Differential microbiota shift on whole romaine lettuce subjected to source or forward processing and on fresh-cut products during cold storage.

Romaine lettuce in the U.S. is primarily grown in California or Arizona and either processed near the growing regions (source processing) or transported long distance for processing in facilities serving distant markets (forward processing). Recurring outbreaks of Escherichia coli O157:H7 implicating romaine lettuce in recent years, which sometimes exhibited patterns of case clustering in Northeast and Midwest, have raised industry concerns over the potential impact of forward processing on romaine lettuce food safety and quality. In this study, freshly harvested romaine lettuce from a commercial field destined for both forward and source processing channels was tracked from farm to processing facility in two separate trials. Whole-head romaine lettuce and packaged fresh-cut products were collected from both forward and source facilities for microbiological and product quality analyses. High-throughput amplicon sequencing targeting16S rRNA gene was performed to describe shifts in lettuce microbiota. Total aerobic bacteria and coliform counts on whole-head lettuce and on fresh-cut lettuce at different storage times were significantly (p < 0.05) higher for those from the forward processing facility than those from the source processing facility. Microbiota on whole-head lettuce and on fresh-cut lettuce showed differential shifting after lettuce being subjected to source or forward processing, and after product storage. Consistent with the length of pre-processing delays between harvest and processing, the lettuce quality scores of source-processed romaine lettuce, especially at late stages of 2-week storage, was significantly higher than of forward-processed product (p < 0.05).

Surveillance of pathogenic bacteria on a food matrix using machine-learning-enabled paper chromogenic arrays.

Global food systems can benefit significantly from continuous monitoring of microbial food safety, a task for which tedious operations, destructive sampling, and the inability to monitor multiple pathogens remain challenging. This study reports significant improvements to a paper chromogenic array sensor - machine learning (PCA-ML) methodology sensing concentrations of volatile organic compounds (VOCs) emitted on a species-specific basis by pathogens by streamlining dye selection, sensor fabrication, database construction, and machine learning and validation. This approach enables noncontact, time-dependent, simultaneous monitoring of multiple pathogens (Listeria monocytogenes, Salmonella, and E. coli O157:H7) at levels as low as 1 log CFU/g with over 90% accuracy. The report provides theoretical and practical frameworks demonstrating that chromogenic response, including limits of detection, depends on time integrals of VOC concentrations. The paper also discusses the potential for implementing PCA-ML in the food supply chain for different food matrices and pathogens, with species- and strain-specific identification.

The effect of mixed culture fermentation of Saccharomyces cerevisiae and Saccharomyces cerevisiae var. diastaticus on fermentation parameters and flavor profile.

Belgian Saisons and Lambics are two well-known examples in the brewing industry of mixed fermentations, combination of two or more yeast and/or bacteria strains. The purpose of this study was to determine the impact different pitch rates of Saccharomyces cerevisiae (traditional brewing yeast) and S. cerevisiae var. diastaticus (a variant associated with Belgian styles) had on the fermentation kinetics and concentration of the volatile compounds in the finished beers. A series of brews were performed utilizing ratios of S. cerevisiae and diastaticus. The fermentations were heavily monitored, and a model was used to fit fermentation variables. It was found that mixed fermentations produced behaviors that were predictable and proportional to the mixture ratios. As expected, the pure cultural fermentations of diastaticus had a slower fermentation midpoint (M) at 45.45 h versus 28.28 h for S. cerevisiae with the mixed ones falling in between the two. Flavor and aroma play a key role in the acceptability of beer. The mixed fermentations showed a combination of the two different yeast strains aromatic profiles. When combined, there was a strong linearity between alcohols (R2  = 0.94), esters (R2  = 0.89), and the overall total (R2  = 0.91) volatile compounds. PRACTICAL APPLICATION: Modeling is a widely utilized tool in several different fields. The purpose of this research is to apply modeling techniques to describe the fermentation speed and flavor profile of a mixed fermentation between S. cerevisiae and diastaticus. The equations from this data can be used by brewers for product development purposes to make beers with certain flavor profiles within a desired timeframe.

Uncovering Interactions between Plant Metabolism and Plant-Associated Bacteria in Huanglongbing-Affected Citrus Cultivars Using Multiomics Analysis and Machine Learning.

