Decontamination of Fresh and Minimally Processed Produce

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Experimental set up of the washing trials of minimally-processed MP lettuce using different treatments. For each treatment, g of lettuce were washed by hand with gentle mixing in a 5 L beaker containing 2.

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Following the washing process, the products were placed in a sieve for 30 s to allow the washing water to drain off. The products were then rinsed with 2. KG, Written, Germany were used. Serial dilutions were made in 0. The supernatant was passed through a 0.

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The appearance of fresh-cut lettuce was assessed visually after 1, 3, 7, and 13 days after treatment. The focus was on discoloration, browning of cut edges and appearance of brown spots on the leaves. The experiments were carried out with four blocks using the same reuterin stock, with each block including washing treatments with TW, CR, ER, and NaClO compare to un-washed lettuce. The decimal reduction of a washing treatment was calculated at each time point by the difference between the treatment and unwashed lettuce count.

To assess the dose dependent effects of acrolein on cell counts, a regression analysis was used with CR and ER data after 1 and 13 days of storage, using SigmaPlot A single stock of reuterin was produced for the four trials carried out over a total period of 7 months. It was observed that the composition of CR prepared from the stock solution changed over time, with a steady decrease of 3-HPA concentration, from However, the acrolein concentration of 1. The concentrations of 3-HPA and acrolein in the solution collected after washing the lettuce were slightly lower than before washing for CR treatments.

Between 0. TABLE 1. Acrolein loss during washing was estimated by the difference of concentration in the solution before and after washing. Acrolein was recently shown to be the main component for antimicrobial activity of reuterin Engels et al. The acrolein titres in ER solutions for the four trials were in the range of 7. The variation in acrolein synthesis in ER solution was due to the oven not able to control the set temperature during incubation.

In case where 7. Between 2—4 mM 3-HPA and 0. The effectiveness of treatments was also expressed by the decimal reduction of the counts for a treatment at a defined time point compare to the unwashed lettuce NW Supplementary Figure 1. Data was grouped based on the acrolein concentration in the ER washing solution: 7. The counts of Enterobacteriaceae steadily increased with storage time to reach similar levels to the unwashed lettuce at day 13 6. In contrast, washing with ER containing The decimal reductions induced by ER treatments The yeasts and molds counts NW, 4.

Washing with NaClO resulted in a 0. ER washing treatments containing The ER inhibition effect was maintained until day 3. At day 7, while yeasts and molds started to regrow, the decimal reduction remained between 1.

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At day 13, the yeasts and molds counts on lettuce washed with ER containing acrolein For NaClO, TW, and CR treatments, growth of yeasts and molds were already noticeable at day 3, and at day 13, the counts of yeasts and molds on the stored lettuce were 5. The total aerobic mesophilic bacteria counts were reduced between 0. TW, CR, and ER treatments resulted in a limited reduction of total aerobic mesophilic bacteria, less than 1 log after 1 day of storage compare to NW.

At day 7, washing with ER containing This effect was maintained until the end of storage in trail 2 A regression analysis was applied to test the dose dependent effect of acrolein on the decimal reduction of Enterobacteriaceae and yeasts and molds which were most affected compare to NW lettuce. Linear regression analysis of decimal reduction of Enterobacteriaceae A,C and yeasts and molds B,D viable cell counts versus acrolein concentration in crude reuterin CR and enhanced reuterin ER treatments after washing and storage for day 1 A,B and day 13 C,D.

Differences in the visual appearance of lettuce washed with ER solutions were observed starting on day 3 for lettuce washed with ER containing The effect was slightly more pronounced with increasing acrolein concentration Figure 4. Lettuce washed with NaClO, TW, and CR showed similar visual quality in all four trials, and hence, we present images from trial 2 to represent all four trials.

Visual evaluation of lettuce after washing with different treatments and during 13 days of storage under modified atmosphere. Selected images of damaged leaves illustrating spoilage of different treatments. The image may not be representative of the entire lettuce. We recently showed that acrolein is the compound of the reuterin system responsible for antimicrobial activity on indicator strains in simple buffered media Engels et al.

Using lettuce as a model vegetable, we confirmed the activity of acrolein as the main antimicrobial of the reuterin system in a complex food matrix, with a dose-dependent activity on Enterobacteriaceae and on yeasts and molds.

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The antimicrobial potential of CR as a food biopreservative was previously investigated against different food-borne pathogens like S. In line with the observation that acrolein is the main antimicrobial component of the reuterin system Engels et al. Most investigations carried out with reuterin in food systems did not provide information on purity of reuterin, and the exact concentrations of 3-HPA and acrolein were not disclosed. Instead reuterin activity was determined using arbitrary activity tests which does not allow comparing results between different studies.

Therefore the exact composition of reuterin, and more specifically the concentration of acrolein in the conditions of the test should be established for rigorous testing of reuterin as a disinfectant or preservative in food.

The sanitation of vegetable produce is intended to reduce the natural microbial load to increase shelf life and ensure safety of raw consumed vegetables. The effect of decontamination solutions used in MP vegetable produce on the natural lettuce microbiota total aerobic mesophilic count was usually reported smaller than that obtained with artificially inoculated bacteria Oliveira et al.

Our results showed that the population of initial total aerobic mesophilic bacteria was less affected by all the treatments applied than Enterobacteriaceae , and yeasts and molds. This may be partly due to biofilm formation by native lettuce microbiota, which may result in a protective effect from the action of antimicrobial compounds Hunter et al. Pathogenic bacteria from the Enterobacteriaceae family such as Salmonella, Shigella , and E. Therefore, the counts of Enterobacteriaceae are of special importance to assess the efficacy of a microbial-reducing process in respect of food safety.

