Commercial wash of leafy vegetables do not significantly decrease bacterial load but leads to shifts in bacterial species composition.

Production of leafy vegetables for the "Ready-to-eat"-market has vastly increased the last 20 years, and consumption of these minimally processed vegetables has led to outbreaks of food-borne diseases. Contamination of leafy vegetables can occur throughout the production chain, and therefore washing of the produce has become a standard in commercial processing. This study explores the bacterial communities of spinach (Spinacia oleracea) and rocket (Diplotaxis tenuifolia) in a commercial setting in order to identify potential contamination events, and to investigate effects on bacterial load by commercial processing. Samples were taken in field, after washing of the produce and at the end of shelf-life. This study found that the bacterial community composition and diversity changed significantly from the first harvest to the end of shelf-life, where the core microbiome from the first to the last sampling constituted <2% of all OTUs. While washing of the produce had no reducing effect on bacterial load compared to unwashed, washing led to a change in species composition. As the leaves entered the cold chain after harvest, a rise was seen in the relative abundance of spoilage bacteria. E. coli was detected after the washing indicating issues of cross-contamination in the wash water.

High-throughput method for detection and quantification of lesions on leaf scale based on trypan blue staining and digital image analysis.

BACKGROUND: Field-grown leafy vegetables can be damaged by biotic and abiotic factors, or mechanically damaged by farming practices. Available methods to evaluate leaf tissue damage mainly rely on colour differentiation between healthy and damaged tissues. Alternatively, sophisticated equipment such as microscopy and hyperspectral cameras can be employed. Depending on the causal factor, colour change in the wounded area is not always induced and, by the time symptoms become visible, a plant can already be severely affected. To accurately detect and quantify damage on leaf scale, including microlesions, reliable differentiation between healthy and damaged tissue is essential. We stained whole leaves with trypan blue dye, which traverses compromised cell membranes but is not absorbed in viable cells, followed by automated quantification of damage on leaf scale. RESULTS: We present a robust, fast and sensitive method for leaf-scale visualisation, accurate automated extraction and measurement of damaged area on leaves of leafy vegetables. The image analysis pipeline we developed automatically identifies leaf area and individual stained (lesion) areas down to cell level. As proof of principle, we tested the methodology for damage detection and quantification on two field-grown leafy vegetable species, spinach and Swiss chard. CONCLUSIONS: Our novel lesion quantification method can be used for detection of large (macro) or single-cell (micro) lesions on leaf scale, enabling quantification of lesions at any stage and without requiring symptoms to be in the visible spectrum. Quantifying the wounded area on leaf scale is necessary for generating prediction models for economic losses and produce shelf-life. In addition, risk assessments are based on accurate prediction of the relationship between leaf damage and infection rates by opportunistic pathogens and our method helps determine the severity of leaf damage at fine resolution.

Leaf mineral content govern microbial community structure in the phyllosphere of spinach (Spinacia oleracea) and rocket (Diplotaxis tenuifolia).

The plant microbiome is an important factor for plant health and productivity. While the impact of nitrogen (N) availability for plant growth and development is well established, its influence on the microbial phyllosphere community structure is unknown. We hypothesize that nitrogen impacts the growth and abundance of several microorganisms on the leaf surface. The bacterial and fungal communities of baby leaf spinach (Spinacia oleracea), and rocket (Diplotaxis tenuifolia) were investigated in a field trial for two years in a commercial setting. Nitrogen fertilizer was tested in four doses (basic nitrogen, basic + suboptimal, basic + commercial, basic + excess) with six replicates in each. Culture-independent (Illumina sequencing) and culture-dependent (viable count and identification of bacterial isolates) community studies were combined with monitoring of plant physiology and site weather conditions. This study found that alpha diversity of bacterial communities decreased in response to increasing nitrogen fertilizer dose, whereas viable counts showed no differences. Correspondingly, fungal communities of the spinach phyllosphere showed a decreasing pattern, whereas the decreasing diversity of fungal communities of rocket was not significant. Plant species and effects of annual variations on microbiome structure were observed for bacterial and fungal communities on both spinach and rocket. This study provides novel insights on the impact of nitrogen fertilizer regime on a nutrient scarce habitat, the phyllosphere.

