Edible films containing carvacrol and cinnamaldehyde inactivate Escherichia coli O157:H7 on organic leafy greens in sealed plastic bags

The antimicrobial effects of apple-, carrot-, and hibiscus-based edible films containing carvacrol and cinnamaldehyde against Escherichia coli O157:H7 on organic leafy greens in sealed plastic bags were investigated. Fresh-cut Romaine and Iceberg lettuce, and mature and baby spinach leaves were inoculated with E. coli O157:H7 and placed into Ziploc® bags. Edible films were then added to the bags, which were stored at 4°C. The evaluation of samples taken at days 0, 3, and 7 showed that on all leafy greens, 3% carvacrol-containing films had the greatest effect against E. coli O157:H7, reducing the bacterial population by about 5 log CFU/g on day 0. All three types of 3% carvacrol-containing films reduced E. coli O157:H7 by about 5 log CFU/g at day 0. The 1.5% carvacrol-containing films reduced E. coli O157:H7 by 1–4 logs CFU/g at day 7. Films with 3% cinnamaldehyde showed reduction of 0.6–3 logs CFU/g on different leafy greens.     

Antimicrobial activity of oregano oil against antibiotic-resistant Salmonella enterica on organic leafy greens at varying exposure times and storage temperatures

The objective of this study was to evaluate the effectiveness of oregano oil on four organic leafy greens (Iceberg and Romaine lettuces and mature and baby spinaches) inoculated with Salmonella Newport as a function of treatment exposure times as well as storage temperatures. Leaf samples were washed, dip inoculated with S. Newport (6-log CFU/ml) and dried. Oregano oil was prepared at 0.1, 0.3, and 0.5% concentrations in sterile phosphate buffered saline (PBS). Inoculated leaves were immersed in the treatment solution for 1 or 2 min, and individually incubated at 4 or 8 °C. Samples were taken at day 0, 1, and 3 for enumeration of survivors. The results showed that oregano oil was effective against S. Newport at all concentrations. S. Newport showed reductions from the PBS control of 0.7–4.8 log CFU/g (Romaine lettuce), 0.8–4.8 log CFU/g (Iceberg lettuce), 0.8–4.9 log CFU/g (mature spinach), and 0.5–4.7 log CFU/g (baby spinach), respectively. The antibacterial activity also increased with exposure time. Leaf samples treated for 2 min generally showed greater reductions (by 1.4–3.2 log CFU/g), than those samples treated for 1 min; however, there was minimal difference in antimicrobial activity among samples stored under refrigeration and abuse temperatures. This study demonstrates the potential of oregano oil to inactivate S. Newport on organic leafy greens.     

Antimicrobial activity of apple, hibiscus, olive, and hydrogen peroxide formulations against Salmonella enterica on organic leafy greens

Salmonella enterica is one of the most common bacterial pathogens implicated in foodborne outbreaks involving fresh produce in the last decade. In an effort to discover natural antimicrobials for use on fresh produce, the objective of the present study was to evaluate the effectiveness of different antimicrobial plant extract-concentrate formulations on four types of organic leafy greens inoculated with S. enterica serovar Newport. The leafy greens tested included organic romaine and iceberg lettuce, and organic adult and baby spinach. Each leaf sample was washed, dip inoculated with Salmonella Newport (10(6) CFU/ml), and dried. Apple and olive extract formulations were prepared at 1, 3, and 5% concentrations, and hibiscus concentrates were prepared at 10, 20, and 30%. Inoculated leaves were immersed in the treatment solution for 2 min and individually incubated at 4°C. After incubation, samples were taken on days 0, 1, and 3 for enumeration of survivors. Our results showed that the antimicrobial activity was both concentration and time dependent. Olive extract exhibited the greatest antimicrobial activity, resulting in 2- to 3-log CFU/g reductions for each concentration and type of leafy green by day 3. Apple extract showed 1- to 2-log CFU/g reductions by day 3 on various leafy greens. Hibiscus concentrate showed an overall reduction of 1 log CFU/g for all leafy greens. The maximum reduction by hydrogen peroxide (3%) was about 1 log CFU/g. The antimicrobial activity was also tested on the background microflora of organic leafy greens, and reductions ranged from 0 to 2.8 log. This study demonstrates the potential of natural plant extract formulations to inactivate Salmonella Newport on organic leafy greens.     

