EU FP7 Veg-i-Trade investigates pre and post-harvest practices influencing microbial quality and safety of leafy greens

Fresh leafy vegetables (e.g. lettuce, spinach, escarole, cabbage and other baby leaves used in salad mixes) are an important part of a healthy diet. Global consumption levels are expected to increase in the future. However, due to recent disease outbreaks and rapid alerts attributed to fresh produce, concerns have emerged with regard to food safety. This was exemplified by the E. coli 0104 outbreak in Europe in Spring 2011. Experts have identified climate change and global trade to present further challenges for assuring food safety.

With the aim of giving an answer to the emerging food safety issues in fresh produce, the European project Veg-i-Trade is assessing the impact of anticipated climate change and globalisation on safety issues concerning fresh produce and derived food products. This multidisciplinary project comprises both fundamental and applied research. It focuses on the economic structure of the fresh produce global market and on the development of control measures for microbiological and chemical hazards. Veg-i-Trade research integrates several tools including sampling and analytical testing methods, field studies on pre and postharvest practices, quality assurance, modelling and simulation, risk assessment and risk communication. Results from the CEBAS-CSIC research team are presented dealing with pre and post-harvest production of leafy vegetables as well as fresh-cut processing practices on microbial and overall quality of leafy greens.

‘Best Practices’ rather than actual HACCP to reduce risk of microbial contamination

Outbreaks associated with leafy greens are increasing because of the increased consumption (more raw and less cooked), large scale production (widespread distribution increasing the size of outbreak), increased sensitive populations, increased public and scientific awareness and greatly improved methodology and ability for the detection of microorganisms. The majority of producerelated outbreaks are associated with lettuce1. Outbreaks associated with Salmonella and E. coli O157:H7 tend to receive the most attention due to the severity of the illness and occurrence of deaths. The last multistate outbreak of E. coli O157:H7 infections linked to romaine lettuce was in December 20112. However, it should be taken into account that the incidence of contamination of leafy vegetables with foodborne pathogens is extremely low (< 0.5-1 per cent).

Fresh leafy vegetables are grown and harvested under a wide range of climatic and geographical conditions using various agricultural inputs and technologies and on farms or in protected culture of varying sizes. Microbial food safety hazards and sources of contamination may vary significantly by the type of crop, production systems and practices and from one particular setting/context to another, even for the same crop3. Within the Veg-i-Trade project, the current state-of-the-art pre and post-harvest preventive measures and interventions along the farm-to-fork continuum to prevent or reduce microbial contamination of leafy greens was reviewed4. The control measures vary from physical (e.g. fencing fields), chemical (e.g. use of sanitisers) to biological interventions (e.g. use of biocontrol). Specific food safety guidelines have been developed for the production of lettuce and leafy greens5. One of the main problems associated with the implementation of the guidelines is that most of the efforts revert to the workers. Awareness and knowledge of the guidelines and procedures are necessary. However, the persistence of existing habits and attitudes may influence compliance with procedures. Moreover, in many production processes of fresh produce, some of the most effective interventions (e.g. irradiation, sanitisers, heat treatment) are neither acceptable to the consumer (or national legislation) nor applicable to ‘ready-to-eat fresh produce’ to eliminate or reduce pathogens to an acceptable level. The application of Hazard Analysis and Critical Control Point (HACCP) principles to primary production, while not broadly applicable, is generally feasible as a springboard starting approach to organising an operational food safety management plan. HACCP, as a formal program, is not feasible due to the absence of strict critical control points and the legal burden of some aspects of record-keeping and documentation. A modified approach of Hazard Analysis and Preventive Controls (HAPC) together with available and evolving guides to good practice should encourage the use of appropriate hygiene practices at farm level. Therefore, ‘best practices’ is highly recommended to reduce the risk of microbial contamination as low as is reasonably achievable from farm-to-fork.