Season and Species: Two Possible Hurdles for Reducing the Food Safety Risk of Escherichia coli O157 Contamination of Leafy Vegetables.

The food safety risk of Shiga toxin-producing Escherichia coli (STEC) infection per serving of leafy vegetables was investigated using a quantitative microbial risk assessment (QMRA) approach. The estimated level of E. coli O157 contamination was based on observed numbers of Enterobacteriaceae and E. coli on leafy vegetables grown and processed in southern Sweden from 2014 to 2016. Samples were collected before harvest, after washing, and at the end of shelf life. The observed counts were combined with data on the ratio of E. coli to E. coli O157 taken from earlier studies to estimate the probability of illness. The risks of STEC infection associated with species, either spinach ( Spinacia oleracea) or rocket ( Diplotaxis tenuifolia), growing season (spring or autumn), and washing (washed or not washed) were then evaluated. The results indicated that leafy vegetable species and growing season could be possible hurdles for reducing the food safety risk of STEC infection. At harvest, the probability of infection was 87% lower when consuming rocket compared with spinach and 90% lower when consuming leafy vegetables grown in spring compared with autumn. These relative risk reductions remained consistent even with other serving sizes and dose-response models. The lowest risk of STEC infection was associated with leafy vegetables early in the production chain, i.e., before harvest, while the risk increased during storage and processing. Consequently, the highest risk was observed when leafy vegetables were consumed at the end of shelf life. Washing had no effect on the food safety risk of STEC infection in this study. To improve the quality of QMRA, there is a need for additional data on the relationship between indicator organisms that can be easily enumerated (e.g., E. coli and Enterobacteriaceae) and E. coli strains that can cause STEC infection (e.g., E. coli O157) but are difficult to identify in food samples such as leafy vegetables.

The Hurdle Approach-A Holistic Concept for Controlling Food Safety Risks Associated With Pathogenic Bacterial Contamination of Leafy Green Vegetables. A Review.

Consumers appreciate leafy green vegetables such as baby leaves for their convenience and wholesomeness and for adding a variety of tastes and colors to their plate. In Western cuisine, leafy green vegetables are usually eaten fresh and raw, with no step in the long chain from seed to consumption where potentially harmful microorganisms could be completely eliminated, e.g., through heating. A concerning trend in recent years is disease outbreaks caused by various leafy vegetable crops and one of the most important foodborne pathogens in this context is Shiga toxin-producing Escherichia coli (STEC). Other pathogens such as Salmonella, Shigella, Yersinia enterocolitica and Listeria monocytogenes should also be considered in disease risk analysis, as they have been implicated in outbreaks associated with leafy greens. These pathogens may enter the horticultural value network during primary production in field or greenhouse via irrigation, at harvest, during processing and distribution or in the home kitchen/restaurant. The hurdle approach involves combining several mitigating approaches, each of which is insufficient on its own, to control or even eliminate pathogens in food products. Since the food chain system for leafy green vegetables contains no absolute kill step for pathogens, use of hurdles at critical points could enable control of pathogens that pose a human health risk. Hurdles should be combined so as to decrease the risk due to pathogenic microbes and also to improve microbial stability, shelf-life, nutritional properties and sensory quality of leafy vegetables. The hurdle toolbox includes different options, such as physical, physiochemical and microbial hurdles. The goal for leafy green vegetables is multi-target preservation through intelligently applied hurdles. This review describes hurdles that could be used for leafy green vegetables and their biological basis, and identifies prospective hurdles that need attention in future research.

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.

Moderate water stress prevents the postharvest decline of ascorbic acid in spinach (Spinacia oleracea L.) but not in spinach beet (Beta vulgaris L.).

