The document reports on several studies that evaluated the antifungal activity and mycotoxin inhibition of essential oil nanoemulsions. Specifically: 1) Limonin-loaded eugenol nanoemulsion effectively inhibited citrus blue mold caused by Penicillium italicum spores and could be an alternative fungicide for citrus fruits. 2) Thymus vulgaris essential oil nanoemulsion showed promising treatment of cutaneous mycoses caused by resistant fungi and further study of its antifungal mechanisms is recommended. 3) Hop essential oil nanoemulsion inhibited the growth and spore germination of Fusarium graminearum isolates and completely prevented mycotoxin production, with its ant

2. Anti-Fungal activity and mycotoxin inhibitory
efficacy of different essential oils based
nanoemulsion under In-vitro and In-vivo
tests
Presenter: Muhammad Imran
Student ID: 8110039004
Advisor: Professor Dr. Yao-Tung Lin
December 7th, 2022

3. Introduction
Objective of the study
Materials and Methods
Results and Discussion
Conclusions
1
2
3
4
5
Outline

4. Introduction
Food waste
• High cost
• High energy input
• Short self-live
• Environmental risk
Traditional food
preservation techniques
• Drying
• Pickling
• Salting
• Canning
• Refrigerating
• Use of chemical
preservatives
Disadvantages
 1.3 billion tonnes – global food waste yearly;
• Food spoilage
• Food safety (essential oils as nanoemulsion)
Source: Google image
4

6. Fig 1. Particle size distribution (A) and microstructure (B,C) of limonin-loaded eugenol nanoemulsion. The mean particle size
was 245.7 nm. Scale bar, 10 µm
Characterization of nanoemulsion
(LI et al., 2021) 6

7. Figure 2. Optical microscopy of the spore germination after treatment with water (CK), eugenol nanoemulsion (EG), and
limonin-loaded eugenol emulsion (EGL). Scale in the graph, 20 µm.
Anti-fungal activity (Inhibition of Spore Germination)
7
(LI et al., 2021)

9. Figure 3. Colony diameters of P. italium after incubation for 3–7 d after different treatments
Anti-fungal activity (Inhibition of Mycelial growth)
9
(LI et al., 2021)

10. Figure 4. Morphology of the mycelia (A–C) and conidia (D–F) of P. italicum, as observed by scanning electron microscopy (SEM),
after treatment with water (A,D), 160 μg/mL EG nanoemulsion (B,E), and 160 μg/mL EGL nanoemulsion (C,F). Scale bar, 10 μm
for mycelia and 1 μm for spore.
Micromorphological analysis by SEM
10
(LI et al., 2021)

11. Figure 5. Influence of the nanoemulsion (at a eugenol concentration of 160 µg/mL) on the extracellular conductivity (A), lipid
peroxidation (B), leakage of nucleic acid (C), and protein (D) of P. italicum. Data are shown as mean ± SD from three replication
and different letter represent a significant difference p < 0.05).
Influence of Nanoemulsions on the Cell Membrane Permeability
- Extracellular Conductivity
11
(LI et al., 2021)

12. • Limonin-loaded eugenol nanoemulsion effectively inhibited the
occurrence of citrus blue mold infection caused by P. italicum
spores.
• Limonin loaded eugenol nanoemulsion, could be considered as
an alternative fungicide to inhibit blue mold in citrus fruits.
Conclusion

13. Figure 3. Colony diameters of P. italium after incubation for 3–7 d after different treatments
.
(Wan et al., 2021) 13
In vitro antifungal activity of Thymus
vulgaris essential oil nanoemulsion

14. Fig. 1. The Zeta potential of Thymus vulgaris Essential Oil
Characterization of TV EO-NEs
(Moazeni et al., 2021) 14

15. Fig. 2. Transmission electron micrograph (TEM) of TVL-NE.
Characterization of TV EO-NEs
15
(Moazeni et al., 2021)

16. Fig. 3. The cytotoxicity activities of different concentrations of both TV EOs and TV EO-NEs on Human Caucasian fetal foreskin
fibroblast (HFFF2) cells: (a) 24 h and (b) 48 h.
Cytotoxicity Effects
16
(Moazeni et al., 2021)

17. Conclusion
(Wan et al., 2021) 17
• The study also highlighted that TV EO-NE is a promising
treatment of cutaneous mycoses especially when the
etiological agents are resistant to conventional
antifungal drugs.
• It is highly recommended to investigate the exact
mechanism of action of the antifungal activity of TV EO-
NE regarding cellular/ molecular approaches.