Crop type affects microbial ecology: the benefits of baby-sized leaves

Intense research is being conducted to understand the influence of pre-harvest factors on the microbial quality and safety of leafy greens. The plant leaf surface is not an ideal environment for the survival of enteric pathogens since the leaf surface is exposed to UV light, shifts in temperature and relative humidity, and presence of available moisture (rain/irrigation). Ephiphytic bacteria do not occur in a uniform pattern. They preferentially colonise at the base of the trichomes, at stomata and along the veins. Foodborne pathogens have been shown to localise near leaf veins, probably because of the greater wettability of the area and higher attachment near the petiole6. Larger numbers of bacteria were found on lower compared to upper leaf surfaces, possibly due to the higher density of stomata or trichomes or a thinner cuticular layer. Different species and cultivars support survival and growth of bacteria better than others, depending on the leaf surface (smooth vs. more complex area)7. The influence of leaf age on the microbiological quality of leafy greens has received little attention. In the Veg-i-Trade project, we compared the fresh-cut product from whole-heads with baby-leaves as immature leaves and multi-leaves as mature ones8. After harvest, the population of bacteria was higher in baby-leaves than in the whole-heads because of the higher surface area of baby-leaves exposure to the environment. However, after cutting, there was a higher leakage of nutrients from the cut edge of the whole-heads, increasing the survival and growth of bacteria during storage. Studies also showed that the new green and red baby-sized leaves are high-quality lettuce, suitable for the fresh-cut market, as they meet all the specific requirements regarding visual quality, low microbial load and high content of phytochemicals occurring naturally in plants. These new baby-sized leaves have been developed recently as high quality lettuce varieties for the fresh-cut market. Some benefits of baby-sized lettuce when compared with whole-head lettuce include: (1) greater efficiency with higher percentage of usable product, (2) easier and faster processing, (3) more attractive presentation in packaging because of the 3-D structure, and (4) minimal oxidation due to smaller stem diameter. Likewise, one benefit of multi-leaf lettuce when compared with baby-leaf is firmer leaves which can confer a longer shelf life.

Overall quality of leafy greens affected by growing season & soil amendments used

Growing conditions such as the differences in climate and environmental factors and growing practices, independently or collectively, impact also the microbiological quality of leafy vegetables. Changes in climatic conditions such as temperature, relative humidity (RH), light intensity and rainfall, influence the cultivation in different production areas under different growing periods. These climatic factors may influence diseases and disorders of leafy vegetables. The disease-induced lesions may promote multiplication of microbial population including foodborne pathogens9. During field studies in Spain in the Veg-i-Trade project, it was shown that seasonality significantly affected the quality of fresh-cut lettuce. During summer, when warmer climate conditions occur, there is a short growing cycle from transplant to harvest and the visual quality of fresh-cut lettuce is reduced by increased browning potential.

Soil amendments are commonly used to add organic and inorganic nutrients to the soil. Global trends have increased the use of organic amendments as fertilisers. In the Veg-i-Trade project, we evaluated the impact of organic soil amendments (urban solid waste and sewage sludge) on microbial quality of rocket leaves10. Sewage sludge increased the crop yield but the non-composted materials such as pig slurry showed the highest microbial risks. In fact, proper composting of fresh organic amend – ments via thermal treatment reduces the risk of potential human pathogen survival. Organic wastes properly composted at optimum doses can be considered suitable as soil amendments because of the increased crop yield without detrimental effects on the natural phyto – chemicals (potential human disease protecting compounds), including vitamin C and poly – phenols. Standard practices with defined time/temperature parameters must be followed to ensure that human pathogens, if present, will be killed.

Irrigation at the optimum dose favours microbial and overall quality

One of the challenges for Mediterranean growers is to reduce water use by improving water management. Because of the climate change, water scarcity is expected to intensify and it may be a limiting factor in many leafy vegetable-production areas. Information on lettuce growth and quality characteristics in relation to water requirements is required. In the Veg-i-Trade project, we evaluated the influence of different irrigation doses on the quality and safety characteristics of freshcut lettuce11. Irrigation treatments were set according to a control treatment (soil at field capacity, FC) and two excess irrigation treatments (FC +50 per cent and FC +25 per cent) and two deficit irrigation treatments (FC -25 per cent, and FC -50 per cent) were evaluated. Water supplied was provided by drip irrigation during the whole growing period. Data from the five irrigation treatments supplied in six different harvest dates and three con – secutive years was examined. The field trials were conducted at the Primaflor experimental farm in Pulpí (Almería, Spain), the commercial co-partner of this research. We observed a decrease in the overall visual quality of the fresh-cut lettuce when the irrigation regime was increased. The population of bacteria increased when the irrigation regime was increased. Irrigation treatments of FC +25 and FC -25 per cent led to lettuce heads of similar size than the control treatment (FC) while excess of irrigation reduced fresh head weight, increased compactness and promoted browning which was positively correlated with an increase in the enzymatic activities. A reduction of about 25 per cent of the irrigation dose regularly applied to Romaine and Iceberg lettuce during cultivation reduced the microbial load, including total coliforms when compared to excess of irrigation. One of the main conclusions of this study is that farmers can reduce the irrigation dose by 25 per cent, saving water without affecting the quality of the whole-head and the fresh-cut product. This study allows lettuce growers to focus on irrigation regimes as a strategy for optimal yield and quality, with the added advantage of prolonging shelf life of the fresh-cut product.

