A longitudinal study on the bacterial quality of baby spinach cultivated in Arizona and California.

In the U.S., baby spinach is mostly produced in Arizona (AZ) and California (CA). Characterizing the impact of growing region on the bacterial quality of baby spinach can inform quality management practices in industry. Between December 2021 and December 2022, baby spinach was sampled after harvest and packaging for microbiological testing, including shelf-life testing of packaged samples that were stored at 4°C. Samples were tested to (i) determine bacterial concentration, and (ii) obtain and identify bacterial isolates. Packaged samples from the Salinas, CA, area (n = 13), compared to those from the Yuma, AZ, area (n = 9), had a significantly higher bacterial concentration, on average, by 0.78 log10 CFU/g (P < 0.01, based on aerobic, mesophilic plate count data) or 0.67 log10 CFU/g (P < 0.01, based on psychrotolerant plate count data); the bacterial concentrations of harvest samples from the Yuma and Salinas areas were not significantly different. Our data also support that an increase in preharvest temperature is significantly associated with an increase in the bacterial concentration on harvested and packaged spinach. A Fisher's exact test and linear discriminant analysis (effect size), respectively, demonstrated that (i) the genera of 2,186 bacterial isolates were associated (P < 0.01) with growing region and (ii) Pseudomonas spp. and Exiguobacterium spp. were enriched in spinach from the Yuma and Salinas areas, respectively. Our findings provide preliminary evidence that growing region and preharvest temperature may impact the bacterial quality of spinach and thus could inform more targeted strategies to manage produce quality. IMPORTANCE: In the U.S., most spinach is produced in Arizona (AZ) and California (CA) seasonally; typically, spinach is cultivated in the Yuma, AZ, area during the winter and in the Salinas, CA, area during the summer. As the bacterial quality of baby spinach can influence consumer acceptance of the product, it is important to assess whether the bacterial quality of baby spinach can vary between spinach-growing regions. The findings of this study provide insights that could be used to support region-specific quality management strategies for baby spinach. Our results also highlight the value of further evaluating the impact of growing region and preharvest temperature on the bacterial quality of different produce commodities.

Programmable RNA detection with a fluorescent RNA aptamer using optimized three-way junction formation.

RNAs play essential roles in various cellular processes and can be used as biomarkers. Hence, it is important to detect endogenous RNA for understanding diverse cellular functions and diagnosing diseases. To construct a low-cost and easy-to-use RNA detection probe, a chemically unmodified RNA aptamer that binds to a pro-fluorophore to increase its fluorescence is desirable. Here, we focused on Broccoli, a superior variant of Spinach, which is a well-known fluorescent RNA aptamer that binds to DFHBI-1T and emits green fluorescence. We experimentally characterized Broccoli and predicted that it forms a G-quadruplex-based DFHBI-1T recognition region sandwiched between two stems. Based on this, we designed a Broccoli-based RNA detection probe (BRD probe) composed of a sequence of destabilized Broccoli fused with complementary sequences against target RNA. The resulting probe with its target RNA formed a stable three-way junction, named the MT2 three-way junction, which contributed to efficient refolding of the Broccoli structure and allowed for programmable RNA detection with high signal-to-noise ratio and sensitivity. Interestingly, the MT2 three-way junction also could be applied to probe construction of a truncated form of Spinach (Baby Spinach). The BRD and Baby Spinach-based RNA detection probes (BSRD probe) exhibited up to 48- and 140-fold fluorescence enhancements in the presence of their target RNAs and detected small amounts of target RNA that were as low as 160 and 5 nM, respectively. Thus, we experimentally characterized the higher order structure of Broccoli and developed structure-switching aptamer probes for highly sensitive, programmable, RNA detection using an MT2 three-way junction.

Neutral electrolyzed oxidizing water is effective for pre-harvest decontamination of fresh produce.

