Francisco López-Gálvez

Fresh-cut product sanitation and wash water disinfection: Problems and solutions

It is well known that fresh-cut processors usually rely on wash water sanitizers to reduce microbial counts in order to maintain quality and extend shelf-life of the end product. Water is a useful tool for reducing potential contamination but it can also transfer pathogenic microorganisms. Washing with sanitizers is important in fresh-cut produce hygiene, particularly removing soil and debris, but especially in water disinfection to avoid cross-contamination between clean and contaminated product. Most of the sanitizing solutions induce higher microbial reduction after washing when compared to water washing, but after storage, epiphytic microorganisms grow rapidly, reaching similar levels. In fact, despite the general idea that sanitizers are used to reduce the microbial population on the produce, their main effect is maintaining the microbial quality of the water. The use of potable water instead of water containing chemical disinfection agents for washing fresh-cut vegetables is being advocated in some European countries. However, the problems of using an inadequate sanitizer or even none are considered in this manuscript. The need for a standardized approach to evaluate and compare the efficiency of sanitizing agents is also presented. Most new alternative techniques accentuate the problems with chlorine suggesting that the industry should move away from this traditional disinfection agent. However, the use of chlorine based sanitizers are presented as belonging to the most effective and efficient sanitizers when adequate doses are used. In this review improvements in water disinfection and sanitation strategies, including a shower pre-washing step and a final rinse of the produce, are suggested.

Prevention of Escherichia coli cross-contamination by different commercial sanitizers during washing of fresh-cut lettuce.

The efficacy of fresh-cut produce sanitizers has mainly been evaluated by measuring microbial reductions on produce. However, its suitability to ensure that pathogens are rapidly killed avoiding cross-contamination of subsequent product also needs to be considered. The efficacy of chlorine, Tsunami, Citrox and Purac on non pathogenic Escherichia coli reductions in processing water and on fresh-cut lettuce were studied. Selection of minimum effective doses was carried out in processing water, which contained a chemical oxygen demand (COD) within the range of 700-1000 mg/l and a total mesophilic load of about 7 log CFU/ml. The processing water was inoculated with two inoculum levels (3 and 5 log CFU/ml). It was observed that 40 mg/l of chlorine and 500 mg/l of Tsunami were effective in reducing the inoculum levels in the processing water to the detection limit (5 and 4 log units). However, Citrox and Purac were not effective in reducing E. coli population even at the highest manufacturers recommended doses. Evaluation of cross-contamination in fresh-cut lettuce was carried out by measuring E. coli transfer from inoculated (~5 log CFU/g) to uninoculated lettuce after washing the contaminated product in the water containing different sanitizing agents. Chlorine and Tsunami were able to inactivate E. coli in wash water, avoiding cross-contamination between contaminated and non-contaminated product. However, Citrox and Purac at the recommended doses did not prevent transfer of E. coli cells between inoculated and uninoculated fresh-cut lettuce and therefore indicating cross-contamination. The results obtained show that chlorine and Tsunami are recommended as water disinfection agents preventing E. coli cross-contamination of produce during processing.

Disinfection potential of ozone, ultraviolet-C and their combination in wash water for the fresh-cut vegetable industry

The purpose of this research was to investigate the disinfection efficacy of ozone (O(3)) and UV-C illumination (UV), and their combination (O(3)-UV) for reducing microbial flora of fresh-cut onion, escarole, carrot, and spinach wash waters collected from the industry. Furthermore, the influence of water physicochemical parameters on the decontamination efficacy and the effect of these technologies on physicochemical quality of wash water were analyzed. O(3), UV, and O(3)-UV were effective disinfection treatments on vegetable wash water, with a maximum microbial reduction of 6.6 log CFU mL(-1) after 60 min treatment with O(3)-UV. However, maximum total microbial reductions achieved by UV and O(3) treatments after 60 min were 4.0 and 5.9 log CFU mL(-1), lower than by O(3)-UV treatment. Furthermore, turbidity of wash water was reduced significantly by O(3) and O(3)-UV treatments, while UV treatment did not affect the physicochemical quality of the water. Conclusions derived from this study illustrate that O(3) and O(3)-UV are alternatives to other sanitizers used in the fresh-cut washing processes. The use of these technologies would allow less frequent changing of spent water and the use of much lower sanitizer doses. Nevertheless, in specific applications such as carrot wash water, where levels of undesirable microbial and chemical constituents are lower than other vegetable wash water, UV treatment could be an appropriate treatment considering cost-effectiveness criteria.

