reveals for the first time that the essence of designing any hydrogel coolant hinges on establishing an ideal structure, which maintains freezable water within a heterogeneously distributed closed-cell matrix crucial for stable superior cooling performance. A mathematical model was developed to define the optimal parameters for the structure of a closed-cell matrix, maximizing its water-retaining and heat-absorbing capabilities. Specifically, we explicitly explained how the size of enclosed chambers varies with the changes in biopolymer molecular size and concentration, as well as freezable water content in the hydrogel. Moreover, scalable one-step chemical crosslinking processes were developed based on the optimized structure provided by the models, enabling controllable gelatin crosslinking in hydrogels to achieve the desired structural features. The JICs demonstrate significant promise for decarbonization efforts in many fields, especially by optimizing packaging efficiency in cold-chain logistics. This research not only bridges a significant gap by applying a theory-driven approach to the development of sustainable hydrogel-based cooling technologies, but also sets a new standard for future innovations in the field.

Environmental monitoring of postharvest facilities is an integral part of microbial food safety management. Contamination of foods traced to the processing or packing environment, due to poor cleaning and sanitation (C&S) or to the presence of resident microbial pathogen populations, is not uncommon. Environmental monitoring commonly performed targets specific microorganisms, either a hazard identified as relevant or an indicator, inevitably ignoring the environmental microbiota. In this review, we discuss how a comprehensive view of the microbiota can actually improve our understanding of the effect of C&S programs, of the prevalence and ecology of foodborne pathogens within processing environments, and assist in the identification of contamination sources.

High microbial counts present on dried laver can be of microbial quality and safety concerns. The current two-step drying, and post-drying decontamination methods had limited effects eliminating microorganisms. This work explored the efficacy of pre-drying decontamination methods for wet laver, including washing wet laver with slightly acidic electrolytic water (SAEW) or ozonated water (OW) before drying, or treating wet laver sheets with ultraviolet (UV) during early stage drying. The average APC and coliform in untreated wet laver were similar to 5.69 and 4.34 Log CFU/g. SAEW (100 ppm, 30 min), OW (1 mg/L, 30 min) and UV (5 cm, 30 min) decreased the APC and coliform of laver by 2.56 and 2.59, 2.95 and 2.84, and 1.03 and 1.22 Log CFU/g, respectively. While SAEW (100 ppm, 30 min) and OW wash (0.7 mg/L, 30 min) caused visible color changes to wet laver, the drying process (right after the treatment) mitigated the changes quite significantly. 16S sequencing results showed that OW significantly reduced Flavobacteriaceae in the Bacteroidota phylum, while the SAEW and UV significantly reduced the Bacteroidota and Proteobacteria phyla. Results showed that decontamination of wet laver before drying or during early drying stage was promising for control microbial loads on dried laver.

The increasing reliance on aquaculture for sustainable protein production highlights the need for responsible antibiotic use to manage bacterial infections, particularly in intensive farming systems. This study investigated the effects of three FDA-approved antibiotics (Aquaflor®, Romet®, Terramycin®) at common fish bacterial disease outbreak temperatures (20 °C, 25 °C, and 30 °C) on the microbiome and resistome of aquaculture water using a catfish model system. Metagenomic analyses evaluated the abundance, diversity, and mobility of antimicrobial resistance genes (ARGs) and antibiotic-resistant bacteria (ARB). The impact of temperature on Aquaflor- and Romet-induced changes in ARG abundance, richness, and resistome composition followed a U-shaped trend, with the least effect observed at 25 °C. Of the three antibiotics tested, Terramycin exerted the most significant influence on the water microbiome and resistome, enriching tetracycline resistance genes and co-selecting for floR, sul, and dfrA genes. Temperature also induced notable shifts in the ARB population, with Mantel tests revealing strong correlations between ARG profiles and changes in the overall bacterial community and ARB populations. While certain ARG classes consistently remained associated with specific host phyla, others shifted, highlighting the potential for horizontal gene transfer (HGT) as a critical mechanism for disseminating resistance genes like tet(C), particularly after antibiotic treatment. This is further supported by the observed reduction in plasmid numbers following treatment, which coincided with increased HGT events. Our findings highlight the pivotal role of temperature in influencing resistome dynamics, emphasizing the importance of accounting for environmental factors when applying antibiotics to effectively mitigate antimicrobial resistance in aquaculture systems.

