## 3 Biological Activities
### Antifungal Activity
\begin{table}
\begin{tabular}{p{142.3 pt} p{142.3 pt}} \hline \hline
**Microorganism** & **Mechanism** & **Ref.** \\ \hline _Saccharomyces cerevisiae_ BY4741 & Loss of membrane and cell wall integrity results in a typical apoptotic/necrotic cell death. However, yeast cells that escape this first cell membrane disruption, particularly evident in sub-lethal concentration, die by metacaspase-mediated apoptosis induced by the accumulation of intracellular ROS. Changes in the morphological characteristics of fungal hyphae, resulting in loss of cell content and distortion of the mycelium. Increase in membrane permeability, with increases in extracellular electrical conductivity and a decrease in soluble protein content. A decrease in the range of ergosterol levels showed that citral altered the physiology of the cell membrane. Reduction in the level of enzymes associated with respiration, resulting in the disruption of energy metabolism. \\ _Aspegillus ochraceus_ & Citral downregulated ochratoxin biosynthetic genes, including _pks_ and _nrps_, but slightly upregulated global regulatory factors _vEA_, _vdB_, and _laeA_. \\ \multirow{4}{*}{_Aspegillus niger_} & Direct damage to the cell membranes of _A. niger_ may explain the antimicrobial activity of citral combined with eugenol. Among the two components, eugenol is mainly responsible for the permeability of damaged cell membranes, whereas citral mainly causes membrane lipid peroxidation, which leads to a burst in ROS. \\ \multirow{4}{*}{_Penicillium roqueforti._} & The combination of citral and eugenol destroyed the integrity of the cell membrane and internal structures and degraded the cell content. The combination induced membrane lipid peroxidation and promoted the ability to destroy the cell membrane. The combined agents eventually caused leakage of cell contents and, ultimately, cell death. \\ \multirow{4}{*}{_Penicillium digitatum_} & Citral can affect the mitochondrial morphology and function of _P. digitatum_, inhibiting the respiratory metabolism, decreasing the activities of TCA-related enzymes, and changing the TCA metabolic abilities. \\ \multirow{4}{*}{_Zigosachanomyces rouxii_} & The antifungal effect can be attributed to the alteration of the integrity and permeability of the cell membrane, which can cause irreversible damage to the cell wall and membrane. They can also destroy yeast proteins and inhibit their synthesis. \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Antifungal mechanisms of action proposed for citral. with different concentrations of citral. The results showed a potent inhibition on the growth of the hyphae of the three fungi studied with an MIC of 0.4 \(\mu\)L/mL. Citral changed the morphological characteristics of the fungal hyphae of _B. dothidea_, resulting in loss of cell content and distortion of the mycelium as observed in the scanning electron microscopy (SEM) and electrical conductivity studies, respectively. Citral increased membrane permeability, increased extracellular electrical conductivity, and decreased soluble protein content in _B. dothidea_. A reduction in the range of ergosterol levels demonstrated that citral altered the physiology of the cell membrane. Furthermore, higher concentrations of citral decrease the level of enzymes associated with respiration, resulting in the disruption of energy metabolism (Figure 3). Importantly, citral also showed positive effects on fruit quality and reduced the decay rate of kiwis. Postharvest treatment with citral also maintained fruit quality and reduced spoilage [82]. In another study, the antifungal activity of 20 monoterenes (including citral) was evaluated against the model yeast _Sacchiuromyces cerevisiae_, finding that oxygenated monoterenes exhibited greater fungistatic and fungicidal activities than hydrocarbons and among the most effective oxygenated monoterenes was citral with an MIC and minimum fungicidal concentration (MFC) of 0.64 mM.