Huanglongbing (HLB) is a highly destructive disease that inflicts significant economic losses on the citrus industry worldwide but with no cure available. However, microbiomes formulated by citrus plants may serve as disease antagonists, increasing the level of HLB tolerance. This study established an integrated analysis of untargeted metabolomics and microbiomics data for different citrus cultivars, providing critical insights into the interactions between plant metabolism and plant-associated bacteria in the development of HLB. Machine learning models were applied to screen important metabolites and bacteria in multiple citrus materials, and the selected metabolites were then analyzed to identify essential pathways enriched in the plant and to correlate with the selected bacteria. Results demonstrated that the regulation of plant pathways, especially ABC transporters and ubiquinone and other terpene-ubiquinone biosynthesis pathways, could affect the microbial community structure, indicating potential solutions for controlling HLB by modulating bacteria in citrus plants or breeding tolerant citrus cultivars.

Nondestructive and multiplex differentiation of pathogenic microorganisms from spoilage microflora on seafood using paper chromogenic array and neural network.

Non-destructive detection of human foodborne pathogens is critical to ensuring food safety and public health. Here, we report a new method using a paper chromogenic array coupled with a machine learning neural network (PCA-NN) to detect viable pathogens in the presence of background microflora and spoilage microbe in seafood via volatile organic compounds sensing. Morganella morganii and Shewanella putrefaciens were used as the model pathogen and spoilage bacteria. The study evaluated microbial detection in monoculture and cocktail multiplex detection. The accuracy of PCA-NN detection was first assessed on standard media and later validated on cod and salmon as real seafood models with pathogenic and spoilage bacteria, as well as background microflora. In this study PCA-NN method successfully identified pathogenic microorganisms from microflora with or without the prevalent spoilage microbe, Shewanella putrefaciens in seafood, with accuracies ranging from 90% to 99%. This approach has the potential to advance smart packaging by achieving nondestructive pathogen surveillance on food without enrichment, incubation, or other sample preparation.

Long-term quality retention and decay inhibition of chestnut using thymol loaded chitosan nanoparticle.

Thymol (TMO) was loaded into chitosan nanoparticles (CSNPs) to inhibit chestnuts decay during storage. Three chestnut treatments were evaluated, including the CK (uncoated control), CSNPs (coated with chitosan nanoparticles), and TMO-CSNPs (coated with thymol-loaded chitosan nanoparticles). Quality assessments of chestnuts were conducted periodically for up to 180 days, which included starch content, amylase activity, water content, respiration rate, weight loss rate, microbiological indicators, decay rate, and quality evaluation. Results indicated that TMO-CSNPs had significantly less nutrient loss in soluble sugar (10.61%) and starch content (27.72%) compared with CK, which was attributed to low metabolic activities as evident in low amylase activity and respiration rate. Moreover, TMO-CSNPs significantly inhibited the growth of mold and yeast (4.17 log CFU g-1 on day 180) and kept the lowest decay rate (5.33%). This study demonstrates the potential of food nanomaterial as an alternative strategy to promote food security and supply chain resilience.

Nanopore Whole Transcriptome Analysis and Pathogen Surveillance by a Novel Solid-Phase Catalysis Approach.

The requirement of a large input amount (500 ng) for Nanopore direct RNA-seq presents a major challenge for low input transcriptomic analysis and early pathogen surveillance. The high RNA input requirement is attributed to significant sample loss associated with library preparation using solid-phase reversible immobilization (SPRI) beads. A novel solid-phase catalysis strategy for RNA library preparation to circumvent the need for SPRI bead purification to remove enzymes is reported here. This new approach leverages concurrent processing of non-polyadenylated transcripts with immobilized poly(A) polymerase and T4 DNA ligase, followed by directly loading the prepared library onto a flow cell. Whole transcriptome sequencing, using a human pathogen Listeria monocytogenes as a model, demonstrates this new method displays little sample loss, takes much less time, and generates higher sequencing throughput correlated with reduced nanopore fouling compared to the current library preparation for 500 ng input. Consequently, this approach enables Nanopore low-input direct RNA-seq, improving pathogen detection and transcript identification in a microbial community standard with spike-in transcript controls. Besides, as evident in the bioinformatic analysis, the new method provides accurate RNA consensus with high fidelity and identifies higher numbers of expressed genes for both high and low input RNA amounts.