BACKGROUND: Babyleaf salads such as spinach (Spinacia oleracea L.) and spinach beet (Beta vulgaris L. subsp. cicla var. cicla) are an important dietary source of antioxidants such as ascorbic acid (vitamin C). Such compounds may be important in disease prevention in consumers but the level of these compounds in leaves frequently declines after harvest. As such, methods to maintain antioxidant levels in fresh produce are being sought. RESULTS: Irrigation deficits were used to apply water stress to S. oleracea and B. vulgaris plants. This treatment prevented postharvest decline of leaf ascorbic acid content in S. oleracea but not in B. vulgaris. Ascorbic acid levels in leaves at harvest were unaffected by the treatment in both species compared to well-watered controls. CONCLUSION: We have shown that restricted irrigation provides a viable means to maintain leaf vitamin content after harvest in S. oleracea, an important finding for producers, retailers and consumers alike. © 2015 Society of Chemical Industry.

High- but not low-intensity light leads to oxidative stress and quality loss of cold-stored baby leaf spinach.

BACKGROUND: Quality management in the fresh produce industry is an important issue. Spinach is exposed to various adverse conditions (temperature, light, etc.) within the supply chain. The present experiments were conducted to investigate the effect of light conditions (dark, low-intensity light (LL) and high-intensity light (HL)) and photoperiod (6 h HL and 18 h dark) on the quality changes of cold-stored spinach. RESULTS: HL exposure resulted in oxidative stress, causing tissue damage and quality loss as evidenced by increased membrane damage and water loss. The content of total ascorbic acid was reduced under HL conditions. On the other hand, storage of spinach under LL conditions gave promising results, as nutritional quality was not reduced, while texture maintenance was improved. No significant differences, with the exception of nutritional quality, were found between spinach leaves stored under continuous (24 h) low-intensity light (30-35 µmol m(-2) s(-1)) and their counterparts stored under the same light integral over 6 h (130-140 µmol m(-2) s(-1)). CONCLUSION: LL extended the shelf-life of spinach. The amount of light received by the leaves was the key factor affecting produce quality. Light intensity, however, has to be low enough not to cause excess oxidative stress and lead to accelerated senescence.

Organically fertilized onions (Allium cepa L.): effects of the fertilizer placement method on quercetin content and soil nitrogen dynamics.

Field-cured onions cv. Hyskin ( Allium cepa L.) supplied with organic nitrogen fertilizer were studied. The fertilizer was applied by broadcasting and harrowing, broadcasting and rotary cultivation, or placement between rows. Nitrogen dynamics were monitored throughout the growing season by soil sampling. Variation in quercetin content in the onion scales was analyzed by HPLC. The organically fertilized onions were compared with inorganically fertilized onions grown in the same field. Inoculation with arbuscular mycorrhizal fungi (AMF) in the row at sowing or during commercial transplant production was tested but did not significantly affect mycorrhizal root colonization levels in the field. Onions that received no fertilizer at all or that had fertilizer placed between rows had better establishment, probably due to more favorable soil nitrogen concentrations for seedling emergence. Broadcast application led to higher nitrogen concentration in the root zone, resulting in fewer but larger individual onions. Quercetin levels were not significantly altered as a result of nitrogen fertilizer source (inorganic or organic), application method, or mycorrhizal inoculation. However, variation between years was significant, with quercetin levels in 2004 almost twice as high as those in 2005.

Quercetin content in field-cured onions (Allium cepa L.): effects of cultivar, lifting time, and nitrogen fertilizer level.

Variation in quercetin content was investigated in field-cured onions (Allium cepa L.) that had been supplied with different nitrogen fertilizer levels and lifted at different developmental stages. Quercetin content varied significantly between years and was well correlated to global radiation in August. Field curing generally resulted in significant increases in quercetin content compared to levels at lifting. Nitrogen fertilizer level did not affect quercetin content, suggesting that nitrogen leakage from soil may be minimized without effects on flavonol content. Lifting time had minor effects on quercetin content in field-cured onions. Cultivar differences in quercetin content were significant but not consistent in all years. Quercetin content increased significantly less in dark environments compared to field curing, but some quercetin synthesis occurred regardless of light. Field curing with or without foliage still attached did not affect quercetin content, suggesting that no transportation from the foliage to the scales occurred.