Inactivation of Listeria monocytogenes on ham and bologna using pectin-based apple, carrot, and hibiscus edible films containing carvacrol and cinnamaldehyde

Edible films can be used as wrapping material on food products to reduce surface contamination. The incorporation of antimicrobials into edible films could serve as an additional barrier against pathogenic and spoilage microorganisms that contaminate food surfaces. The objective of this study was to investigate the antimicrobial effects of carvacrol and cinnamaldehyde, incorporated into apple, carrot, and hibiscus-based edible films against Listeria monocytogenes on contaminated ham and bologna. Ham or bologna samples were inoculated with L. monocytogenes and dried for 30 min, then surface wrapped with edible films containing the antimicrobials at various concentrations. The inoculated, film-wrapped samples were stored at 4 °C. Samples were taken at day 0, 3, and 7 for enumeration of surviving L. monocytogenes by plating on appropriate media. Carvacrol films showed better antimicrobial activity than cinnamaldehyde films. Compared to control films without antimicrobials, films with 3% carvacrol induced 1 to 3, 2 to 3, and 2 to 3 log CFU/g reductions on ham and bologna at day 0, 3, and 7, respectively. Corresponding reductions with 1.5% carvacrol were 0.5 to 1, 1 to 1.5, and 1 to 2 logs, respectively. At day 7, films with 3% cinnamaldehyde reduced L. monocytogenes population by 0.5 to 1.5 and 0.5 to 1.0 logs on ham and bologna, respectively. Inactivation by apple films was greater than that by carrot or hibiscus films. Apple films containing 3% carvacrol reduced L. monocytogenes population on ham by 3 logs CFU/g on day 0 which was 1 to 2 logs greater than that by carrot and hibiscus films. Films were more effective on ham than on bologna. The food industry and consumers could use these films to control surface contamination by pathogenic microorganisms. PRACTICAL APPLICATION: Antimicrobial edible, food-compatible film wraps prepared from apples, carrots, and hibiscus calyces can be used by the food industry to inactivate Listeria monocytogenes on widely consumed ready to eat meat products such as bologna and ham. This study provides a scientific basis for large-scale application of edible fruit- and vegetable-based antimicrobial films on foods to improve microbial food safety.     

Effectiveness of Active Packaging on Control of Escherichia Coli O157:H7 and Total Aerobic Bacteria on Iceberg Lettuce

Contaminated leafy green vegetables have been linked to several outbreaks of human gastrointestinal infections. Antimicrobial interventions that are adoptable by the fresh produce industry for control of pathogen contamination are in great demand. This study was undertaken to evaluate the efficacy of sustained active packaging on control of Escherichia coli O157:H7 and total aerobic bacteria on lettuce. Commercial Iceberg lettuce was inoculated with a 3‐strain mixture of E. coli O157:H7 at 10² or 10⁴ CFU/g. The contaminated lettuce and un‐inoculated controls were placed respectively in 5 different active packaging structures. Traditional, nonactive packaging structure was included as controls. Packaged lettuce was stored at 4, 10, or 22 °C for 3 wk and sampled weekly for the population of E. coli O157:H7 and total aerobic bacteria. Results showed that packaging structures with ClO₂ generator, CO₂ generator, or one of the O₂ scavengers effectively controlled the growth of E. coli O157:H7 and total aerobic bacteria under all storage conditions. Packaging structure with the ClO₂ generator was most effective and no E. coli O157:H7 was detected in samples packaged in this structure except for those that were inoculated with 4 log CFU/g of E. coli O157:H7 and stored at 22 °C. Packaging structures with an oxygen scavenger and the allyl isothiocyanate generator were mostly ineffective in control of the growth of the bacteria on Iceberg lettuce. The research suggests that some of the packaging structures evaluated in the study can be used to control the presence of foodborne pathogens on leafy green vegetables.     

