Progress report for GNE24-329
Project Information
In the last two decades, tomatoes have been implicated in over 170 foodborne outbreaks in the US. Among the different foodborne pathogens, Salmonella enterica has been associated with over 37% of tomato-related outbreaks. Fresh tomatoes can get contaminated from via multiple routes including irrigation water, soil, and manure. In addition, since fresh tomatoes are consumed raw, they pose an increased risk for food safety and public health. Thus, improving the microbial safety of tomatoes continues to be a pressing need for the produce industry. However, while chemical methods do not appeal to consumer preference for natural foods, physical methods tend to be expensive and cannot provide the sustainable antimicrobial effect. Hence, it is critical to develop effective, safe, consumer and environmentally friendly antimicrobial strategies to control foodborne pathogens in tomatoes. Toward this, probiotics including lactic acid bacteria can be ideal candidates for application in produce safety. Probiotics exert their antimicrobial ability by producing lactic acid, antimicrobial peptides, and via bacterial antagonism. Further, they occupy the same niche as pathogens and hence can provide a sustained antimicrobial effect against pathogens including Salmonella. Thus, this study will investigate the efficacy of probiotic spray application in controlling S. enterica contamination on tomatoes. Overall, the successful completion of the study is anticipated to develop an effective, natural, user and environmentally friendly antimicrobial strategy to improve the microbial safety of tomatoes.
The overall objective of this proposed study is to improve food safety and public health while also benefiting the produce industry. Specifically, this study aims to develop an effective, environmentally friendly, natural, and sustainable antimicrobial intervention to reduce foodborne pathogen contamination of tomatoes. The hypothesis of this research is that spray application of probiotics can reduce S. enterica contamination in tomatoes following harvest and during subsequent storage, thereby improving the microbial safety of tomatoes.
The specific objectives for this study are:
To determine the efficacy of probiotics to reduce S. enterica on tomatoes
- when applied as a post-harvest spray, and
- during refrigerated storage
In the last two decades, tomatoes have been implicated in over 170 foodborne outbreaks in the US. Among the different foodborne pathogens, Salmonella enterica has been associated with over 37% of tomato-related outbreaks. Fresh tomatoes can get contaminated via multiple routes including irrigation water, soil, and manure. In addition, since fresh tomatoes are consumed raw, they pose an increased risk for food safety and public health. Thus, improving the microbial safety of tomatoes continues to be a pressing need for the produce industry. However, while chemical methods do not appeal to consumer preference for natural foods, physical methods tend to be expensive and cannot provide the sustainable antimicrobial effect. Hence, it is critical to develop effective, safe, consumer and environmentally friendly antimicrobial strategies to control foodborne pathogens in tomatoes. Toward this, probiotics, including lactic acid bacteria, can be ideal candidates for application in produce safety. Probiotics exert their antimicrobial ability by producing lactic acid, antimicrobial peptides, and via bacterial antagonism. Further, they occupy the same niche as pathogens and hence can provide a sustained antimicrobial effect against pathogens including Salmonella. Thus, this study will investigate the efficacy of probiotic spray applications in controlling S. enterica contamination on tomatoes. Overall, the successful completion of the study is anticipated to develop a safe and effective, antimicrobial strategy to improve the microbial safety of tomatoes.
Research
Bacterial cultures: A five-strain cocktail of S. enterica (SE; S. Newport, S. Montevideo, S. Baildon, S. Braenderup, S. Poona -fresh produce and human outbreak isolates) was used in this study. All pathogen strains were induced for resistance to nalidixic acid (NA, 50mg/ml) to facilitate selective enumeration of the inoculated pathogen (Gurtler et al., 2018; Danyluk et al., 2014). Each strain was cultured separately in 10 ml of sterile tryptic soy broth with NA (TSB-NA, 50 µg/ml) at 37°C for 16-18 h. The overnight cultures were centrifuged at 3500 x g for 10 min at 4°C and washed twice with sterile phosphate buffered saline (PBS, pH 7.0). Equal portions from each strain were combined to make the S. enterica cocktail. The bacterial population in the cocktail was determined by plating appropriate dilutions on Xylose Lysine Deoxycholate agar (XLD-NA) followed by incubation at 37°C for 24 h (Danyluk et al., 2014; Gurtler et al., 2018; Ren et al., 2020; Fay et al., 2023). Appropriate dilutions of the five-strain mixture in PBS were used to obtain the desired level of inoculum (5 log CFU/ tomato). Probiotic bacteria including Lactobacillus rhamnosus NRRL- B-442 (LR), L. paracasei DUP-13076 (LP), Lactococcus lactis B-23802 (LL1), and L. lactis B-23804 (LL2) from the lab culture collection were used in this study. The probiotic cultures were grown separately in de Man Rogosa and Sharpe broth (MRS) at 37°C for 18-20 h. Overnight cultures were centrifuged (3500 x g, 10 min, 4°C), washed and resuspended in sterile water to obtain the desired inoculum (9 log CFU/ml). The probiotic population in the inoculum was determined following serial dilution and plating on MRS agar (Muyyarikkandy and Amalaradjou, 2017).