Protected culture system versus open field production: a difficult choice

Production systems and the interaction between enteric pathogens and epiphytic microbes have often been discussed. However, more experiments need to be conducted that better reflect conditions encountered in the real world to study the microbial risks associated with different production systems and the interaction between enteric pathogens and epiphytic microorganisms. Within the Veg-i- Trade project, different production systems were evaluated. Protected culture system increased yield, allows off-season production, controls the abiotic factors and facilitates pest management. However, open field production provided more resistance to crops because of the adverse weather conditions and long growing cycle. Field research on the variability of crop production systems on visual quality and microbial contamination of crops and fresh-cut lettuce demonstrated that higher quality and microbiologically safer leafy vegetables can be provided by using the soilless system as a new growing system although the obtained results depended on the variety and the season12. In open field, we studied the influence of soil and soilless growing system on the microbiological quality of three lettuce types (lollo rosso, red oak leaf and butterhead). We observed that the soilless growing system was a good production system because of the lower populations of bacteria compared with soil. However, because of the accessibility of nutrients in the soilless system, the shorter growing cycle in soilless than in soil (60 vs. 100 days in winter) may reduce the shelf life of some varieties.

Harvest and post-harvest handling before processing

Lettuce and other leafy greens may be harvested by hand or mechanically depending on the market. Lettuce for fresh-cut are usually harvested by hand. In California, generally lettuce are de-cored in the field, (removal of the core and outer leaves), sprayed with a sanitising wash, bagged and boxed in the field. Sanitation and design of lettuce coring knives are essential to avoid cross-contamination. Baby lettuce and young leaves for use in bagged salads are machine harvested, dispensed into bins, placed into a refrigerated truck and then transported to the processing facility.

After harvest, leafy greens should be refrigerated as soon as possible to remove the respiratory heat, to decrease respiration and increase shelf life, and also to limit the ability of microorganisms to grow. Leafy greens are generally cooled under forced air, but vacuum cooling is a common practice in the leafy greens industry. However, some studies suggest that the process can promote infiltration of pathogens into the tissue13. In addition, the RH around the leafy greens after harvest can have a dramatic effect on microbial safety of the product. In the Veg-i-Trade project, we recently studied the influence of different RH on the microbial populations of baby leaves14. Baby spinach was exposed at 99, 85 and 72 per cent RH conditions for 36 hours at 15°C. After processing, hydration of samples exposed to low RH recovered and freshness, brightness and crispiness improved. Baby spinach exposed to low RH showed the highest stiffness which helped to withstand the minimal process. However, baby spinach exposed to high RH was more fragile, affecting membrane integrity and increasing microbiological counts. Psychrophilic bacteria and Pseudomonas counts of samples exposed to high RH were one log higher than those exposed to low and medium RH. To minimise the impact of leaf damage, baby spinach should be processed at medium–low hydration levels. This study shows that controlled RH after harvest is critical as it can influence the microbiological population and the maintenance of acceptable visual quality of leafy vegetables.

Equipment design, sanitation, appropriate packaging and storage

At the processing facility, various steps are including in the preparation of fresh ready to eat leafy vegetables. Each unit operation must be performed properly to ensure that finished product quality, shelf life and food safety are satisfactory. The product may be sliced mechanically before it goes into the agitating tank containing weakly chlorinated water for washing. The cleaning occurs in the second tank or flume containing a sanitising agent. The final wash is a rinse step to remove chlorine. When leafy greens are dumped into water flumes containing sanitising water, leaves may float on the surface, called ‘lily padding’, which limit the exposure of the entire leaf to the antimicrobial contained in the water. Equipment design is critical and it has to agitate the water and completely submerges the leaves by, for instance, air jets or Jacuzzi.