Inactivation of Salmonella enterica serovar Typhimurium and Quality Maintenance of Cherry Tomatoes Treated with Gaseous Essential Oils

The antimicrobial activity of the essential oils (EOs) from cinnamon bark, oregano, mustard, and of their major components cinnamaldehyde, carvacrol, and allyl isothiocyanate (AIT) was evaluated as a gaseous treatment to reduce Salmonella enterica serovar Typhimurium in vitro and on tomatoes. In vitro tests showed that mustard EO and AIT had the greatest inhibition of Salmonella, followed by cinnamon EO and cinnamaldehyde, while oregano and carvacrol showed the least inhibition. Scanning electron microscopy images of S. Typhimurium on tomatoes suggest that the EOs and their major components damaged the bacteria, and the damage was more obvious after posttreatment storage at 10 °C for 4 and 7 d. Salmonella on inoculated tomatoes was reduced by more than 5 log colony forming units (CFU)/g by mustard EO and AIT, by 4.56 and 3.79 log CFU/g following cinnamon EO and cinnamaldehyde treatments, respectively, and 1.54 and 3.37 log CFU/g after oregano EO and carvacrol treatments, respectively. Mustard EO and AIT induced discoloration, softening, and loss of the vitamin C and lycopene during 21 d of storage at 10 °C, while treatment with cinnamon EO and cinnamaldehyde did not result in significant changes in tomato quality. Tomatoes treated with oregano EO had better quality than nontreated samples after storage. Therefore, treatment with cinnamon and oregano EO and their major components appeared to be feasible for inactivation of Salmonella on tomatoes and maintaining quality.     

Edible Apple Film Wraps Containing Plant Antimicrobials Inactivate Foodborne Pathogens on Meat and Poultry Products

ABSTRACT:  Apple-based edible films containing plant antimicrobials were evaluated for their activity against pathogenic bacteria on meat and poultry products.  Salmonella enterica  or  E. coli  O157:H7 (10⁷ CFU/g) cultures were surface inoculated on chicken breasts and  Listeria monocytogenes  (10⁶ CFU/g) on ham. The inoculated products were then wrapped with edible films containing 3 concentrations (0.5%, 1.5%, and 3%) of cinnamaldehyde or carvacrol. Following incubation at either 23 or 4 °C for 72 h, samples were stomached in buffered peptone water, diluted, and plated for enumeration of survivors. The antimicrobial films exhibited concentration-dependent activities against the pathogens tested. At 23 °C on chicken breasts, films with 3% antimicrobials showed the highest reductions (4.3 to 6.8 log CFU/g) of both  S. enterica  and  E. coli  O157:H7. Films with 1.5% and 0.5% antimicrobials showed 2.4 to 4.3 and 1.6 to 2.8 log reductions, respectively. At 4 °C, carvacrol exhibited greater activity than did cinnamaldehyde. Films with 3%, 1.5%, and 0.5% carvacrol reduced the bacterial populations by about 3, 1.6 to 3, and 0.8 to 1 logs, respectively. Films with 3% and 1.5% cinnamaldehyde induced 1.2 to 2.8 and 1.2 to 1.3 log reductions, respectively. For  L. monocytogenes  on ham, carvacrol films induced greater reductions than did cinnamaldehyde films at all concentrations tested. In general, the reduction of  L. monocytogenes  on ham at 23 °C was greater than at 4 °C. Added antimicrobials had minor effects on physical properties of the films. The results suggest that the food industry and consumers could use these films as wrappings to control surface contamination by foodborne pathogenic microorganisms.     

Salmonella sensitivity to sodium hypochlorite and citric acid in washing water of lettuce residues