Zinc Exposure Promotes Commensal-to-Pathogen Transition in Pseudomonas aeruginosa Leading to Mucosal Inflammation and Illness in Mice.

The opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) is associated gastrointestinal (GI) inflammation and illness; however, factors motivating commensal-to-pathogen transition are unclear. Excessive zinc intake from supplements is common in humans. Due to the fact that zinc exposure enhances P. aeruginosa colonization in vitro, we hypothesized zinc exposure broadly activates virulence mechanisms, leading to inflammation and illness. P. aeruginosa was treated with excess zinc and growth, expression and secretion of key virulence factors, and biofilm production were determined. Effects on invasion, barrier function, and cytotoxicity were evaluated in Caco-2 cells co-cultured with P. aeruginosa pre-treated with zinc. Effects on colonization, mucosal pathology, inflammation, and illness were evaluated in mice infected with P. aeruginosa pre-treated with zinc. We found the expression and secretion of key virulence factors involved in quorum sensing (QS), motility (type IV pili, flagella), biosurfactants (rhamnolipids), toxins (exotoxin A), zinc homeostasis (CzcR), and biofilm production, were all significantly increased. Zinc exposure significantly increased P. aeruginosa invasion, permeability and cytotoxicity in Caco-2 cells, and enhanced colonization, inflammation, mucosal damage, and illness in mice. Excess zinc exposure has broad effects on key virulence mechanisms promoting commensal-to-pathogen transition of P. aeruginosa and illness in mice, suggesting excess zinc intake may have adverse effects on GI health in humans.

Effect of Protein Adsorption on Air Plastron Behavior of a Superhydrophobic Surface.

Protein fouling on critical biointerfaces causes significant public health and clinical ramifications. Multiple strategies, including superhydrophobic (SHP) surfaces and coatings, have been explored to mitigate protein adsorption on solid surfaces. SHP materials with underwater air plastron (AP) layers hold great promise by physically reducing the contact area between a substrate and protein molecules. However, sustaining AP stability or lifetime is crucial in determining the durability and long-term applications of SHP materials. This work investigated the effect of protein on the AP stability using model SHP substrates, which were prepared from a mixture of silica nanoparticles and epoxy. The AP stability was determined using a submersion test with real-time visualization. The results showed that AP stability was significantly weakened by protein solutions compared to water, which could be attributed to the surface tension of protein solutions and protein adsorption on SHP substrates. The results were further examined to reveal the correlation between protein fouling and accelerated AP dissipation on SHP materials by confocal fluorescent imaging, surface energy measurement, and surface robustness modeling of the Cassie-Baxter to Wenzel transition. The study reveals fundamental protein adsorption mechanisms on SHP materials, which could guide future SHP material design to better mitigate protein fouling on critical biointerfaces.

Nondestructive multiplex detection of foodborne pathogens with background microflora and symbiosis using a paper chromogenic array and advanced neural network.

We have developed an inexpensive, standardized paper chromogenic array (PCA) integrated with a machine learning approach to accurately identify single pathogens (Listeria monocytogenes, Salmonella Enteritidis, or Escherichia coli O157:H7) or multiple pathogens (either in multiple monocultures, or in a single cocktail culture), in the presence of background microflora on food. Cantaloupe, a commodity with significant volatile organic compound (VOC) emission and large diverse populations of background microflora, was used as the model food. The PCA was fabricated from a paper microarray via photolithography and paper microfluidics, into which 22 chromogenic dye spots were infused and to which three red/green/blue color-standard dots were taped. When exposed to VOCs emitted by pathogens of interest, dye spots exhibited distinguishable color changes and pattern shifts, which were automatically segmented and digitized into a ΔR/ΔG/ΔB database. We developed an advanced deep feedforward neural network with a learning rate scheduler, L2 regularization, and shortcut connections. After training on the ΔR/ΔG/ΔB database, the network demonstrated excellent performance in identifying pathogens in single monocultures, multiple monocultures, and in cocktail culture, and in distinguishing them from the background signal on cantaloupe, providing accuracy of up to 93% and 91% under ambient and refrigerated conditions, respectively. With its combination of speed, reliability, portability, and low cost, this nondestructive approach holds great potential to significantly advance culture-free pathogen detection and identification on food, and is readily extendable to other food commodities with complex microflora.