Pre-harvest sanitization of irrigation water has potential for reducing pathogen contamination of fresh produce. We compared the sanitizing effects of irrigation water containing neutral electrolyzed oxidizing water (EOW) or sodium hypochlorite (NaClO) on pre-harvest lettuce and baby spinach leaves artificially contaminated with a mixture of Escherichia coli, Salmonella Enteritidis and Listeria innocua (~1 × 108 colony-forming units/mL each resuspended in water containing 100 mg/L dissolved organic carbon, simulating a splash-back scenario from contaminated soil/manure). The microbial load and leaf quality were assessed over 7 days, and post-harvest shelf life evaluated for 10 days. Irrigation with water containing EOW or NaClO at 50 mg/L free chlorine significantly reduced the inoculated bacterial load by ≥ 1.5 log10, whereas tap water irrigation reduced the inoculated bacterial load by an average of 0.5 log10, when compared with untreated leaves. There were no visual effects of EOW or tap water irrigation on baby spinach or lettuce leaf surfaces pre- or post-harvest, whereas there were obvious negative effects of NaClO irrigation on leaf appearance for both plants, including severe necrotic zones and yellowing/browning of leaves. Therefore, EOW could serve as a viable alternative to chemical-based sanitizers for pre-harvest disinfection of minimally processed vegetables.

A pH-neutral electrolyzed oxidizing water significantly reduces microbial contamination of fresh spinach leaves.

There are growing demands globally to use safe, efficacious and environmentally friendly sanitizers for post-harvest treatment of fresh produce to reduce or eliminate spoilage and foodborne pathogens. Here, we compared the efficacy of a pH-neutral electrolyzed oxidizing water (Ecas4 Anolyte; ECAS) with that of an approved peroxyacetic acid-based sanitizer (Ecolab Tsunami® 100) in reducing the total microbial load and inoculated Escherichia coli, Salmonella Enteritidis and Listeria innocua populations on post-harvest baby spinach leaves over 10 days. The impact of both sanitizers on the overall quality of the spinach leaves during storage was also assessed by shelf life and vitamin C content measurements. ECAS at 50 ppm and 85 ppm significantly reduced the bacterial load compared to tap water-treated or untreated (control) leaves, and at similar levels (approx. 10-fold reduction) to those achieved using 50 ppm of Ecolab Tsunami® 100. While there were no obvious deleterious effects of treatment with 50 ppm Tsunami® 100 or ECAS at 50 ppm and 85 ppm on plant leaf appearance, tap water-treated and untreated leaves showed some yellowing, bruising and sliming. Given its safety, efficacy and environmentally-friendly characteristics, ECAS could be a viable alternative to chemical-based sanitizers for post-harvest treatment of fresh produce.

Isolation, characterization, and application of a lytic bacteriophage SSP49 to control Staphylococcus aureus contamination on baby spinach leaves.

Staphylococcus aureus, a major foodborne pathogen, is frequently detected in fresh produce. It often causes food poisoning accompanied by abdominal pain, diarrhea, and vomiting. Additionally, the abuse of antibiotics to control S. aureus has resulted in the emergence of antibiotics-resistant bacteria, such as methicillin resistant S. aureus. Therefore, bacteriophage, a natural antimicrobial agent, has been suggested as an alternative to antibiotics. In this study, a lytic phage SSP49 that specifically infects S. aureus was isolated from a sewage sample, and its morphological, biological, and genetic characteristics were determined. We found that phage SSP49 belongs to the Straboviridae family (Caudoviricetes class) and maintained host growth inhibition for 30 h in vitro. In addition, it showed high host specificity and a broad host range against various S. aureus strains. Receptor analysis revealed that phage SSP49 utilized cell wall teichoic acid as a host receptor. Whole genome sequencing revealed that the genome size of SSP49 was 137,283 bp and it contained 191 open reading frames. The genome of phage SSP49 did not contain genes related to lysogen formation, bacterial toxicity, and antibiotic resistance, suggesting its safety in food application. The activity of phage SSP49 was considerably stable under various high temperature and pH conditions. Furthermore, phage SSP49 effectively inhibited S. aureus growth on baby spinach leaves both at 4 °C and 25 °C while maintaining the numbers of active phage during treatments (reductions of 1.2 and 2.1 log CFU/cm2, respectively). Thus, this study demonstrated the potential of phage SSP49 as an alternative natural biocontrol agent against S. aureus contamination in fresh produce.

Metagenetic characterization of bacterial communities associated with ready-to-eat leafy vegetables and study of temperature effect on their composition during storage.