Salmonella spp. is one of the main lettuce pathogens and should be inactivated during the disinfection of these vegetables before consumption. In minimally processed vegetable industries, residues of organic matter can prevent the inactivation of this pathogen by disinfectants. The objective of the present work was to evaluate the inactivation of Salmonella isolated from organic lettuce to sodium hypochlorite (25 and 50 ppm) and citric acid (0.5 and 1%) in washing water added with lettuce residues. To do so, a washing water with lettuce residues was elaborated, and Salmonella was added in the order of 10⁶ CFU/ml. Thereafter, each sanitizer was added separately to evaluate its effect on reducing Salmonella counts. After 1, 2, 3, 4, 5, 10, and 15 min of contact with the sanitizers, serial dilutions using neutralizer (0.5% sodium thiosulfate) were performed and each dilution was sown in Xylose-Lysine-Desoxycholate medium. Total aerobic mesophilic counts of wash water with lettuce residues before testing (without Salmonella) and after 15 min of exposure to each sanitizer (with Salmonella) were also performed. In addition, the free chlorine still present in the samples after the contact of sodium hypochlorite with lettuce residues for 15 min. The results demonstrated that 50 and 25 ppm sodium hypochlorite could reduce 6 log CFU/ml of Salmonella in 1 and 3 min of contact, respectively, while 0.5 and 1% citric acid was able to reduce 1.26 and 1.74 log CFU/ml respectively from the same microorganism within 15 min of contact. The total aerobic mesophilic counts of the wash water before being tested were, on average, 1.5 log CFU/ml. After addition of Salmonella, with 15 min of contact with the sanitizer, the results of total counts showed the same magnitude as the Salmonella counts. Organic matter may have reacted with the free chlorine present, reducing chlorine concentrations, since values of 30.4 ppm were observed when the initial concentration should be 50 and 17.1 ppm when the initial concentration should be 25 ppm. Based on the results, sodium hypochlorite demonstrated a greater microbial reduction capacity in wash water with lettuce residues, indicating that it is more appropriate to avoid cross-contamination between batches during sanitation of lettuce in washing tanks.     

Inactivation of Escherichia coli O157:H7, Salmonella typhimurium,and Listeria monocytogenes on Stored Iceberg Lettuce by Aqueous Chlorine Dioxide Treatment

ABSTRACT: Inactivation of Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes in iceberg lettuce by aqueous chlorine dioxide (ClO₂) treatment was evaluated. Iceberg lettuce samples were inoculated with approximately 7 log CFU/g of E. coli O157:H7, S. typhimurium, and L. monocytogenes. Iceberg lettuce samples were then treated with 0, 5, 10, or 50 ppm ClO₂ solution and stored at 4 °C. Aqueous ClO₂ treatment significantly decreased the populations of pathogenic bacteria on shredded lettuce (P < 0.05). In particular, 50 ppm ClO₂ treatment reduced E. coli O157:H7, S. typhimurium, and L. monocytogenes by 1.44, 1.95, and 1.20 log CFU/g, respectively. The D₁₀‐values of E. coli O157:H7, S. typhimurium, and L. monocytogenes in shredded lettuce were 11, 26, and 42 ppm, respectively. The effect of aqueous ClO₂ treatment on the growth of pathogenic bacteria during storage was evaluated, and a decrease in the population size of these pathogenic bacteria was observed. Additionally, aqueous ClO₂ treatment did not affect the color of lettuce during storage. These results suggest that aqueous ClO₂ treatment can be used to improve the microbial safety of shredded lettuce during storage.     

Antimicrobial edible apple films inactivate antibiotic resistant and susceptible Campylobacter jejuni strains on chicken breast

Abstract: Campylobacter jejuni is the leading cause of bacterial diarrheal illness worldwide. Many strains are now becoming multidrug resistant. Apple‐based edible films containing carvacrol and cinnamaldehyde were evaluated for bactericidal activity against antibiotic resistant and susceptible C. jejuni strains on chicken. Retail chicken breast samples inoculated with D28a and H2a (resistant strains) and A24a (a sensitive strain) were wrapped in apple films containing cinnamaldehyde or carvacrol at 0.5%, 1.5%, and 3% concentrations, and then incubated at 4 or 23 °C for 72 h. Immediately after wrapping and at 72 h, samples were plated for enumeration of viable C. jejuni. The antimicrobial films exhibited dose‐ and temperature‐dependent bactericidal activity against all strains. Films with ≥1.5% cinnamaldehyde reduced populations of all strains to below detection at 23 °C at 72 h. At 4 °C with cinnamaldehyde, reductions were variable for all strains, ranging from 0.2 to 2.5 logs and 1.8 to 6.0 logs at 1.5% and 3.0%, respectively. Films with 3% carvacrol reduced populations of A24a and H2a to below detection, and D28a by 2.4 logs at 23 °C and 72 h. A 0.5‐log reduction was observed for both A24a and D28a, and 0.9 logs for H2a at 4 °C at 3% carvacrol. Reductions ranged from 1.1 to 1.9 logs and 0.4 to 1.2 logs with 1.5% and 0.5% carvacrol at 23 °C, respectively. The films with cinnamaldehyde were more effective than carvacrol films. Reductions at 23 °C were greater than those at 4 °C. Our results showed that antimicrobial apple films have the potential to reduce C. jejuni on chicken and therefore, the risk of campylobacteriosis. Possible mechanisms of antimicrobial effects are discussed. Practical Application: Apple antimicrobial films could potentially be used in retail food packaging to reduce C. jejuni commonly present on food.     

