# The Role of Fucoidan in Controlling _Listeria monocytogenes_ Biofilms on Seafood-Contact Surfaces
## 2 Materials and Methods
### Biofilm Inhibition and the Detachment of Biofilm Process  
The biofilm inhibition test was performed using a previous published methodology [26]. The MIC of fucoidan against _L. monocytogenes_ was found to be 150 \(\upmu\)g/mL in this investigation. Although not directly bactericidal, inhibition of biofilm was assessed at sub-MIC levels, which could include affected bacterial virulence characteristics. A control group devoid of fucoidan and test groups, which were administered fucoidan at 1/8, 1/4, and 1/2 MIC, were part of the experimental design.  
In 50 mL Falcon tubes containing food-contact surface samples, 10 mL of TSB was mixed with fucoidan at the specified doses (0, 1/8, 1/4, and 1/2 MIC) to promote the development of biofilms. A vortex mixer (Scientific Industries, SI-0256, Bohemia, NY, USA) was used to homogenize the liquid after a 100 \(\upmu\)L aliquot of microbial suspension (10\({}^{5}\) Log CFU/mL) was added. To enable the formation of biofilm, the samples were subsequently incubated for 24 h at 30 \({}^{\circ}\)C under static conditions.  
After incubation, they were washed two times with sterile DW to eliminate any loosely attached bacteria. To remove the adhering bacteria, the surfaces were vortexed for 2 min after being submerged in 10 mL of peptone water (PW), including ten sterile glass beads. To count the colony, the recovered microbial solution was serially diluted and cultured on TSA. The colony-forming units (CFU) were counted following a 24 h incubation at 30 \({}^{\circ}\)C.  
The antibiofilm effectiveness of fucoidan was measured by calculating the decrease in microbial populations (Log CFU/cm\({}^{2}\)) in comparison to the control.  
RNA Extraction, Complementary DNA (cDNA) Synthesis, and Real-Time Quantitative PCR (RT-qPCR) Analysis  
This study was carried out with slight modifications from established methods [27; 28; 29] to assess the influence of fucoidan on QS and virulence-related gene expression in _L. monocytogenes_. Bacterial suspensions treated with fucoidan (10\({}^{5}\) log CFU/mL) were inoculated in 10 mL of TSB within Falcon tubes and incubated at 30 \({}^{\circ}\)C for 24 h to promote biofilm development. Following incubation, the bacterial cells were harvested by centrifugation, and total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hidden, Germany).  
A NanoDrop spectrophotometer (BioTek Instruments, Chicago, IL, USA) was used for measuring absorbance at 260/280 nm and 260/230 nm ratios to evaluate the concentrationand purity of RNA. The Maxime RT PreMix (Ran-Damon Primer) kit (iNtRON Biotechnology Co., Ltd., Seoul, Republic of Korea) was used to synthesize cDNA. Table 1 lists the primer sequences for RT-qPCR, using 16S rRNA as the housekeeping reference gene.  
A 20 \(\upmu\)L RT-qPCR reaction was performed using 1 \(\upmu\)L of cDNA template, gene-specific primers, and Power SYBR Green PCR Master Mix (Applied Biosystems, Thermo Fisher Scientific, Warrington, UK). Amplification was performed on a CFX Real-Time PCR System (Bio-Rad, Hercules, CA, USA) under the following cycle conditions: initial denaturation at 95 \({}^{\circ}\)C for 20 s, annealing at 50 \({}^{\circ}\)C for 20 s, and extension at 72 \({}^{\circ}\)C for 20 sec. A melting curve analysis was used to establish product specificity. The 2\({}^{-\Delta\Delta\text{Ct}}\) technique was used to determine relative gene expression levels.