Ready-to-eat (RTE) and fresh-cut vegetables meet the current needs for healthy and easy-to-prepare food. However, raw vegetables are widely known to harbor large and diverse bacterial communities promoting spoilage and reducing their shelf-life. A better understanding of their bacterial community and the impact of various environmental factors on its composition is essential to ensure the production of high-quality fresh-cut produce. Therefore, a metagenetic amplicon approach, based on gyrB sequencing, was applied for deciphering the bacterial communities associated with the spoilage of RTE rocket and baby spinach and monitoring the changes occurring in their composition during storage at different temperatures. Our results indicated that Pseudomonas genus was the main spoilage group for both leafy vegetables. Specifically, Pseudomonas viridiflava was dominant in most samples of rocket, while a new Pseudomonas species as well as, Pseudomonas fluorescens and/or Pseudomonas fragi were highly abundant in baby spinach. A significant variability on bacterial species composition among different batches of each vegetable type was observed. In the case of baby spinach, the impact of temperature and/or storage time on bacterial microbiota was not explicitly revealed at batch-level. Concerning rocket, the storage time was the most influential factor resulting in the reduction of Pseudomonas species' abundances and the parallel increase of lactic acid bacteria abundances. The results suggest that a large-scale sampling and further investigation of the various environmental factors shaping the microbiota are needed for gaining deeper knowledge of the diverse bacterial communities on RTE leafy vegetables and thus, enhance the quality of these products.

Antimicrobial action of octanoic acid against Escherichia coli O157:H7 during washing of baby spinach and grape tomatoes.

We investigated the antimicrobial efficacy of octanoic acid (OA) against Escherichia coli O157:H7 inoculated on the surface of baby spinach and grape tomatoes during simulated washing processes. 3 mM OA at 45 °C achieved >6 log CFU/g reduction from the surface of tomatoes within 2 min. However, washing baby spinach with 6 mM OA at 5 °C resulted in <1 log CFU/g reduction, highlighting the role of surface properties in inactivation efficacy. OA significantly (p < 0.05) reduced the risk of cross-contamination during washing of spinach as well as tomatoes. Also, total mold and yeast population on surface of spinach was significantly reduced immediately after OA wash and inhibited during following 14 days. Baby spinach and grape tomatoes washed with OA did not cause significant (p > 0.05) difference in color compared to the control and no residual OA was detected in most cases following rinsing of produce in water. OA at the concentrations above 2 mM and temperature higher than 25 °C induced severe membrane damage along with release of ATP and other intracellular constituents resulting in bacterial death. OA can be an attractive natural decontamination agent for washing fresh produce.

Towards the Next-Generation Disinfectant: Composition, Storability and Preservation Potential of Plasma Activated Water on Baby Spinach Leaves.

Plasma activated water (PAW) has rapidly emerged as a promising alternative to traditional sanitizers applied in the fresh produce industry. In the present study, PAW chemistry and storage stability were assessed as a function of plasma operating conditions. Increasing plasma exposure time (5, 12.5, 20 min) and power (16, 26, 36 W) led to a significant drop in pH (2.4) and higher nitrates and nitrites levels (320 and 7.2 mg/L, respectively) in the PAW. Non-detectable hydrogen peroxide concentration, irrespective of the treatment conditions, was attributed to its instability in acidic environments and the remote PAW generation mode. pH, nitrates and nitrites levels in the PAW remained unaffected after two weeks at 4 °C. The potential of PAW for microbial inactivation and quality retention was demonstrated on baby spinach leaves. Rinsing steps influenced colour development during chilled storage to a greater extent than PAW treatment itself. About 1 log reduction in total bacterial counts (5 log CFU/g) was achieved through PAW rinsing, with no variability after eight days at 4 °C (typical shelf-life at retailers). Moreover, microbial levels on PAW-treated samples after storage were significantly lower than those on control samples, thus contributing to extended product shelf-life and reduced food waste generation.

Impact of Sweet Potato Starch-Based Nanocomposite Films Activated With Thyme Essential Oil on the Shelf-Life of Baby Spinach Leaves.

Salmonella Typhimurium (S. Typhi) and Escherichia coli (E. coli) have been responsible for an increasing number of outbreaks linked to fresh produce, such as baby spinach leaves, in the last two decades. More recently, antimicrobial biodegradable packaging systems have been attracting much attention in the food packaging industry as eco-friendly alternatives to conventional plastic packaging. The objective of this study was to evaluate the effect of antibacterial nanocomposite films on inoculated spinach leaves and on the sensory properties of these leaves during eight days of refrigerated storage. In this study, an antibacterial film comprised of sweet potato starch (SPS), montmorillonite (MMT) nanoclays and thyme essential oil (TEO) as a natural antimicrobial agent was developed. Our results showed that the incorporation of TEO in the film significantly (p < 0.05) reduced the population of E. coli and S. Typhi on fresh baby spinach leaves to below detectable levels within five days, whereas the control samples without essential oil maintained approximately 4.5 Log colony forming unit (CFU)/g. The sensory scores for spinach samples wrapped in films containing TEO were higher than those of the control. This study thus suggests that TEO has the potential to be directly incorporated into a SPS film to prepare antimicrobial nanocomposite films for food packaging applications.