Decontamination of Salmonella,Shigella, and Escherichia coli O157:H7 from Leafy Green Vegetables Using Edible Plant Extracts

Fresh cilantro, parsley, and spinach are products that are regularly consumed fresh, but are difficult to decontaminate, as a result, they are common vehicles of transmission of enteropathogenic bacteria. In this study, the efficacy of plant extracts as alternatives for disinfection of cilantro, parsley, and spinach that were artificially contaminated with Salmonella, Escherichia coli O157:H7, and Shigella sonnei was determined. Edible plant extracts obtained using ethanol as the extraction solvent were tested to determine the minimum bactericidal concentration (MBC) and those that exhibited the lowest MBC were selected for further studies. Leaves of fresh greens were washed with sterile water and dried. For seeding, leaves were submerged in suspensions of 2 different concentrations of bacteria (1.5 × 10⁸ and 1 × 10⁵), dried, and then stored at 4 °C until use. To determine the effects of the extracts, inoculated leafy greens were submerged in a container and subjected to treatments with chlorine, Citrol®, or selected plant extracts. Each treatment type was stored at 4 °C for 0, 1, 5, and 7 d, and the bacterial counts were determined. From the 41 plant extracts tested, the extracts from oregano leaves and from the peel and pulp of limes were found to be as effective as chlorine or Citrol® in reducing by > 2 logs, the population of pathogenic bacteria on leafy greens and therefore, may be a natural and edible alternative to chemicals to reduce the risk of Salmonella, E. coli O157:H7 and S. sonnei contamination on leafy vegetables. Practical Application: The antimicrobial efficacy of the extracts of Mexican lime and oregano was clearly demonstrated on cilantro, parsley, and spinach. The extracts of Mexican lime and oregano provide alternatives to chlorine to significantly reduce bacterial pathogens that have been associated with outbreaks from contaminated leafy green vegetables. A simple, low cost, and labor‐saving extraction system for production of the extracts was used.     

Nanoencapsulation and immobilization of cinnamaldehyde for developing antimicrobial food packaging material

Use of antimicrobial coatings on food packaging is one of the important technologies of active packaging for improving food safety. There is growing demand for natural antimicrobials because of fear of adverse health effects of synthetic preservatives. The objectives of this study were to compare antibacterial properties of free and nanoencapsulated cinnamaldehyde in solution; polylactic acid (PLA) surfaces cast with cinnamaldehyde; and glass and PLA surfaces coated with cinnamaldehyde nano-liposomes. Cinnamaldehyde was nano-encapsulated by lipid bilayers of polydiacetylene – N-hydroxysuccinimide (PDA–NHS) nano liposomes and immobilized on glass slides and PLA films. Glass surfaces immobilized with nano-encapsulated cinnamaldehyde showed significant antibacterial activity against Escherichia coli W1485 and Bacillus cereus ATCC 14579, with reductions of 2.56 log₁₀ CFU/ml and 1.59 log₁₀ CFU/ml respectively in 48 h. PLA films cast with cinnamaldehyde also showed significant antibacterial activities against E. coli W1485 (2.01 log₁₀ CFU/ml reduction) and B. cereus (4.81 log₁₀ CFU/ml reduction). However, when the liposomal encapsulated cinnamaldehyde was immobilized on PLA films, it did not show any antibacterial activity. Glass surfaces coated with nano-encapsulated cinnamaldehyde may be used as an active packaging material in preserving liquid foods; however, further study is required to improve antimicrobial activities of PLA surfaces.     