BACKGROUND:Florfenicol is a broad-spectrum antimicrobial approved in many countries for treating bacterial infections in production animals. Although florfenicol has been widely used in the US catfish industry, its impact on the native microbiota within catfish tissues remains largely unknown. Florfenicol treatment is followed by a mandatory withdrawal period to ensure drug residues fall below regulatory limits before harvest. This interval also allows for the potential recovery of the native microbiota. In particular, the skin and gill microbiota have often been overlooked in aquaculture microbiome research. Moreover, the dynamics of microbial communities and resistome profiles following drug withdrawal are still poorly understood, despite their ecological significance. RESULTS:A significant increase in intestinal microbial diversity was observed at the end of the withdrawal period. The highest alpha diversity (Shannon index) was observed in catfish intestines. This increase indicated the restoration of the normal microbiota in catfish intestine. The predominant bacterial phyla shared among catfish gill, intestine, and skin are Proteobacteria (62%), Bacteroidetes (18%), Actinobacteriota (12%), Firmicutes (3%), Patescibacteria (2%), and Verrucomicrobiota (1%). Florfenicol application can have lasting effects through the withdrawal period, particularly altering the intestinal microbial community. CONCLUSION:The result of this study underscores the impact of florfenicol treatment on the bacterial landscape and antibiotic resistance in catfish, highlighting significant changes in microbial composition in the catfish intestine and at the end of the withdrawal period. These findings address the need for monitoring and managing antibiotic resistance in fish farming environments.

Dark leafy green vegetable juices (DLGVJs), prepared with vegetables such as kale and chard, have shown substantial market growth in the past decade due to its profound health benefits. Unfortunately, as an innovative product, there was limited research focusing on DLGVJs. This study aimed to bridge key critical knowledge gaps associated with the microbial quality and shelf life of DLGVJs and elucidate the changes in both physicochemical and microbial properties of different organic DLGVJs during refrigerated storage. Culture-dependent and cultureindependent analytical methods were applied to profile the bacterial and fungal populations in three organic DLGVJs (kale, chard, and collard greens) during refrigeration. Plate count results showed that the initial total aerobic plate counts (APC) ranged from 6.49 f 0.03 to 6.74 f 0.08 Log CFU/mL, and the initial total fungal counts ranged from 5.19 f 0.06 to 6.75 f 0.04 Log CFU/mL in fresh DLGVJs. During refrigerated storage the pH of DLGVJs decreased as the storage time increased and the redness of the juices increased as well. The culturedependent analysis revealed bacterial counts initially increased and then declined, while fungal counts showed an initial decrease followed by an increase. By the end of the storage, the highest APC and fungal population were observed in kale juice and chard juice (7.03 f 0.05 and 6.70 f 0.03 Log CFU/mL) respectively. 16S rRNA sequencing results showed that the dominant bacterial genera of spoiled DLGVJs at the end of storage included Serratia, Leuconostoc, and Hafnia-obesumbacterium, and the major spoilage fungal were composed of Mrakia, Filobasidium, and Vishniacozyma. Results of this study provide insights into the microbial populations present in DLGVJs and the dominant spoilage genera during refrigerated storage, laying the foundation for the development of intervention strategies in the future.