Fate of Listeria monocytogenes , Pathogenic Yersinia enterocolitica , and Escherichia coli O157:H7 gfp+ in Ready-to-Eat Salad during Cold Storage: What Is the Risk to Consumers?

In this study, we investigated the fate of Listeria monocytogenes , pathogenic Yersinia enterocolitica , and Escherichia coli O157:H7 gfp+ inoculated in low numbers into ready-to-eat baby spinach and mixed-ingredient salad (baby spinach with chicken meat). Samples were stored at recommended maximum refrigerator temperature (8°C in Sweden) or at an abuse temperature (15°C) for up to 7 days. Mixed-ingredient salad supported considerable growth when stored at 15°C during shelf life (3 days), with populations of L. monocytogenes , pathogenic Y. enterocolitica , and E. coli O157:H7 gfp+ increasing from less than 2.0 log CFU/g on day 0 to 7.0, 4.0, and 5.6 log CFU/g, respectively. However, when mixed-ingredient salad was stored at 8°C during shelf life, only L. monocytogenes increased significantly, reaching 3.0 log CFU/g within 3 days. In plain baby spinach, only pathogenic Y. enterocolitica populations increased significantly during storage for 7 days, and this was exclusively at an abuse temperature (15°C). Thus, mixing ready-to-eat leafy vegetables with chicken meat strongly influenced levels of inoculated strains during storage. To explore the food safety implications of these findings, bacterial numbers were translated into risks of infection by modeling. The risk of listeriosis (measured as probability of infection) was 16 times higher when consuming a mixed-ingredient salad stored at 8°C at the end of shelf life, or 200,000 times higher when stored at 15°C, compared with when consuming it on the day of inoculation. This indicates that efforts should focus on preventing temperature abuse during storage to mitigate the risk of listeriosis. The storage conditions recommended for mixed-ingredient salads in Sweden (maximum 8°C for 3 days) did not prevent growth of L. monocytogenes in baby spinach mixed with chicken meat. Manufacturers preparing these salads should be aware of this, and recommended storage temperature should be revised downwards to reduce the risk of foodborne disease.

Emerging microbiota during cold storage and temperature abuse of ready-to-eat salad​.

Introduction: Ready-to-eat (RTE) leafy vegetables have a natural leaf microbiota that changes during different processing and handling steps from farm to fork. The objectives of this study were (i) to compare the microbiota of RTE baby spinach and mixed-ingredient salad before and after seven days of storage at 8°C or 15°C; (ii) to explore associations between bacterial communities and the foodborne pathogens Listeria monocytogenes, pathogenic Yersinia enterocolitica, and pathogen model organism Escherichia coli O157:H7 gfp+ when experimentally inoculated into the salads before storage; and (iii) to investigate if bacterial pathogens may be detected in the 16S rRNA amplicon dataset. Material and methods: The microbiota was studied by means of Illumina 16S rRNA amplicon sequencing. Subsets of samples were inoculated with low numbers (50-100 CFU g-1) of E. coli O157:H7 gfp+, pathogenic Y. enterocolitica or L. monocytogenes before storage. Results and discussion: The composition of bacterial communities changed during storage of RTE baby spinach and mixed-ingredient salad, with Pseudomonadales as the most abundant order across all samples. Although pathogens were present at high viable counts in some samples, they were only detected in the community-wide dataset in samples where they represented approximately 10% of total viable counts. Positive correlations were identified between viable counts of inoculated strains and the abundance of Lactobacillales, Enterobacteriales, and Bacillales, pointing to positive interactions or similar environmental driver variables that may make it feasible to use such bacterial lineages as indicators of microbial health hazards in leafy vegetables. The data from this study contribute to a better understanding of the bacteria present in RTE salads and may help when developing new types of biocontrol agents.​.