Cross-contamination of fresh-cut lettuce after a short-term exposure during pre-washing cannot be controlled after subsequent washing with chlorine dioxide or sodium hypochlorite

Chlorine dioxide (ClO(2)) has been postulated as an alternative to sodium hypochlorite (NaClO) for fresh-cut produce sanitization to avoid risks associated with chlorination by-products. Experiments were performed to determine the prevention of cross-contamination of fresh-cut lettuce by Escherichia coli using chlorine dioxide (3 mg/L) or sodium hypochlorite (100 mg/L) as sanitation agents. The efficacy of these sanitation solutions was evaluated simulating as much as possible the conditions of a fresh-cut processing line. Thus, to evaluate the potential risk of cross-contamination during pre-washing, inoculated fresh-cut lettuce was pre-washed and after that non-inoculated lettuce was then pre-washed in the same water. After this pre-washing, non-inoculated lettuce was cross-contaminated, changing from 0 to 3.4 log units of E. coli cells. During washing with sanitizers, none of the tested sanitation agents significantly reduced E. coli counts in both inoculated and cross-contaminated lettuce. These results suggest that when cross-contamination occurs, even if the event is recent, subsequent sanitation steps are inefficient for inactivating E. coli cells on the vegetable tissue. However, chlorine dioxide and sodium hypochlorite solutions were able to inactivate most E. coli cells that passed from inoculated product to wash water. Therefore, they might be able to avoid cross-contamination between clean and contaminated product during the washing step. Scanning electron microscopy micrographs indicated that bacterial cells were mainly located in clusters or tissue stomata where they might be protected, which explains the low efficacy of sodium hypochlorite and chlorine dioxide solutions observed in this study.

Impact of Wash Water Quality on Sensory and Microbial Quality, Including Escherichia coli Cross-Contamination, of Fresh-Cut Escarole

The influence of wash water quality on the microbial load and sensory quality of fresh-cut escarole was evaluated. Additionally, the degree of Escherichia coli cross-contamination between inoculated and uninoculated products after washing was also studied. Three types of wash water, i.e., potable water, diluted recirculated water, and recirculated water, containing different microbial counts and organic loads, were used. Results showed that microbial load (P > or = 0.02) and sensory quality (P > 0.625) of the product were not influenced by the water quality after washing and storage. Cross-contamination between inoculated and uninoculated products was observed after washing, as there was significant transmission of E. coli cells from the product to the wash water (P < 0.001). When fresh-cut escarole was contaminated at a high inoculum level (5.1 log CFU/g), wash water quality influenced the level of cross-contamination, as the highest E. coli load (P < 0.001) was shown in uninoculated fresh-cut escarole washed with recirculated water. However, when fresh-cut escarole was contaminated at a low inoculum level (3.2 log CFU/g), the wash water quality did not influence the level of cross-contamination, as E. coli slightly increased, although not at a statistically significant level, after the uninoculated product was washed with recirculated water (P > 0.035). Therefore, the contamination level may impact the effectiveness of water quality to reduce pathogen concentrations. It was clearly observed that cross-contamination of fresh-cut escarole with E. coli occurs, thereby suggesting that small amounts of contamination could impact the overall product and indicating the necessity of using wash water sanitizers to eliminate pathogens.

Electrochemical disinfection: An efficient treatment to inactivate Escherichia coli O157:H7 in process wash water containing organic matter

The efficacy of an electrochemical treatment in water disinfection, using boron-doped diamond electrodes, was studied and its suitability for the fresh-cut produce industry analyzed. Tap water (TW), and tap water supplemented with NaCl (NaClW) containing different levels of organic matter (Chemical Oxygen Demand (COD) around 60, 300, 550 ± 50 and 750 ± 50 mg/L) obtained from lettuce, were inoculated with a cocktail of Escherichia coli O157:H7 at 10⁵ cfu/mL. Changes in levels of E. coli O157:H7, free, combined and total chlorine, pH, oxidation-reduction potential, COD and temperature were monitored during the treatments. In NaClW, free chlorine was produced more rapidly than in TW and, as a consequence, reductions of 5 log units of E. coli O157:H7 were achieved faster (0.17, 4, 15 and 24 min for water with 60, 300, 500 and 750 mg/L of COD, respectively) than in TW alone (0.9, 25, 60 min and 90 min for water with 60, 300, 600 and 800 mg/L of COD, respectively). Nonetheless, the equipment showed potential for water disinfection and organic matter reduction even without adding NaCl. Additionally, different mathematical models were assessed to account for microbial inactivation curves obtained from the electrochemical treatments.

Characterization of Escherichia coli from raw poultry in Belgium and impact on the detection of Campylobacter jejuni using Bolton broth.