The role of disinfection is to prevent introduction and to minimise re-distribution of plant and human microbial pathogens in the water. Reduction of surface microbial load is secondary15. Scanning electron microscopy micrographs revealed that cross-contamination of E. coli on fresh-cut lettuce occurred after a short-term exposure during pre-washing even in the presence of sodium hypochlorite16. A sanitiser is therefore needed to maintain the quality of the wash water, to reduce the number of renewals, to avoid cross-con – tamination and possible infiltration process. The efficacy of various wash/sanitising systems to remove or inactivate microorganisms associated with leafy greens has been studied broadly. Chemical sanitisers generally provide a 1 to 2-log reduction in viable bacteria. However, after storage, the same microbial counts are achieved17.

The final operation in the processing of fresh-cut leafy vegetables takes place in the assembly and packaging room. After inserting the correct amount of product into the package, the packs are sealed. Un-perforated, polymeric films are used in an effort to maintain product quality, while extending shelf life and slowing microbial growth rate. The role of Modified Atmosphere Packaging (MAP) generated inside the package reduces respiration rate and metabolic activities. The correct combination of packaging material, produce weight and gas composition within packaging are critical components, which must be determined for each product. Packaging cannot correct unsanitary product handling, temperature abuse or poor-quality raw materials. For optimal MAP, films of good thermal sealing, appropriate permeability, avoiding browning and anaerobic respiration are needed.

Proper control of storage will extend the shelf life of fresh-cut leafy vegetables. Fresh-cut leafy vegetables must be held continuously at refrigeration temperature and guarded from light exposure. In the Veg-i-Trade project, we observed that light exposure during storage caused browning and quality loss of fresh-cut lettuce18. Light exposure during storage positively influenced the number of open stomata (74 per cent in light vs. 24 per cent in darkness) which contributed slightly to weight loss. In fact, uncontrolled light conditions during storage and commercial distribution can contribute to induction of the browning process which can cause the product to be unsalable, particularly in some lettuce types more sensitive to browning such as Romaine. Taking into account these factors, processors can offer a more standardised quality product.

Significance of the Veg-i-Trade project

Improvement in the microbiological safety of leafy greens is dependent on the understanding of the interaction of enteric foodborne pathogens with plant tissue. This knowledge will aid in the development of methods for detection and identification of target microorganisms, facilitate the development of novel strategies for reducing or eliminating target pathogens, and permit interaction of GAP, GMP and HACCP programs to ensure that leafy greens are handled appropriately from the field to the consumers’ home.

Veg-i-Trade unifies 23 international partners from universities, research institutes, SMEs and large industrial partners. It is a highly visible project, widely known within the scientific community, industry and competent auth – orities, in the EU and throughout the world. Veg-i-Trade research will support the development of management strategies in the fresh produce supply chain. It was noted from literature reviews on i) pre and post-harvest preventive measures and ii) on the quality of water and its sources and water treatment technologies applied in the fresh produce supply chain that it is important to establish more tailored interventions, taking into account specific contexts and/or demands. The output of Veg-i-Trade research will support the development of technological control measures.

For updated information on research results and dissemination events of EU FP7 Veg-i-Trade research, please refer to the Veg-i-Trade website (www.Veg-i-Trade.org).

References

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14. Medina, M.S., Tudela, J.A., Marín, A., Allende, A., Gil, M.I. 2012. Short postharvest storage under low relative humidity improves quality and shelf life of minimally processed baby spinach (Spinacia oleracea L.). Postharvest Biol. Technol., 67, 1-9

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17. Allende, A., Selma, M.V., López-Gálvez, F., Villaescusa, R., Gil, M.I. 2008. Role of commercial sanitizers and washing systems on epiphytic microorganisms and sensory quality of fresh-cut escarole and lettuce”. Postharvest Biol. Technol., 49, 155-163

18. Martínez-Sánchez, A., Tudela, J.A., Luna, C., Allende, A., Gil, M.I. 2011b. Low oxygen levels and light exposure affect quality of fresh-cut Romaine lettuce. Postharvest Biol. Technol., 59, 34-42