THE ANTIBACTERIAL EFFECT OF CINNAMALDEHYDE, THYMOL, CARVACROL AND THEIR COMBINATIONS AGAINST THE FOODBORNE PATHOGEN SALMONELLA TYPHIMURIUM

The aim of this study was to investigate the antimicrobial activity of cinnamaldehyde, thymol and carvacrol alone or their combinations against Salmonella Typhimurium. The results showed that the lowest concentrations of cinnamaldehyde, thymol and carvacrol inhibiting the growth of S. Typhimurium significantly were 200, 400 and 400 mg/L, respectively. In a system combining different antimicrobials, treatments with cinnamaldehyde/thymol, cinnamaldehyde/carvacrol and thymol/carvacrol revealed significantly less population of S. Typhimurium and had synergy effect compared with samples treated with cinnamaldehyde, thymol or carvacrol alone. By means of their paired combinations, the lowest addition of cinnamaldehyde, thymol and carvacrol could be decreased from 200, 400 and 400 mg/L to 100, 100 and 100 mg/L, respectively.     

Reduction of Salmonella Typhimurium and Listeria monocytogenes on produce by trisodium phosphate

The application of trisodium phosphate (TSP) on produce against food-borne bacteria has not been extensively evaluated. This research studied the effect of 20 and 50 mg/ml TSP in reducing Salmonella Typhimurium (ST) and Listeria monocytogenes (Lm) on model produce (lettuce and peppers). Washed and air-dried Iceberg lettuce (3 × 3 cm²) and Jalapeno peppers (25–30 g) were spiked with S. Typhimurium or L. monocytogenes (～7 log₁₀ CFU/ml). Samples were treated with 20 or 50 mg/ml TSP, 200 mg/L sodium hypochlorite (for comparison with traditional washes) or water (control rinse) for 15 or 30 s. Treatments were immediately neutralized with trypticase soy broth (TSB) containing 30 mg/ml beef extract, serially diluted, plated on Xylose Lysine Tergitol 4 (XLT4) agar for ST and Tryptic Soy Agar (TSA) for Lm and incubated at 37 °C for 24–48 h. Results showed that 20 mg/ml TSP and 200 mg/L sodium hypochlorite caused 5–6 log₁₀ CFU/ml reduction in ST on both lettuce and peppers after 15 s and 30 s, while 50 mg/ml TSP reduced ST to undetectable levels on both produce at both contact times. For overnight Lm cultures, a mere reduction of 0.21 and 0.28 log₁₀ CFU/ml was obtained using 20 and 50 mg/ml TSP after even 5 min, while 200 mg/L sodium hypochlorite decreased Lm by ～1 log₁₀ CFU/ml after 30 s and 1 min on lettuce, and decreased pure culture Lm to undetectable levels after 3 min. Thus, 50 mg/ml TSP appears effective against ST with negligible effects against Lm, whose mode of action needs to be understood.     

Effectiveness of organic acid,ozonated water and chlorine dippings on microbial reduction and storage quality of fresh‐cut iceberg lettuce

BACKGROUND: The comparative effects of organic (citric and lactic) acids, ozone and chlorine on the microbiological population and quality parameters of fresh-cut lettuce during storage were evaluated. RESULTS: Dipping of lettuce in 100 mg L⁻¹ chlorine solution reduced the numbers of mesophilic and psychrotrophic bacteria and Enterobacteriaceae by 1.7, 2.0 and 1.6 log₁₀ colony-forming units (CFU) g⁻¹ respectively. Treatment of lettuce with citric (5 g L⁻¹) and lactic (5 mL L⁻¹) acid solutions and ozonated water (4 mg L⁻¹) reduced the populations of mesophilic and psychrotrophic bacteria by 1.7 and 1.5 log₁₀ CFU g⁻¹ respectively. Organic acid dippings resulted in lower mesophilic and psychrotrophic counts than ozonated water and chlorine dippings during 12 days of storage. Lactic acid dipping effectively reduced (by 2.2 log₁₀ CFU g⁻¹) and maintained low populations of Enterobacteriaceae on lettuce for the first 6 days of storage. No significant (P > 0.05) changes were observed in the texture and moisture content of lettuce samples dipped in chlorine, organic acids and ozonated water during storage. Colour, β-carotene and vitamin C values of fresh-cut iceberg lettuce did not change significantly (P > 0.05) until day 8. CONCLUSION: Lactic and citric acid and ozonated water dippings could be alternative treatments to chlorine dipping to prolong the shelf life of fresh-cut iceberg lettuce. Copyright © 2007 Society of Chemical Industry     