18. .
(Wan et al., 2021) 18
Antifungal activity, mycotoxin
inhibitory efficacy, and mode of
action of hop essential oil
nanoemulsion against Fusarium
graminearum

19. Physical stability of hop essential oil-in-water nanoemulsions
(Jiang et al., 2022) 19
Fig. 1. (A) Impact of medium chain triglycerides (MCT) content in total oil phase (5 wt%) on mean particle diameter
of hop essential oil-in-water (HEO) nanoemulsion (0.5 wt% of tween 80 and 94.5 wt% of 10 mM phosphate buffer,
pH 7.0; average particle size of various wt% of MCT in oil phase with different lowercase letters were significantly
different from each other at p < 0.05); (B) storage stability of HEO nanoemulsion over a course of 7 days storage
time at 25 ℃ (size values under various days with different lowercase letters were significantly different from each
other at p < 0.05); and (C) particle size distribution of HEO nanoemulsion at the selected storage time.

20. Fig. 2. (A) Effect of hop essential oil concentration in nanoemulsion on mycelial growth inhibition rate of F.
graminearum 10–124-1 and F. graminearum 10–125-1 (different lowercase letters indicate statistically significant
intraspecies differences at p < 0.05); and (B) scanning electron microscope (SEM) observation of F.
graminearum hyphae treated by water (control) and HEO nanoemulsion
Anti-fungal - Mycelial growth inhibition rate (MGI)
20
(Jiang et al., 2022)

21. Fig. 3. (A) Effect of hop essential oil concentration in nanoemulsion on spore germination inhibition rate of F.
graminearum 10–124-1 and F. graminearum 10–125-1 (different lowercase letters indicate statistically significant
intraspecies differences at p < 0.05); and (B) scanning electron microscope (SEM) observation of F.
graminearum spores treated by water (control) and HEO nanoemulsion.
Spore Germination Inhibition
21
(Jiang et al., 2022)

22. Fig. 4. Inhibition of mycotoxins production in F. graminearum 10–124-1 and F. graminearum 10–125-1 inoculated
rice culture treated by HEO nanoemulsion for 5 days of incubation (water treatment was used as control; different
lowercase letters indicate statistically significant intraspecies differences at p < 0.05; n.d. denoting not detected).
The effect of hop essential oil-in-water nanoemulsions on
mycotoxin production
22
(Jiang et al., 2022)

23. Fig. 5. (A) Effect of HEO nanoemulsion on total lipid content of outer cell membrane of F. graminearum isolates.
(different lowercase letters indicate statistically significant intraspecies differences at p < 0.05); and (B) confocal
laser scanning microscopy (CLSM) images of outer cell membrane of F. graminearum isolates after incubation for
10 h in potato dextrose broth (chitin was stained by calcofluor white dye and shown in blue color). (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of this article.)
Antifungal mode of action of hop essential oil-in-water
nanoemulsions
3.5.1. Total lipid content
23
(Jiang et al., 2022)

24. Fig. 6. Confocal laser scanning microscopy (CLSM) images of cytoplasmic membrane of F. graminearum isolates
simultaneously stained by fluorescein diacetate and propidium iodide after incubation for 10 h in potato dextrose
broth.
Permeability of cytoplasmic membrane
24
(Jiang et al., 2022)

25. Conclusions
25
(Jiang et al., 2022)
• Regarding the antifungal activity, HEO nanoemulsion could
inhibit the mycelial growth and spore germination of the two
chemotypes F. graminearum isolates.
• In addition to their antifungal activity, HEO also displayed the
capability to completely hinder biosynthesis
of Fusarium mycotoxin.
• HEO-in-water nanoemulsion acts as natural antifungal and
detoxifying agents in the malting and food industry.