Novel wash water formulation from sustainable resources to improve safety and shelf-life of leafy greens

Abstract

In the past two decades, multiple outbreaks occurred due to consumption of fresh leafy-greens contaminated with Shiga toxin-producing Escherichia coli (STECs). There is, therefore, a large need to find effective intervention strategies to control microbial contamination in fresh produce. Current practices include the use of potable water or at best with a wash-water sanitizer, commonly peracetic acid, to clean debris, reduce microbial load and extend shelf-life. Surface characteristics of produce as well as other factors (e.g., organic load) limit the efficacy of existing technologies. Recent studies have analyzed the combination of essential oils with surface-active ingredients to improve antimicrobial effectiveness. The amphiphilic structure of surface-active antimicrobials allows them to penetrate the phospholipid membrane, altering the membrane structure and fluidity. This increases cell permeability and can disrupt metabolic processes. Oxidizers are effective antimicrobials due to the oxidizing effect on enzymes, cell membrane, structural proteins and other cellular functions depending on the type of oxidizer. Combining a surface-active antimicrobial (e.g., lauric arginate) and an oxidizer (e.g., organic acids), can create synergistic effects to offer a solution as sustainable ingredients with enhanced antimicrobial effectiveness at reduced concentrations. The objectives of this study were to: 1) evaluate the in vitro effectiveness and stability of individual and combined treatments of surface-active (lauric arginate (LAE) and LAE and cinnamaldehyde (CA) emulsions) and oxidizing sanitizers (lactic acid (LA) and peracetic acid (PAA) against pathogenic E. coli as a function of time and concentrations; 2) assess the antimicrobial effectiveness of treatments (lactic acid, peracetic acid, lauric arginate and lauric arginate emulsion) applied individually or sequentially to E. coli-inoculated spinach in a pilot-washing system as a function of time and organic load (1% and 10% v/v); 3) implement effective treatments from pilot-wash system for use in field washing trials to compare the shelf-life (yeast and mold growth, aerobic plate counts and visual quality) as a function of leafy green type (lettuce, spinach and green mix) and treatment type (LAE (1%) + CA (1%) individually and LAE (1%) + CA (1%) then PAA and SaniDate 5.0 control). To evaluate the in vitro effectiveness and stability of individual and combined treatments of surface-active and oxidizing sanitizers food grade emulsions were created using LAE (1%) + CA (1 and 2%) with or without coconut oil (CO) (8%) as a carrier. Emulsions were characterized for particle size, surface charge and hydrophobicity. Treatments (1mL) also included lactic acid (LA) solution (1%), LAE solution (1%) and their combinations (0.5mL LA with 0.5mL LAE) and peracetic acid (PAA) (100ppm). Treatments were applied to a STEC E. coli cocktail (O157:H7, O26 and O121) (10⁸ CFU/mL) that was grown overnight. Solutions were neutralized and enumerated at 0.08, 0.16, 0.5, 1, 3, 5 and 24 hours. The most effective single treatment was LAE (1%) + CA (1%) with a 3.78 CFU/mL log reduction after 5 minutes contact. Positive (PAA) and negative (water) controls had a 0.22 CFU/mL log and 0.08 CFU/mL log reduction, respectively. LAE (1%) + CA (1%) had a positive surface charge and an average particle size of 192.7nm. Therefore, LAE (1%) + CA (1%) emulsions are proven effective against E. coli and show potential for use in the food industry. To determine the antimicrobial effectiveness of treatments (lactic acid, peracetic acid, lauric arginate and lauric arginate emulsion) applied individually or sequentially, spinach (10 g) was inoculated with an overnight STEC cocktail (final concentration of 10⁶ CFU/g). Spinach was then washed in a pilot setup (1 L treatment) with the following antimicrobial treatments: LA solution (1%), LAE solution (1%), and LAE (1%) + CA (1%) emulsions. PAA (100 ppm) and water were used as positive and negative controls, respectively. Treatments were applied individually (5 or 10 min) or sequentially (2.5+2.5 or 5+5 min) with or without organic load (OL) (1% and 10%). The samples were then neutralized, and E. coli population enumerated. Spinach color was measured before and after washing in the treatments. The most effective individual treatment was LAE (1%) + CA (1%) no OL with 2.23 CFU/g log reduction after 10 minutes contact time. Positive (PAA) and negative (water) control had a 1.33 CFU/g and 0.22 CFU/g log reduction, respectively. The most effective sequential treatment was LA (1%) followed by LAE (1%) + CA (1%) with no OL. However, at 10% OL, sequential treatment of LAE (1%) + CA (1%) and PAA was the most effective treatment with a 1.83 CFU/g log reduction. Overall, combinations of surface-active antimicrobials (LAE), essential oils (CA) and organic acids can provide sustainable wash-water solutions to effectively control and reduce STECs contamination on leafy greens. To determine the effectiveness of new treatments on shelf-life in field washing trials, fresh lettuce, leafy green mix and spinach were washed at a commercial produce farm in northeast Kansas using the best performing treatments at 10% OL (LAE (1%) + CA (1%) emulsion individually and sequentially followed by PAA) to compare to their existing washing treatment (SaniDate 5.0). Shelf-life of the products were predicted by leafy green color, yeast and mold counts (CFU/g) and total aerobic plate counts (CFU/g) at varying days after harvest and washing (0, 2, 4, 7 and 10 days). After 10 days, for spinach, mixed greens and lettuce, respectively, control (no treatment) showed yeast and mold counts of 6.05 CFU/g, 4.37 CFU/g and 4.02 CFU/g and aerobic plate counts of 6.92 CFU/g, 6.90 CFU/g and 7.40 CFU/g. LAE (1%) + CA (1%) had yeast and mold counts of 3.22 CFU/g, 1.52 CFU/g and 3.48 CFU/g and aerobic plate counts of 4.06 CFU/g, 6.51 CFU/g and 7.12 CFU/g. LAE (1%) + CA (1%) followed by PAA had yeast and mold counts of 2.79 CFU/g, 2.98 CFU/g and 3.59 CFU/g and aerobic plate counts of 3.70 CFU/g, 6.47 CFU/g and 5.41 CFU/g. However, sequential application showed the greatest change in green color (*a) after 10 days. Spinach washed in LAE (1%) + CA (1%) retained green color (*a) the best. LAE (1%) + CA (1%) individually and sequentially with PAA improved the shelf-life of leafy greens by having the lowest yeast and mold and aerobic plate counts. In some cases, sequential application of improved visual quality. A survey was developed using field washing trial results and given to produce growers at farmers’ markets to determine their willingness to implement and pay for the new wash-water treatments. Most farmers (79%) believed that spinach washed in the new treatment showed a visual difference after 7 days compared to control. Most (83%) also said they would be willing to pay more for this treatment and 26% would pay up to $0.30 more per unit to use this new treatment. These studies conclude that when combined with or used sequentially, lauric arginate and cinnamaldehyde emulsions are effective at inactivating E. coli and prolonging shelf-life by reducing yeast, mold and aerobic plate counts on leafy greens. This technology can also be used for other types of produce and has been found effective in other food matrixes. Lauric arginate and essential oil emulsions can provide the food industry with effective antimicrobials that are both sustainable and natural. These treatments can be used by the produce industry to improve the efficacy of their current antimicrobial interventions for fresh produce and reduce the potential for future foodborne outbreak events.