Plant Antimicrobials: Inactivation of Foodborne Pathogens or Induction into the VBNC State and Their Effects on the Sensory Properties of Organic Romaine Lettuce

Abstract

This project consisted of three studies: the first study investigated whether plant-based antimicrobials inactivate foodborne pathogens or induce them to go into the VBNC state in vitro and on romaine lettuce; the second study focused on sensory analysis of organic leafy greens treated with plant antimicrobials in the wash water; and the third study examined the novel concept of applying antimicrobials via edible films. The inert metabolic state of Viable but Non-Culturable (VBNC) that some pathogens can go into is gaining importance in the food and health industries. It is a state in which bacteria do not grow on lab based media but can cause disease in a human host. Foodborne pathogens that can go into VBNC are of immense concern since they can result in false negatives, leading to an outbreak if consumed by the public. The state of VBNC can be triggered by the presence of stress factors that includes chemicals such as sanitizers and antimicrobials. The objective of the first study was to determine whether plant-based antimicrobials inactivate the foodborne pathogens Salmonella enterica serovar Newport, Escherichia coli O157:H7 and Listeria monocytogenes or induce them to go into the VBNC state in vitro and on romaine lettuce. This was done by using selective media and fluorescence microscopy to confirm the viability of the pathogens. Organic iceberg lettuce inoculated with S. enterica, E. coli O157:H7 or L. monocytogenes was dip-treated in one of the following solutions: 0.1%, 0.3%, and 0.5% of essential oils or their active components; 3 and 5% of one of the plant extracts, hydrogen peroxide or chlorine for 2 min and stored at refrigeration temperature (4°C). Samples were taken on days 0, 1, and 3, for enumeration of survivors. The reductions in bacterial populations ranged from 2-6 log CFU/g. Oregano oil and its active component carvacrol were seen to be most effective in reducing the bacterial populations to below detectable limits on day 0. All the treatments reduced Listeria populations to below detectable limits by day 1. It was generally seen that citral showed more potent antimicrobial activity as compared to lemongrass oil against Listeria. The fluorescence microscopy results also correlate with the plating results. The dead cells fluoresced orange/red and the viable cells fluoresced green. The second study focused on sensory analysis of organic leafy greens treated with plant antimicrobials in the wash water. The objective of the second study was to a) evaluate panelists’ responses to changes in the sensory attributes of romaine lettuce treated with plant antimicrobials added to the wash water b) identify preference liking of the panelists to leafy greens washed with various essential oils and plant extracts and c) identify and evaluate the effects of these antimicrobials on the color and texture of the treated leafy greens. Organic romaine lettuce was washed with various plant antimicrobials in tap water for 2 min and stored at 4°C for 20-24 h before performing sensory evaluation and measuring changes in physical properties (color and texture) of leafy greens. A randomized block design was used with 75 panelists. The sensory attributes of the samples, which included pungency, browning, bitterness, off-odor, and sourness were evaluated using a 5 point hedonic scale. The preference liking was evaluated for aroma, color, freshness, mouthfeel, flavor, and overall acceptability using a 9-point hedonic scale where 9 was extremely liked and 1 not liked at all. The color analysis was done using the CIE L*a*b* coordinates and the texture was analyzed using a texture analyzer. Lettuce treated with 7% olive extract and 3% apple extract had a higher likelihood of being purchased and the least likely to be purchased treatments were oregano oil and a combination of oregano oil and grapeseed extract. As per the preference liking, lettuce treated with 7% olive extract and 0.1% citral had a higher overall acceptability. The least acceptable treatments were those of oregano oil and clove bud oil. The color of the samples was affected the least by olive extract and lemongrass oil, with oregano oil and carvacrol showing changes in the color. Similarly, for the texture analysis, it was seen that lettuce treated with 0.1% citral (890.0±79.5 N) was the least affected and 0.1% oregano treated samples were the most affected requiring less force (635 N) to crush the samples. We know that essential oils are well known for their potent fragrances and flavor imparting properties. The second study also indicated that the direct exposure of leafy greens to the antimicrobials may be less preferred and so the novel concept of applying antimicrobials via edible films was experimented with in the third study. Edible films are thin layer films made using the pulp of edible plant parts or biopolymers such as chitosan. Our films were made of fruit and vegetable pulp and contained the active components of plant antimicrobials. The objective of the third study was to a) evaluate panelists’ responses to changes in the sensory attributes of romaine lettuce treated with plant antimicrobials added to edible films b) identify preference liking of the panelists to leafy greens treated with various edible films and c) identify and evaluate the effects of these edible films on the color and texture of the treated leafy greens. The edible films were added to bagged lettuce. Edible films were made from tomato, apple, or carrot pulp which included 0.5%, and 1.5% of carvacrol or cinnamaldehyde. Similar parameters were evaluated with these samples. Romaine lettuce treated with apple films had a higher likelihood of being purchased in comparison to other films, with lettuce treated with films containing 1.5% cinnamaldehyde and the control films being the most to be purchased. The least likely to be purchased treatments were those of the tomato films, specifically the films containing 1.5% cinnamaldehyde. The lettuce treated with carrot films had a higher acceptance rating as compared to other films, with films containing 1.5% cinnamaldehyde and the control films being the highest. The color analysis results indicate that the films containing cinnamaldehyde had the least adverse effects. The cinnamaldehyde containing films also needed lower force to crush the leaves, indicating more firmness or crispiness as compared to those containing carvacrol. The results from the three studies are useful in not only helping to decide which plant antimicrobials can be used as potential sanitizers in the organic industry but also provide insight into which treatments are preferred by consumers and will not affect the marketability of the leafy greens.