Microbial safety considerations of flooding in primary production of leafy greens: A case study

This study evaluated the effects of a flood event, floodplain and climatic parameters on microbial contamination of leafy greens grown in the floodplains. Additionally, correlations between pathogenic bacteria and levels of indicator microorganisms have been also determined. To diagnose the microbial contamination after the flood event, sampling was carried out in weeks 1, 3, 5 and 7 after the flooding in four flooded lettuce fields. To assess the impact of flooding on the microbial contamination of leafy greens, indicator microorganisms (coliforms, Escherichia coli and Enterococcus) and pathogenic microorganisms (Salmonella spp., VTEC (E. coli O157:H7 and other verocytotoxin producing E. coli, O26, O103, O111, O145) and Listeria monocytogenes) were evaluated. Irrigation water, soil and lettuce samples showed levels of coliforms and E. coli higher than 5 and 3 log cfu/g or 100 mL, respectively when sampled 1 week after flooding. However, bacterial counts drastically declined three weeks after the flooding. Climatic conditions after flooding, particularly the solar radiation (6–8 MJ/m²), affected the survival of bacteria in the field. L. monocytogenes was not detected in lettuce samples, except for 2 samples collected 3 weeks after the flooding. The presence of Salmonella was detected in irrigation water, soil and lettuce by multiplex PCR one week after the flooding, but only 2 samples of soil and 1 sample of water were confirmed by colony isolation. Verotoxigenic E. coli was detected in soil and lettuce samples by multiplex PCR. Therefore, the implication of flood water as the source of VTEC contamination of soil and lettuce was not clear. E. coli counts in irrigation water were positively correlated with those in lettuce. A significant correlation (P < 0.005) was found between the presence of pathogens and E. coli counts, highlighting a higher probability of detection of pathogens when high levels of E. coli are found. The results obtained in the present study confirm previous knowledge which defined flooding as a main risk factor for the microbial contamination of leafy greens.     

Whole-Leaf Wash Improves Chlorine Efficacy for Microbial Reduction and Prevents Pathogen Cross-Contamination during Fresh-Cut Lettuce Processing

Abstract: Currently, most fresh-cut processing facilities in the United States use chlorinated water or other sanitizer solutions for microbial reduction after lettuce is cut. Freshly cut lettuce releases significant amounts of organic matter that negatively impacts the effectiveness of chlorine or other sanitizers for microbial reduction. The objective of this study is to evaluate whether a sanitizer wash before cutting improves microbial reduction efficacy compared to a traditional postcutting sanitizer wash. Romaine lettuce leaves were quantitatively inoculated with E. coli O157:H7 strains and washed in chlorinated water before or after cutting, and E. coli O157:H7 cells that survived the washing process were enumerated to determine the effectiveness of microbial reduction for the 2 cutting and washing sequences. Whole-leaf washing in chlorinated water improved pathogen reduction by approximately 1 log unit over traditional cut-leaf sanitization. Similar improvement in the reduction of background microflora was also observed. Inoculated “Lollo Rossa” red lettuce leaves were mixed with noninoculated Green-Leaf lettuce leaves to evaluate pathogen cross-contamination during processing. High level (96.7% subsamples, average MPN 0.6 log CFU/g) of cross-contamination of noninoculated green leaves by inoculated red leaves was observed when mixed lettuce leaves were cut prior to washing in chlorinated water. In contrast, cross-contamination of noninoculated green leaves was significantly reduced (3.3% of subsamples, average MPN ≤−0.3 log CFU/g) when the mixed leaves were washed in chlorinated water before cutting. This result suggests that whole-leaf sanitizing washes could be a practical strategy for enhancing the efficacy of chlorine washes for pathogen reduction and cross-contamination prevention. Practical Application: Freshly cut leafy greens release large amount of organic matter that negatively impact the chlorine washing efficacy. Implementing the primary antimicrobial intervention step of chlorine washing prior to cutting can significantly improve the efficacy of microbial reduction and minimize pathogen cross-contamination.     