A comparative study examining Bolton broth and Preston broth for enrichment and reliable detection of Campylobacter jejuni (both healthy and freeze stressed cells) was performed. Tested as pure cultures, Bolton broth enabled faster resuscitation and growth of C. jejuni compared to Preston broth. When C. jejuni was co-incubated with extended-spectrum-beta-lactamase (ESBL) producing Escherichia coli isolated from Belgian poultry meat preparations, the latter dominated in the Bolton enrichment broth and crowded the mCCDA plates. This resulted in the inability to recover C. jejuni by ISO 10272-1:2006 standard method. Preston broth did not support the growth of the ESBL E. coli isolates, but showed longer detection time of C. jejuni compared to Bolton broth. The use of the same antibiotic (sodium cefoperazone) in Bolton broth and in mCCDA plates may explain the problems encountered for detection of C. jejuni, as high numbers of ESBL E. coli present after enrichment in Bolton broth, also caused overgrowth and masked the few C. jejuni colonies present on the mCCDA plates. The use of Campylobacter spp. specific real-time PCR circumvented these problems and enabled rapid detection of the pathogen after 24h enrichment in both Bolton and Preston broth, for both healthy and freeze stressed cells.

Modelling growth of Escherichia coli O157:H7 in fresh-cut lettuce submitted to commercial process conditions: Chlorine washing and modified atmosphere packaging

Fresh-cut iceberg lettuce inoculated with Escherichia coli O157:H7 was submitted to chlorine washing (150 mg/mL) and modified atmosphere packaging on laboratory scale. Populations of E. coli O157:H7 were assessed in fresh-cut lettuce stored at 4, 8, 13 and 16 °C using 6-8 replicates in each analysis point in order to capture experimental variability. The pathogen was able to grow at temperatures ≥8 °C, although at low temperatures, growth data presented a high variability between replicates. Indeed, at 8 °C after 15 days, some replicates did not show growth while other replicates did present an increase. A growth primary model was fitted to the raw growth data to estimate lag time and maximum growth rate. The prediction and confidence bands for the fitted growth models were estimated based on Monte-Carlo method. The estimated maximum growth rates (log cfu/day) corresponded to 0.14 (95% CI: 0.06-0.31), 0.55 (95% CI: 0.17-1.20) and 1.43 (95% CI: 0.82-2.15) for 8, 13 and 16 °C, respectively. A square-root secondary model was satisfactorily derived from the estimated growth rates (R(2) > 0.80; Bf = 0.97; Af = 1.46). Predictive models and data obtained in this study are intended to improve quantitative risk assessment studies for E. coli O157:H7 in leafy green products.

Modeling growth of Escherichia coli O157:H7 in fresh-cut lettuce treated with neutral electrolyzed water and under modified atmosphere packaging.

The purpose of this study was to evaluate and model the growth of Escherichia coli O157:H7 in fresh-cut lettuce submitted to a neutral electrolyzed water (NEW) treatment, packaged in passive modified atmosphere and subsequently stored at different temperatures (4, 8, 13, 16°C) for a maximum of 27 days. Results indicated that E. coli O157:H7 was able to grow at 8, 13, and 16°C, and declined at 4°C. However at 8°C, the lag time lasted 19 days, above the typical shelf-life time for this type of products. A secondary model predicting growth rate as a function of temperature was developed based on a square-root function. A comparison with literature data indicated that the growth predicted by the model for E. coli O157:H7 was again lower than those observed with other disinfection treatments or packaging conditions (chlorinated water, untreated product, NEW, etc.). The specific models here developed might be applied to predict growth in products treated with NEW and to improve existing quantitative risk assessments.

Heterogeneous photocatalytic disinfection of wash waters from the fresh-cut vegetable industry.

The effectiveness of photocatalytic disinfection for control of natural and potentially pathogenic microflora in wash waters used for fresh-cut vegetables was evaluated. Wash waters for lettuce, escarole, chicory, carrot, onion, and spinach from a fresh-cut vegetable processing plant were treated with a titanium dioxide (TiO2) photocatalytic system. The vegetable wash waters were impelled out with a pump at a flow rate of 1,000 liters/h and conducted through a stainless steel circuit to the filtration system to reach the TiO2 photocatalyst fiber, which was illuminated with a 40-W UV-C lamp. The microbial and physicochemical qualities of the wash water were analyzed. Heterogeneous photocatalysis was an effective disinfection method, reducing counts of bacteria, molds, and yeasts. Most of the treated wash waters had total bacteria reductions of 4.1 +/- 1.3 to 4.8 +/- 0.4 log CFU/ml after 10 min of treatment when compared with untreated water. Higher decontamination efficacy was observed in carrot wash water (6.2 +/- 0.1-log reductions), where turbidity and organic matter were lower than those in the wash waters for other vegetables. The tested heterogeneous photocatalytic system also was effective for reducing water turbidity, although chemical oxygen demand was unaffected after the treatments. The efficacy of the photocatalytic system for reducing microbial load depended on the physicochemical characteristics of the wash water, which depended on the vegetable being washed. The conclusions derived from this study illustrate that implementation of a heterogeneous photocatalytic system in the fresh-cut vegetable washing processes could allow the reuse of wash water.