## 3 Biological Activities
### Antibacterial Activity
The _bcs_ operons are partially regulated by _AgfD_, a thin fimbtia that increases the production of cellulose and curli [76]. As mentioned above, cellulose and other extracellular polymeric substances (\(\beta\)-1,6-N-acetyl-D-glucosamine and colonic acid) are key components during the biofilm formation process of _E. coli_ O157:H7 [77]. Citral, in combination with carvacrol, demonstrated antib biofilm activity against _C. sakazakii_, showing that the biofilm formation rate of the samples treated with carvacrol (1/2 MIC) and citral (1/2 MIC) for 48 h decreased from 100% to 81.66% and 59.62%, respectively. On the other hand, after treatments with the combinations of carvacrol and citral (1/4 MIC and 1/2 MIC) for 48 h, the rate of biofilm formation was reduced to 33.41% and 6.28%, respectively. Biofilm formation was almost completely suppressed at 37 \({}^{\circ}\)C when the combination of citral 1/2 MIC and carvacrol was used. These data revealed that carvacrol and citral had a synergistic inhibitory effect on the biofilm formation of _C. sakazakii_. [55]. Additionally, the authors propose that the genes associated with biofilm formation in _C. sakazakii_ were generally downregulated under treatment with the combination of citral, revealing that citral significantly downregulated the expression of _bcsA_ and _bcsG_ that encode the cellulose biosynthesis operon. Citral may influence the biosynthesis of components associated with EPS and consequently change the structure of biofilm, making it more susceptible. It has been proposed that essential oils may affect the growth of flagella and thereby inhibit the formation of biofilms [78]. Citral was combined with other terpene compounds (carvacrol, thymol, and caprylic acid), and the growth inhibition of _C. sakazakii_ in reconstituted infant formulas was evaluated. Results showed that only the combination of citral with carvacrol demonstrated a synergistic effect among the evaluated combinations. This combination had a stronger growth inhibition effect than treatment with citral or carvacrol alone, leading to more portions of cells in the propidium iodide fluorescent region and causing more severe cell damage. Citral and carvacrol also showed potent and promising antibiofilm activity against _C. sakazakii_, which was evaluated by the crystal violet assay, field emission scanning electron microscopy, and qRT-PCR. Gentle heat treatment combined with citral and carvacrol at a concentration of 2 or 3 MIC completely eradicated _C. sakazakii_ in the reconstituted infant \begin{table}
\begin{tabular}{l l l l} \hline \hline
**Microorganism** & **Dose** & **Effect** & **Ref.** \\ \hline _Staphylococcus aureus_ (MRSA) & 100 \(\mu\)g/mL & Citral inhibited the biofilm formation of human severe pathogen MRSA without affecting the growth. & [7] \\  & 25 \(\mu\)g/mL & The percentage of biofilm inhibition by a synergistic combination of citral & \\  & 25 \(\mu\)g/mL & + thymol was 91\%, while 22\% and 28\% of biofilm inhibition were observed for individual application, respectively. & [74] \\ _S. aureus_ DMST 4745 & & _S. aureus_ DMST 4745 & _S. aureus_ DMST 4745 was more susceptible than _S. aureus_ clinical isolate to citral, showing biofilm reductions of 74.6 and 46.5\%, respectively. & [48] \\ _C. sakazakii_ & 0.8 mg/mL & 1/2 MIC for 48 h decreased biofilm formation by 59.62\%. & [55] \\  & 225 \(\mu\)M & The biofilm formation was inhibited by 67.1\%, 69.5\%, and 70.1\% at the tested concentration after treatment at 25 \({}^{\circ}\)C for 24, 48, and & [75] \\  & & 72 h, respectively. & \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Antibiofilm activity of citral against bacteria. formula. Furthermore, the combination at 2 MIC had no significant effect on the color and aroma scores of the reconstituted infant formulas [55]. In a similar study, [PERSON], [PERSON] [75] investigated the antibiofilm efficacy of citral on C. _sakazakii_ ATCC 29544. Bacteria were grown at 12 \({}^{\circ}\)C and 25 \({}^{\circ}\)C for 24, 48, and 72 h on microtiter plates.