The application of enhanced reuterin washing with acrolein concentration of Our results are in agreement with previous finding suggesting that Gram-negative bacteria are more sensitive to reuterin than Gram-positive bacteria Chung et al. The role of yeasts and molds in fresh-cut vegetables spoilage and mycotoxin production was previously reported by Tournas In our study, between 1. The MIC of reuterin on several yeasts and filamentous fungi were previously reported between 0. Sanitizing agents of fresh-cut produce may be considerably more effective against yeasts and molds than against aerobic mesophilic microbiota, because the later include several Gram-positive spore-forming bacteria of the Bacillus genus, known to be more resistant to chemical sanitizing agents Brackett and Splittstoesser, After an initial reduction after washing with ER, Enterobacteriaceae , and yeasts and molds counts increased steadily, eventually reaching equal levels of the other treatments and the unwashed produce at the end of the 13 day storage period.

This also emphasizes that a decontamination process may not be aimed at increasing the shelf-life of the product but rather enhance its safety during the normal storage life. Many studies have identified that chlorine is the most popular disinfection method for fresh produce Shen et al. Sodium hypochlorite NaClO is the source of chlorine commonly used by the food industry for sanitizing both products and equipments Gil et al. For efficacy and stability, the pH should be kept in the range of 6. Using 50— ppm of hypochloride and 1—2 min contact time at this pH results in a maximum of 1—2 log reduction of the initial total aerobic microbes in many commodities Parish et al.

Pezzuto et al. In our study the NaClO washing solution at uncontrolled pH of approximately 9. The lack of efficiency could be associated with difference in microbiota composition of the lettuce used in trial 4, evident by high numbers of initial counts of yeasts and molds in trial 4, compare to other trials. Since the ban of the use of chlorine for decontamination of fresh produce in some European countries, most fresh-cut producers currently use tap water for washing. Our results indicate that washing fresh-cut produce with tap water alone was not sufficient to significantly reduce the initial microbial load on the fresh-cut lettuce 0.

Similar results were obtained by Gonzalez et al. Additionally, Luo did not find differences in the aerobic mesophilic growth of Romaine lettuce after washing with potable tap water. In our study, washing fresh-cut produce with reuterin treatments containing acrolein in the range between The main antimicrobial component of the reuterin system, acrolein, is highly volatile, colorless, and may be present in many food, sometimes at high levels of more than 4.

Acrolein is considered a highly cytotoxic compound after a single exposure, hence a tolerable daily intake of 0. In our study, between 0.

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We therefore tested acrolein in the rinsing water and in washed lettuce using the high sensitive IC-PAD method developed by Engels et al. We did not detect acrolein in the macerated lettuce and rinsing water after washing with ER solutions, with the detection limit of the method of 4. Because acrolein is a volatile compound, we speculate that part of unrecovered acrolein in the wash solution may have been lost by volatization.

Aside volatilization and reaction with microbes and plant components, reversible, first-order hydration of acrolein to 3-HPA which is enhanced at low temperatures may be a significant pathway for the elimination Engels et al. However, little is known about reactions and degradation products of acrolein which is a very reactive component Engels et al. Taken together, the absence of detectable acrolein in the treated lettuce suggest safety of reuterin washing at effective acrolein concentrations, but additional testing may be needed to identify potential by-products of acrolein. Quality preservation is, after safety, the most important attribute of minimally processed vegetables, since purchasing decisions often depends on consumers satisfaction in terms of visual, textural and flavor quality of the product Allende et al.

In this study, we observed increased brown discoloration of the fresh-cut lettuce after washing with ER containing acrolein concentrations in the range from 7.

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  • The dark green color of the cut-lettuce started to decrease from day 3 and continued to decline throughout the remaining storage period. Interestingly, the point of discoloration of lettuce after washing with ER corresponds to regrowth of Enterobacteriaceae and yeast and molds. We speculate that these microbes were able to multiply faster once the tissue has been damage. Unwashed and NaClO washed samples maintained fresh appearance during 13 day storage. This is in agreement with previous studies where washing fresh-cut lettuce with chlorinated water reduced browning Baur et al.

    The major enzyme controlling oxidative discolouration of cut lettuce has been reported to be polyphenol oxidase PPO Hunter et al. Acrolein induces oxidative stress in cells, due to the oxidation of phenolic compounds Schaefer et al. We speculate that this deterioration of produce washed with enhanced reuterin is due to acrolein oxidation of the phenolic compounds in the cut lettuce. Further studies are required to understand the phenomenon for the increase browning on lettuce washed with enhance reuterin on a molecular level.

    Our study shows that in food system as for laboratory test conditions, acrolein is the main antimicrobial component of the reuterin system because only the application of ER containing between However, at effective acrolein concentration, reuterin may not be used to extend the shelf-life of fresh-cut lettuce, due to chemical reactions changing the visual appearance. The application of reuterin for the decontamination and biopreservation of other vegetable less sensitive to oxidative stresses than lettuce, such as carrot, apples, beets, and radish, should be tested. Furthermore the reuse and recycling ER washing solutions containing high levels of acrolein should be investigated to limit environmental impact.

    All authors read and approved the final manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors extend their acknowledgments to Prof. Selcuk Yildirim and Dr. The authors are also grateful to Dr.