Standardizing pathogen load on tomatoes: Roma Tomatoes were purchased from local farms and immediately transported to the lab, stored at 4°C, and were used within 3 days. A day before the experiment, tomatoes were transferred to room temperature for tempering prior to use (Penteado et al., 2004a, b). Tomatoes were sprayed using 70% ethanol and wiped. Then, samples were inoculated with the pathogen cocktail [droplet inoculation; 50μl (stem scar) – (9 log/ml, 8 log/ml, and 7.5 log/ml)] using a micropipette to obtain ~ 5 log CFU/tomato of S. enterica. Inoculated tomatoes were dried for 1 or 2 h in the biosafety hood. (Yun et al., 2015; Yuk et al., 2017; Wei et al., 1995). Then, samples were packed in sterilized stomacher bag and stored at 4°C for 3 days. The tomatoes were sampled as described below to determine SE recovery and surviving pathogen populations.
Screening of potential lactic acid bacteria for antimicrobial effect: Inoculated tomatoes were sprayed with (LR, LP, LL1, LL2 - ~9 log CFU/ml) or water (control). Each tomato was sprayed in a biosafety hood using dual action siphon feed airbrush sprayer or electrostatic sprayer to examine the effectivity of spray methods. After spraying, tomatoes were dried for 1h or 2h in the biosafety cabinet, then samples were packed in sterilized stomach bags and stored at 4°C for 1 day (Mortada et al., 2020; Yun et al., 2015).
Bacterial enumeration: Surviving SE and probiotic populations (n=3 fruits/treatment/time) were enumerated at day 0, 1, and 3. Sampling on day 0 helped determine the antimicrobial effect of probiotics when applied as a post-harvest spray application. Sampling at subsequent times was to determine the sustained effect of probiotics during subsequent storage and against potential contamination post-harvest. At each sampling time, each tomato was homogenized in 20 ml PBS in a stomacher for 1 min ribbing. Then, serial dilutions will be plated on appropriate media for bacterial enumeration. For LAB populations, appropriate dilutions will be plated on MRS agar and incubated at 37°C for 24-48 h to enumerate surviving probiotic population (Muyyarikkandy and Amalaradjou, 2017). For SE, samples were plated on XLD-NA agar followed by incubation at 37°C for 24 h (Gurtler et al., 2018; Ren et al., 2020; FDA BAM, 2023; Mathew et al., 2018).
In the current project period, experiments were performed to standardize involution load, drying time, and probiotic antimicrobial efficacy. Specifically, we determined the processing details for SE inoculation on tomatoes to achieve the expected pathogen load (5 log CFU/tomato). We observed that using ~7.5 log CFU/ml cocktail of SE and 1h drying time led to a pathogen recovery level of ~5 log CFU/tomato on day 0. Further, we observed that refrigerated storage of the inoculated tomatoes led to a 1 log reduction in SE populations by day 3 of storage. In addition, our results revealed that using the electrostatic sprayer was better at uniform deposition of probiotics on the tomato surface. Further, initial screening revealed that all probiotics tested were able to significantly reduce SE populations on tomatoes when compared to the control. Given this, in the next project period, we will conduct trials as proposed in the project to validate the results of our preliminary findings and perform longer storage trials. Also, we will perform experiments to evaluate the effect of LAB spraying on tomato quality.
Education & Outreach Activities and Participation Summary
Participation Summary:
The graduate student will collaborate with Dr. Indu Upadhyaya to engage with stakeholders in the region. The intended audience for the outreach initiative will encompass produce farmers, scientists, and extension professionals. Our aim is to inform produce farmers about the benefits of utilizing probiotic cultures to enhance the microbial safety of tomatoes. Furthermore, we will disseminate the findings of our study to the produce industry through extension conferences and gatherings such as the regional produce safety meeting, CT small fruit and vegetable producer conference, and the NECAFS annual conference. These presentations will utilize PowerPoint slides and supporting literature to effectively convey the data. Additionally, we will share our results on the University of Connecticut food safety webpage and Naturally@UConn website to enhance accessibility for producers. Moreover, we plan to present our outcomes at local and national meetings including the Microbiology Symposium at the University of Connecticut, as well as events hosted by the International Association for Food Protection (IAFP), Institute of Food Technologist (IFT), and American Society for Microbiology (ASM). Furthermore, the project's data will be submitted for publication in peer-reviewed journals.