The presence of Vibrio parahaemolyticus ( Vp ) at various stages of seafood production has adversely affected public health and threatened the sustainability of the industry. To address the critical public health threats posed by this prevalent seafood-borne pathogen, this research applied advanced machine learning (ML) and deep learning (DL) algorithms to predict the pathogenic potential of Vp using pangenome data. Utilizing comprehensive pangenomic assemblies and sophisticated ML/DL models, this study achieved robust and precise pathogenic potential prediction of Vp based on source attribution, which provides a novel reliable diagnostic tool facilitating conventional serotyping and virulence gene combination approaches. Based on results, non-core regions in Vp pangenome exhibited useful signals ML models can utilize in pathogenic potential determination process. Tree-based ensemble learning methods (Random Forest and Gradient Boosting Trees) have shown the distinguished performance with AUC score 0.97 based on selected pangenome matrix. Furthermore, Convolutional Neural Network successfully predicted the pathogenic potential of isolates with slightly better performance with AUC score 0.98 based on full pangenome. Critical biological insights revealing critical pathogenic potential-associated genes were retrieved from established ML/DL models: the gene feature weight analysis from Random Forest revealed the importance of accessory genes during Vp evolution (similarly highlighted by Gram-cam analysis of Convolutional Neural Network), which provided potential guidance for future research direction. ### Competing Interest Statement The authors have declared no competing interest.

Dark leafy green vegetables (DLGVs), including kale, chard, and collard greens, have gained increasing attention for their rich nutrient profiles and health benefits, driving the development of derivative products such as dark leafy green vegetable juices (DLGVJs). However, fresh DLGVJs are susceptible to contamination by foodborne pathogens due to inadequate hygiene, improper handling, and cross-contamination. Additionally, temperature abuse during storage and distribution may further compromise their safety. This study investigated the fate of Salmonella, pathogenic Escherichia coli, and Listeria monocytogenes in non-acidified and acidified kale, chard, and collard greens juices when being stored at 4, 10, and 23 °C. Changes in pH and indigenous microbial populations (total aerobic plate count [APC]) were also assessed. Results showed that pH remained stable in acidified DLGVJs at all temperatures, while significant pH declines were observed in non-acidified juices at elevated temperatures. The growth rate of APC increased with rising temperatures, with greater increases observed in non-acidified juices. At 4 °C, all pathogens survived in all juices for over 168 h. At 23 °C, growth of pathogens was observed, particularly in non-acidified chard juice. Temperature abuse could compromise the food safety benefit brought by acidification. Based on results from DMFit, the Baranyi and Roberts model effectively described the fate of most pathogens with R2 ranging from 0.474 to 0.997. These findings highlight the importance of acidification and temperature management in maintaining the microbial safety of DLGVJs and provide insights for developing food safety guidelines to mitigate food safety risks associated with fresh DLGVJs.

Waxing of fresh produce in packing houses ensures product quality, safety, and extended shelf life. However, limited data on the cleaning methods and efficiency of waxing roller brushes, which are critical food contact surfaces, make effective cleaning practices challenging, raising food safety concerns. This study aims to guide cleaning practice through the understanding of structural and material properties of tufted roller brushes (fibered with nylon or horsehair/polyethylene) and the physicochemical properties of representative formulated waxes (carnauba-, vegetable oil-, and mineral oil-based) along with their interactions. Cleaning methods incorporating chemical treatments (neutral cleaner, pH 8.56; alkaline cleaner, pH 12.97) with or without physical treatments (50 degrees C, ultrasound) were evaluated for their effectiveness in removing wet, dried, and buildup wax residues. Results revealed that frequent water rinsing removes wet wax effectively, but dried wax often requires combined treatments. This study also provides critical insights to enhance sustainability, cost efficiency, and food safety in produce packaging and suggests future advancements.