Machine learning-enabled non-destructive paper chromogenic array detection of multiplexed viable pathogens on food.

Fast and simultaneous identification of multiple viable pathogens on food is critical to public health. Here we report a pathogen identification system using a paper chromogenic array (PCA) enabled by machine learning. The PCA consists of a paper substrate impregnated with 23 chromogenic dyes and dye combinations, which undergo colour changes on exposure to volatile organic compounds emitted by pathogens of interest. These colour changes are digitized and used to train a multi-layer neural network (NN), endowing it with high-accuracy (91-95%) strain-specific pathogen identification and quantification capabilities. The trained PCA-NN system can distinguish between viable Escherichia coli, E. coli O157:H7 and other viable pathogens, and can simultaneously identify both E. coli O157:H7 and Listeria monocytogenes on fresh-cut romaine lettuce, which represents a realistic and complex environment. This approach has the potential to advance non-destructive pathogen detection and identification on food, without enrichment, culturing, incubation or other sample preparation steps.

Direct Metatranscriptome RNA-seq and Multiplex RT-PCR Amplicon Sequencing on Nanopore MinION - Promising Strategies for Multiplex Identification of Viable Pathogens in Food.

Viable pathogenic bacteria are major biohazards that pose a significant threat to food safety. Despite the recent developments in detection platforms, multiplex identification of viable pathogens in food remains a major challenge. A novel strategy is developed through direct metatranscriptome RNA-seq and multiplex RT-PCR amplicon sequencing on Nanopore MinION to achieve real-time multiplex identification of viable pathogens in food. Specifically, this study reports an optimized universal Nanopore sample extraction and library preparation protocol applicable to both Gram-positive and Gram-negative pathogenic bacteria, demonstrated using a cocktail culture of E. coli O157:H7, Salmonella enteritidis, and Listeria monocytogenes, which were selected based on their impact on economic loss or prevalence in recent outbreaks. Further evaluation and validation confirmed the accuracy of direct metatranscriptome RNA-seq and multiplex RT-PCR amplicon sequencing using Sanger sequencing and selective media. The study also included a comparison of different bioinformatic pipelines for metatranscriptomic and amplicon genomic analysis. MEGAN without rRNA mapping showed the highest accuracy of multiplex identification using the metatranscriptomic data. EPI2ME also demonstrated high accuracy using multiplex RT-PCR amplicon sequencing. In addition, a systemic comparison was drawn between Nanopore sequencing of the direct metatranscriptome RNA-seq and RT-PCR amplicons. Both methods are comparable in accuracy and time. Nanopore sequencing of RT-PCR amplicons has higher sensitivity, but Nanopore metatranscriptome sequencing excels in read length and dealing with complex microbiome and non-bacterial transcriptome backgrounds.

Is "nano safe to eat or not"? A review of the state-of-the art in soft engineered nanoparticle (sENP) formulation and delivery in foods.

With superior physicochemical properties, soft engineered nanoparticles (sENP) (protein, carbohydrate, lipids and other biomaterials) are widely used in foods. The preparation, functionalities, applications, transformations in gastrointestinal (GI) tract, and effects on gut microbiota of sENP directly incorporated for ingestion are reviewed herein. At the time of this review, there is no notable report of safety concerns of these nanomaterials found in the literature. Meanwhile, various beneficial effects have been demonstrated for the application of sENP. To address public perception and safety concerns of nanoscale materials in food, methodologies for evaluation of physiological effects of nanomaterials are reviewed. The combination of these complementary methods will be useful for the establishment of a comprehensive risk assessment system.

An immune magnetic nano-assembly for specifically amplifying intercellular quorum sensing signals.

