## 3 Biological Activities
### Antibacterial Activity
Other authors have mentioned that unsaturated alpha and beta aldehydes have a broad antimicrobial spectrum and show similar activity against Gram-positive and Gram-negative microorganisms [62]. Several authors have reported some of these mechanisms of action against different pathogenic bacteria (Table 3). Geraniol, a monoterepenic alcohol, can affect cytoplasmic membranes and cause intracellular leakage due to the solvation of phospholipids from the membrane. On the other hand, geraniol may cause a loss of structural organization of cytoplasmic membranes for a short period before protein denaturation of the cytoplasm occurs [48]. EOs are being explored as a natural disinfection alternative, and their effectiveness depends mainly on the diverse constituents they may present [63; 64]. The EO of the _C. citratus_ plant, rich in terpenes such as citral (85%) and geraniol (1.5%), was effective in inhibiting the growth of _E. coli_, _Salmonella_ spp., _L. monocytogenes_, and _S. aureus_[64; 65; 66]. However, several studies evaluated the antimicrobial activity on planktonic cells, that is, against dispersed individual bacterial cells, which differs from the form of bacterial aggregates (biofilms), the true survival mechanism [67]. As observed, citral possesses a remarkable antibacterial activity against different pathogenic bacteria, and in this sense, more studies evaluating their combination with antibiotics are needed. This might provide a more effective treatment for infections caused by antibiotic-resistant strains and address the growing interest in natural and alternative therapies for bacterial infections. Antibiofilm Activity of Citral against Bacteria  
The persistence and resistance of some microorganisms to disinfection processes are associated with their ability to form biofilms on surfaces. A biofilm is a sessile bacterial community of cells attached to each other and surfaces [68]. Biofilm adhesion and formation on surfaces are cross-contamination and public health problems [69]. Biofilms can also form on food industry surfaces such as pipes, pumps, and containers used to harvest, transport, and display food [70; 71; 72]. It has been reported that different bacteria can form biofilms on stainless steel, where insufficient control of surface growth can lead to the detachment of cells responsible for the dispersion of contamination [73]. The foregoing highlights the importance of studying bacterial aggregates' characteristics to ensure the disinfection processes' efficiency and consequent assurance of food safety. In this way, citral has shown the capacity to affect biofilm formation (Table 4). \begin{table}
\begin{tabular}{l l l} \hline \hline
**Microorganism** & **Mechanism** & **Ref. ** \\ \hline _C. sakazkii_ & Citral affected the cell membrane of _C. sakazakii_, as demonstrated by the & [60] \\ _C. sakazakii_ & decrease in intracellular ATP concentration, the reduction in pH, and the & [60] \\ _Salmonella enterica_ serovar Typhimurium & _enteritidis_ cell membranes, increasing the proportion of saturated & [53] \\ _E. coli_ MG1655 & & \\ _E. The resistance of _E. coli_ O157:H7 to disinfection is associated with its ability to form biofilms, consisting mainly of glucans produced by glucosyltransferases [6]. Citral has shown antibacterial activity against planktonic _E. coli_, showing that citral decreases the production of glucans. However, there is insufficient information on its efficacy and mode of action against _E. coli_ biofilms. Furthermore, citral acts as a non-competitive inhibitor of the activity of the enzyme glucosyltransferase. Evidence gathered by coupling analysis indicated that citral could interact with the helix finger of the glucosyltransferase responsible for polymer production [6]. Cellulose synthesis in _E. coli_ O157:H7 is regulated by the _bcs_ (bacterial cellulose synthesis) genes. The four _bcs_ genes are called _bcsA_, _bcsB_, _bcsZ_, and _bcsC_ and are organized like an operon.