Inhibition of Listeria monocytogenes and Salmonella by Combinations of Oriental Mustard,Malic Acid,and EDTA

The antimicrobial activities of oriental mustard extract alone or combined with malic acid and EDTA were investigated against Salmonella spp. or Listeria monocytogenes at different temperatures. Five strain Salmonella or L. monocytogenes cocktails were separately inoculated in Brain Heart Infusion broth containing 0.5% (w/v) aqueous oriental mustard extract and incubated at 4 °C to 21 °C for 21 d. For inhibitor combination tests, Salmonella Typhimurium 02:8423 and L. monocytogenes 2–243 were individually inoculated in Mueller Hinton broth containing the mustard extract with either or both 0.2% (w/v) malic acid and 0.2% (w/v) EDTA and incubated at 10 °C or 21 °C for 10 to 14 d. Mustard extract inhibited growth of the L. monocytogenes cocktail at 4 °C up to 21 d (2.3 log₁₀ CFU/mL inhibition) or at 10 °C for 7 d (2.4 log₁₀ CFU/mL inhibition). Salmonella spp. viability was slightly, but significantly reduced by mustard extract at 4 °C by 21 d. Although hydrolysis of sinigrin in mustard extract by both pathogens was 2 to 6 times higher at 21 °C than at 4 °C to 10 °C, mustard was not inhibitory at 21 °C, perhaps because of the instability of its hydrolysis product (allyl isothiocyanate). At 21 °C, additive inhibitory effects of mustard extract with EDTA or malic acid led to undetectable levels of S. Typhimurium and L. monocytogenes by 7 d and 10 d, respectively. At 10 °C, S. Typhimurium was similarly susceptible, but combinations of antimicrobials were not more inhibitory to L. monocytogenes than the individual agents.     

Combined effect of ultrasound and organic acids to reduce Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes on organic fresh lettuce

This study was performed to compare the effectiveness of individual treatments (ultrasound and organic acids) and their combination on reducing foodborne pathogens on organic fresh lettuce. Lettuce leaves were inoculated with a cocktail of three strains each of Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes and treated with ultrasound (40 kHz) alone, organic acids (0.3, 0.5, 0.7, 1.0, and 2.0% — malic acid, lactic acid, and citric acid) alone and combined with ultrasound and organic acids for 5 min. For all 3 pathogens, the combined treatment of ultrasound and organic acids resulted in additional 0.8 to 1.0 log reduction compared to individual treatments, without causing significant quality change (color and texture) on lettuce during 7 day storage. The maximum reductions of E. coli O157:H7, S. Typhimurium, and L. monocytogenes were 2.75, 3.18, and 2.87 log CFU/g observed after combined treatment with ultrasound and 2% organic acid for 5 min, respectively. Our results suggest that the combined treatment of ultrasound with organic acids was effective at increasing pathogen reduction compared to individual treatments without significantly affecting quality, and demonstrates its potential as a novel method to increase the microbial safety on organic fresh lettuce.     

Spatial persistence of Escherichia coli O157:H7 flowing on micropatterned structures inspired by stomata and microgrooves of leafy greens

This study investigated the persistence of Escherichia coli O157:H7 on the surface of leafy greens, and penetration into intercellular spaces through stomatal pores. This was done by determining the role of leafy green-mimicking microgrooves (normal and fully open stomata and micro-sized crevices) and the roles of pore size and orientation on proliferation of E. coli. Microgroove-embedded microfluidics were fabricated using soft-lithography. Natural wax-polydimethylsiloxane (PDMS) substrates were used for mimicking the surface of leafy greens, such as spinach and lettuce. Spatial microcolonization of E. coli was characterized after cell suspensions (10⁹ CFU/mL) were exposed to static and laminar flow conditions for 36 h. Under fluid shear, higher numbers of microcolonies were seen at vortices and edges, particularly located on downstream areas of individual stomata microstructures. With crevice arrays, colonies existed predominantly on crevices at the sides of arrays rather than those in center-lines, due to higher fluid shear at these locations. These results suggested that spatial changes of bacterial colonization in response to shear flow can affect contamination of leafy greens and show the need and possibilities to design irrigation and washing systems to mitigate such growth.