Quorum sensing (QS) enables intercellular communication after bacterial cells sense the autoinducers have reached or exceeded a critical concentration. Selectively amplifying specific bacterial "quorum" activity at a lower cell density is still a challenge. Here, we propose a novel platform of immune magnetic nano-assembly to amplify specific bacterial QS signaling via improving the bioavailability of autoinducers-2 (AI-2, furanosyl borate) from sender (wide-type, WT cells) to receiver (reporter cells). Antibody coated magnetic nanoparticle (MNPAB) was fabricated with an average diameter of 12 nm and a specific surface area of 96.5 m2/g. The distribution efficiency of the antibody on the surface was 25.8 µg/m2 of magnetic nanoparticles. It was found that more than 3 × 108 of K12 serotype Escherichia coli (E. coli) reporter or WT cells were collected using 1 mg fabricated MNPAB at a saturated condition. The MNPAB not only captured E. coli WT cells but also brought them into proximity of E. coli (CT104, pCT6+pET-DsRed) reporter cells via magnetic attraction. The amplified QS signaling of the reporter cells by this immune magnetic nano-assembly was approximately 3 times higher than the nature QS signaling in cell suspension at optical density (OD) 0.08. This study foresees potential applications in amplifying specific biological QS signals based on a preprogrammed design.

Alternatives to carcinogenic preservatives in Chinese Sausage - Sorbic acid-loaded chitosan/tripolyphosphate nanoparticles.

Preservatives in processed meat raise significant concerns associated with bowel cancer and diabetes, and implicate various public health issues. This introduces the need for safer preservatives to uphold public health standards. However, developing safer alternatives to these preservatives poses a significant challenge to food industry. A potential solution to this issue is the use of loaded nanoparticles as preservative agents. This study investigated antimicrobial and antioxidant effects of sorbic acid-loaded chitosan/tripolyphosphate nanoparticles (SAN) in Chinese Sausage. SAN were prepared through ionic gelation, followed by natural air-drying for 20 days. After preparation, the antimicrobial and antioxidant activities of various treatment groups were analyzed intermittently during storage at room temperature. SAN-treated samples had significantly lower levels of surviving bacteria, molds, and yeasts than the blank control (p < 0.05) over the entire 72 days of storage. Additionally the SAN-treated samples also had lower levels of surviving bacteria than the chitosan/tripolyphosphate samples after 31-56 days of storage (p < 0.05). The thiobarbituric acid value and pH of the SAN-treated samples were also significantly lower than the blank control (p < 0.05). These results indicated that SAN could be a good intervention strategy to retard lipid oxidation and inhibit microbial growth in Chinese Sausage.

Focusing quorum sensing signalling by nano-magnetic assembly.

Quorum sensing (QS) exists widely among bacteria, enabling a transition to multicellular behaviour after bacterial populations reach a particular density. The coordination of multicellularity enables biotechnological application, dissolution of biofilms, coordination of virulence, and so forth. Here, a method to elicit and subsequently disperse multicellular behaviour among QS-negative cells is developed using magnetic nanoparticle assembly. We fabricated magnetic nanoparticles (MNPs, ∼5 nm) that electrostatically collect wild-type (WT) Escherichia coli BL21 cells and brings them into proximity of bioengineered E. coli [CT104 (W3110 lsrFG- luxS- pCT6 + pET-DsRed)] reporter cells that exhibit a QS response after receiving autoinducer-2 (AI-2). By shortening the distance between WT and reporter cells (e.g., increasing local available AI-2 concentrations), the QS response signalling was amplified four-fold compared to that in native conditions without assembly. This study suggests potential applications in facilitating intercellular communication and modulating multicellular behaviours based on user-specified designs.

Facile and template-free solvothermal synthesis of mesoporous/macroporous metal-organic framework nanosheets.

A facile and template-free solvothermal method was developed as a bottom-up approach to synthesize mesoporous/macroporous MOF nanosheets in a simple and scalable way. It was found that starting coordination complexes of different copper(ii)-ligand compounds mediated the controlled growth and morphology of MOF crystals. By controlling the size and shape of the MOF crystals, the possibility to adjust and tailor the structure and performances of the assemblies was demonstrated. This work provides a bottom-up approach to synthesize MOF films and nanosheets in a simple and scalable way, which may have potential in energy and biomedical applications.

Association between bacterial survival and free chlorine concentration during commercial fresh-cut produce wash operation.