After finishing waxes are applied, citrus fruits are typically dried at 32 to 60°C for 2 to 3 min before final packing. The survival of Listeria monocytogenes, Salmonella, and Enterococcus faecium NRRL B-2354 was evaluated under laboratory conditions on lemons after applying one of four finishing waxes (F4, F6, F8, and F15) followed by an ambient hold or heated (50 or 60°C) drying step. The reduction of inoculated microorganisms during drying was significantly influenced by wax type and temperature with greater reductions at higher temperatures. Greater reductions after waxing and drying at 60°C were observed with L. monocytogenes (2.84–4.44 log) than with Salmonella (1.65–3.67 log), and with Salmonella than with E. faecium (0.99–2.93 log). The survival of Salmonella inoculated at 5.8–5.9 log/fruit on lemons and oranges after applying wax F6 and drying at 60°C was evaluated during storage 4 and 22°C. The reductions of Salmonella after waxing and drying were 1.7 log; additional reductions during storage at 4 or 22°C were 1.40–1.43 or 0.18–0.29 log, respectively, on waxed lemons, and 0.56–1.02 or 0.54–0.57 log, respectively, on waxed oranges. Under pilot scale packinghouse conditions with wax F4, mean and minimum reductions of E. faecium ranged from 2.15 to 2.89 and 1.64 to 2.12 log, respectively. However, E. faecium was recovered by whole-fruit enrichment (limit of detection: 0.60 log CFU/lemon) but not by plating (LOD: 1.3 log CFU/lemon) from uninoculated lemons run with or after the inoculated lemons. The findings should provide useful information to establish and implement packinghouse food safety plans.

Abstract: Worldwide, food losses and waste exert a substantial negative environmental im-pact. The harvesting of Romaine lettuce, for instance, generates significant waste as outer leaves are typically removed from the cores during harvest and left to decompose in the field. Upcycling technologies offer innovative methods to convert these leaves into valuable prod-ucts, enhancing sustainability in food systems. This approach not only mitigates waste during the Romaine lettuce harvest but also reduces contaminants. Furthermore, Romaine lettuce is rich in nutritional value and its phenolic compounds are known for their antioxidant, anti-inflammatory, antilipemic, antidiabetic, and antihypertensive properties. In this review, we ex-plore opportunities for upcycling Romaine lettuce outer leaves and the environmental benefits with consideration of potential contaminants. Understanding the nutritional value and health benefits of Romaine lettuce underscores the importance of sustainable practices in agriculture and food management.

Background Fungal pathogens significantly impact the quality of fruits and vegetables at different stages of the supply chain, leading to substantial food losses. Understanding how these persistent fungal infections occur and progress in postharvest conditions is essential to developing effective control strategies. Results In this study, we developed a reliable and consistent inoculation protocol to simulate disease spread from infected fruits to adjacent healthy fruits during postharvest storage. We tested different combinations of relevant fruit commodities, including oranges, tomatoes, and apples, against impactful postharvest pathogens such as Penicillium digitatum, Penicillium italicum, Botrytis cinerea, and Penicillium expansum. We assessed the efficacy of this protocol using fruits treated with various postharvest methods and multiple isolates for each pathogen. We optimized the source of infected tissue and incubation conditions for each fruit-pathogen combination. Disease incidence and severity were quantitatively evaluated to study infection success and progression. At the final evaluation point, 80% or higher disease incidence rates were observed in all trials except for the fungicide-treated oranges inoculated with fungicide-susceptible Penicillium spp. isolates. Although disease incidence was lower in that particular scenario, it is noteworthy that the pathogen was still able to establish itself under unfavorable conditions, indicating the robustness of our methodology. Finally, we used multispectral imaging to detect early P. digitatum infections in oranges before the disease became visible to the naked eye but after the pathogen was established. Conclusions We developed a non-invasive inoculation strategy that can be used to recreate infections caused by contact or nesting in postharvest. The observed high disease incidence and severity values across fruit commodities and fungal pathogens demonstrate the robustness, efficacy, and reproducibility of the developed methodology. The protocol has the potential to be tailored for other pathosystems. Additionally, this approach can facilitate the study of fruit-pathogen interactions and the assessment of innovative control strategies.