By Tanmaie Kalapala, University of Arkansas
Researchers from University of Connecticut in U.S. conducted this research study to investigate the efficacy of Trans-Cinnamaldehyde Nano Emulsion (TCNE) as a natural antimicrobial for controlling biofilms formed by Salmonella Enteritidis on common food processing plants and farm-contact surfaces. Salmonella Enteritidis is one of the leading causes of foodborne illnesses that are commonly associated with consumption or handling poultry meats and/or eggs. This pathogen is responsible for several outbreaks globally and causes huge economic losses to the poultry industry every year. Its ability to form persistent biofilms on stainless steel, plastic, and even on eggshells with increased tolerance to regular cleaning and sanitization, increases the chances of cross-contamination in poultry processing environments. Currently used chemical based disinfectants, such as chlorine, peracetic acid, and hydrogen peroxide, had limited success against mature biofilms, especially in the presence of organic matter. This limitation highlights the need for more sustainable and safe antimicrobial strategies to control S. Enteritidis biofilm in farm and processing environments. Phytochemicals can be used as safe and sustainable antimicrobials that are generally secondary metabolites produced as a defense mechanism to protect plants from pathogenic microorganisms. Previous research showed their antimicrobial effects on various food borne pathogens. Among the currently available phytochemicals, Trans-cinnamaldehyde (TC), extracted from cinnamon bark and recognized as Generally Recognized as Safe (GRAS) by the FDA, has shown strong antimicrobial activity against different pathogens. However, due to its low solubility, it has not been widely used as water-based disinfectant in poultry industries. To overcome this challenge, the present study developed a stable nanoemulsion of TC and evaluated its efficacy in inhibiting and inactivating S. Enteritidis biofilms on polystyrene and stainless-steel surface, as well as its impact on the expression of biofilm-associated genes.
The researchers prepared an oil-in-water nanoemulsion of TC by mixing with Tween 80 that acts as surfactant by using sonication, a high energy method to achieve nano sized droplets. The nanoemulsion showed a mean particle size of approximately 112 nanometers, with a zeta potential of ~5 mV showing its stability at 4°C for two months. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and sub-inhibitory concentration (SIC) of TC and Transcinnamaldehyde Nano emulsion against S. Enteritidis strain SE21 that’s isolated from chicken intestine were determined. The MIC and MBC were found to be 0.03% and 0.06%, respectively, whereas 0.01% was identified as the SIC, a concentration that does not inhibit bacterial growth but can modulate physiological processes including biofilm formation. These concentrations were applied to study biofilm inhibition and inactivation on both polystyrene and stainless-steel surfaces.
At the sub-inhibitory concentration, TCNE significantly reduced biofilm formation. When S. Enteritidis was allowed to form biofilms in the presence of 0.01% TCNE at 25°C, biofilm development was reduced by approximately 45% on polystyrene surface and 75% on stainless steel after 48 hours, compared to untreated controls. Even at 0.5% and 1% concentration of TCNE biofilm forming S. Enteritidis were reduced significantly when compared to TC. These results suggest that the non-emulsified TC oil was not as effective as TCNE in inhibition of biofilm formation on steel surfaces, suggesting that nanoemulsion enhanced the bioavailability and surface interaction of the compound. In the inactivation experiments, S. Enteritidis were allowed to form biofilm for 48 hours at 25°C and were exposed to different concentrations of 0.5% and 1% TC and TCNE for 1, 5, and 15 minutes. Both treatments resulted in significant reductions in viable Salmonella counts, but the nanoemulsion consistently showed higher efficacy when compared to TC. Within one minute of exposure, 0.5% TCNE reduced bacterial counts by about 1.5 log CFU/mL, after 5 mins S. Enteritidis was reduced further by 3 log CFU/mL and by 15 minutes, further reduction by 6 log CFU/mL was achieved on both polystyrene and steel surfaces. The enhanced antimicrobial activity was attributed to the small droplet size and high surface area-to-volume ratio of the nanoemulsion, which improved interaction with bacterial membranes and facilitated penetration into the biofilm matrix. Confocal laser scanning microscopy further confirmed that TCNE disrupted the biofilm architecture and cell membranes, as shown by the predominance of red fluorescence that depicts dead cells in treated samples compared to green-stained viable cells in untreated controls.
To understand the molecular mechanism of biofilm inhibition, the researchers analyzed the expression of several S. Enteritidis genes that were known to regulate adhesion, motility, and biofilm matrix formation. Treating the biofilms at sub-inhibitory concentrations of TC significantly downregulated multiple genes. Among these, sipA, sipB, sipC, sopB, hilA, and hilC, which are involved in host cell invasion and surface attachment, were reduced by over threefold. The flagellar motility gene flhD and the quorum-sensing regulator sdiA were also suppressed, indicating reduced bacterial motility and communication. Similarly, csgA and csgD, key regulators of curli fiber formation and biofilm matrix structure, were downregulated by approximately threefold. Interestingly, rpoS, a general stress response gene, was not changed, suggesting that TC specifically targeted biofilm-related pathways rather than general stress adaptation mechanisms. These findings indicate that TC disrupts the genetic regulatory networks essential for biofilm establishment, further validating its antibiofilm potential.
Overall, the study provides strong evidence that trans-cinnamaldehyde nanoemulsion can be used an effective natural antimicrobial formulation that’s capable of both preventing and inactivating S. Enteritidis biofilms. The improved dispersion and stability of TCNE enhanced its antimicrobial efficacy compared to TC oil alone, while its mode of action involves direct structural disruption of bacterial cell membranes and transcriptional repression of genes associated with virulence and biofilm formation. The research highlights TCNE’s potential as a GRAS-based alternative to conventional sanitizers for use in poultry farms and food processing facilities. Implementing such natural antimicrobials could reduce the reliance on chemical-based antimicrobials thereby reducing antimicrobial resistance and improve food safety through reduced pathogen persistence on industrial surfaces. Future studies could focus on evaluating the correlation between a reduction in S. Enteritidis biofilm counts and corresponding reductions in pathogen load on carcass under commercial processing conditions, including its interaction with organic matter and various microbial communities, and assessing the synergistic effects with other available phytochemicals. In conclusion, trans-cinnamaldehyde nanoemulsion has the potential to be used as a natural, safe, and environmentally friendly formulation to control biofilm in the poultry industry, with broad implications for sustainable food safety management.
The full paper can be found in Volume 104, Issue 5 of Poultry Science and is available online at https://doi.org/10.1016/j.psj.2025.105086
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