Determining the minimal effective free chlorine (FC) concentration for preventing pathogen survival and cross-contamination during produce washing is critical for developing science- and risk-based food safety practices. The correlation between dynamic FC concentrations and bacterial survival was investigated during commercial washing of chopped Romaine lettuce, shredded Iceberg lettuce, and diced cabbage as pathogen inoculation study during commercial operation is not feasible. Wash water was sampled every 30 min and assayed for organic loading, FC, and total aerobic mesophilic bacteria after chlorine neutralization. Water turbidity, chemical oxygen demand, and total dissolved solids increased significantly over time, with more rapid increases in diced cabbage water. Combined chlorine increased consistently while FC fluctuated in response to rates of chlorine dosing, product loading, and water replenishment. Total bacterial survival showed a strong correlation with real-time FC concentration. Under approximately 10 mg/L, increasing FC significantly reduced the frequency and population of surviving bacteria detected. Increasing FC further resulted in the reduction of the aerobic plate count to below the detection limit (50 CFU/100 mL), except for a few sporadic positive samples with low cell counts. This study confirms that maintaining at least 10 mg/L FC in wash water strongly reduced the likelihood of bacterial survival and thus potential cross contamination of washed produce.

Investigation on chlorine-based sanitization under stabilized conditions in the presence of organic load.

Chlorine, the most commonly used sanitizer for fresh produce washing, has constantly shown inferior sanitizing efficacy in the presence of organic load. Conventionally this is attributed indirectly to the rapid chlorine depletion by organics leading to fluctuating free chlorine (FC) contents. However, little is known on whether organic load affects the sanitization process directly at well-maintained FC levels. Hereby, a sustained chlorine decay approach was employed to study the inactivation of Escherichia coli O157:H7 under stabilized washing conditions. Chlorine solution was first incubated with organic load for up to 4h, modeling the chlorination in produce washing lines. The FC level was then stabilized at five targeted values for sanitization study. Our study showed decreased sanitizing efficacy as the organic load increased. At 5s residence time and pH6.5, a minimum of 0.5 and 7.5mg/L FC were needed to achieve a 5 log reduction at 0 and 900mg/L chemical oxygen demand (COD), respectively. The decrease was more pronounced at lower FC, higher COD, higher pH, and shorter residence time values. The organics-associated interference with FC measurement and disruption of chlorine/bacteria interaction, together with the chlorine demand of concentrated inoculum per se, collectively resulted in inadequate sanitization. Finally, our results were compared with existing studies conducted under dynamic conditions in the context of different experimental settings. This study provided a feasible method for studying the bacteria/sanitizer interaction while ruling out the confounding effect from fluctuating FC levels, and it indicated the direct, negative impact of organic load.

Development of Metal-Organic Framework for Gaseous Plant Hormone Encapsulation To Manage Ripening of Climacteric Produce.

Controlled ripening of climacteric fruits, such as bananas and avocados, is a critical step to provide consumers with high-quality products while reducing postharvest losses. Prior to ripening, these fruits can be stored for an extended period of time but are usually not suitable for consumption. However, once ripening is initiated, they undergo irreversible changes that lead to rapid quality loss and decay if not consumed within a short window of time. Therefore, technologies to slow the ripening process after its onset or to stimulate ripening immediately before consumption are in high demand. In this study, we developed a solid porous metal-organic framework (MOF) to encapsulate gaseous ethylene for subsequent release. We evaluated the feasibility of this technology for on-demand stimulated ripening of bananas and avocados. Copper terephthalate (CuTPA) MOF was synthesized via a solvothermal method and loaded with ethylene gas. Its crystalline structure and chemical composition were characterized by X-ray diffraction crystallography, porosity by N2 and ethylene isotherms, and morphology by electron microscopy. The MOF loaded with ethylene (MOF-ethylene) was placed inside sealed containers with preclimacteric bananas and avocados and stored at 16 °C. The headspace gas composition and fruit color and texture were monitored periodically. Results showed that this CuTPA MOF is highly porous, with a total pore volume of 0.39 cm(3)/g. A 50 mg portion of MOF-ethylene can absorb and release up to 654 µL/L of ethylene in a 4 L container. MOF-ethylene significantly accelerated the ripening-related color and firmness changes of treated bananas and avocados. This result suggests that MOF-ethylene technology could be used for postharvest application to stimulate ripening just before the point of consumption.