## 3 Results
### TEM Analysis  
The untreated negative control cells (Figure 7a,b) showed characteristics similar to the cells exposed to 6.25% EEP at 3 and 6 months of fermentation respectively (Figure 8a-d). The cells exhibited an intact cell membrane and a smoothly outlined nucleus with an intact nucleus membrane, containing nucleoli (Nu) and heterochromatin (HChr) deposited along the nuclear membrane. The cytoplasm showed intact organelles, including mitochondria (indicated by a white arrow) with preserved cristae, endoplasmic reticulum (indicated by a yellow arrow) and minimal cytoplasmic vacuolisation (V).  
Figure 6: Live and Dead Cell assay of MC3T3-E1 cells exposed to EEP at 3.13%, 1.57% and 0.78%, observed under fluorescence microscopy (\(\times 100\)): (**a**) 3 M 3.13% EEP; (**b**) 6 M 3.13% EEP; (**c**) 3 M 1.57% EEP; (**d**) 6 M 1.57% EEP; (**e**) 3 M 0.78% EEP; (**f**) 6 M 0.78% EEP. 3 M: 3 months of fermentation; 6 M: 6 months of fermentation. Green: viable cells.  
Figure 8: _Cont._  
Figure 7: Representative images via transmission electron microscopy of the effect of control groups on MC3T3-E1 cells. (**a**) Untreated negative control (\(\times\)2200); (**b**) enlargement of image (**a**) (\(\times\)3000). CM: cell membrane; N: nucleus; Nu: nucleoli; NM: nuclear membrane; HChr: heterochromatinatin; LC: low cytoplasmic density; white arrow: mitochondria; yellow arrow: endoplasmic reticulum; red arrow: cell fragments. Scale bar of (**a**): 2 \(\upmu\)m; scale bar of (**b**): 1 \(\upmu\)m.  
On the other hand, cells exposed to EEP 12.5% at both 3 and 6 months of fermentation showed evidence of cell death, as depicted in Figure 9a-d. The cells demonstrated cell surface changes with plasma membrane blebs and the formation of numerous apoptotic bodies (ApBs) that were seen detaching from the plasma membrane (Figure 9a,c). At the nuclear level, the nucleus lost its regular shape and membrane integrity. The chromatin (Chr) became densely condensed with the disappearance of the nucleolus. Additionally, there were numerous nucleus aggregates (yellow arrows) and cytoplasmic aggregates (white arrows), as observed in Figure 9a-d. These aggregates were small, round or oval-shaped fragments evident in both the nucleus and cytoplasm.  
Furthermore, noticeable morphological alterations were observed in the cytoplasm and organelles. The cytoplasm exhibited an increased number of fragmented organelles (red arrow) and autophagic vacuoles (V) containing cell debris. In addition, the mitochondria (M) lost their regular shape, and the internal cristae were collapsed and not discernible.  
Figure 8: Representative images via transmission electron microscopy of the effect of 3 M 6.25% EEP and 6 M 6.25% EEP on MC3T3-E1 cells: (**a**) 3 M 6.25% EEP (\(\times\)2400); (**b**) an enlargement of image (**a**) (\(\times\)4000); (**c**) 6M 6.25% EEP (\(\times\)2000); (**d**) an enlargement of image (**c**) (\(\times\)3200). CM: cell membrane; N: nucleus; Nu: nucleoli; NM: nuclear membrane; HChr: heterochromatin; V: vacuoilsation; white arrow: mitochondria; yellow arrow: endoplasmic reticulum. Scale bar of (**a,c**): 2 \(\upmu\)m; scale bar of (**b,d**): 1 \(\upmu\)m.