Antifungal Activity Of Essential Oils Against Botrytis And Penicillium Fungi

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i.What did your research discover or show?

ii.What kind of problem(s) did you work on?

What did you learn from this lab?
 

Experimental Design

Economic losses (Hocking, 1998) due to food spoilage and reduction in food quality (Baratta,1998) has forced the usage of preservatives. The negative effects of the synthetic preservatives especially sodium content has forced to fall for organic alternatives. The study focussed on evaluating the antifungal activity of two fungi, based on two types of essential oils (EOs). A laboratory experiment performed for analysing the fungi static activity of essential oils (from plants extract) on varied fungal organism.   Mycelium growth inhibition (MGI) values recorded and studied for varied fungal organism as per application of different concentration of essential oils. The synthetic fungicides used to control the growth of fungi are not safe for indoor application or for food products.  The adverse effects of these additives on the health of living organisms have encouraged the use of antimicrobial plant products.  

For the purpose of this study, essential oils extracted from varied plants have been used as Eugonel (73% pure extract from clove oil) and Menthol (50% pure active component of peppermint oil). These oils are known to have antiviral, antifungal and antibacterial properties as they slow down fungal growth by lowering mycotoxin production. The study will conduct the antifungal analysis on botrytis and penicillium fungus. 

The study of antifungal properties of essential oils is due to the food safety and longevity of fruits and packaged food items. In the situation of film-forming dispersions when treated with 3% lemon essential oil, the properties of the strawberries such as acidity, pH and soluble solid content were preserved throughout storage (Perdones, A., et al (2012)). Earlier Matricaria chamomilla L. flower essential oil was used against Aspergillus Niger. Remarkable inhibition of fungal growth was noticed. Positive effect of M. chamomilla L. essential oil in preventing fungal growth was noticed. It helped in food storage in organic way (Tolouee, Marziyeh, et al (2010)). The extracts of M. piperita oil, Mentha spicata oil , Chamomilla oil, Ocimum basilicum oil also exhibited anti fungal properties and showed prominent results (Sokovi?, Marina, et al,2010). Based on the previous knowledge the experiment was performed with two essential oils.

In this study or experiment, there are various null hypotheses were assumed. : The concentration level of clove oil has no effect on the control of Botrytis fungal growth.

Null hypothesis2:: The concentration level of peppermint oil has no effect on the control of Botrytis fungal growth.

Null hypothesis3:: The concentration level of clove oil has no effect on the control of penicillium fungal growth.

Results

 Null hypothesis4: : The concentration level of peppermint oil has no effect on the control of penicillium fungal growth.

The study was performed to determine the effect of essential oils on fungi growth. Research for new antimicrobial agents from varied sources for combating microbial resistance (Balouiri, Sadiki and Ibnsouda (2016)) is already in demand. Methods for in vitro for analysing antimicrobial activity are a matter of interest. Hua, Xing, Selvaraj, Wang, Zhao, Zhou, Liu and Liu (2014) article reviews role of essential oils as a form of bio control for reducing fungal contamination of Ochratoxin A (OTA) (Hua,2014). Tian, Ban, Zeng, He, Chen and Wang (2012) article analyses antifungal action from essential oil on Aspergillus flavus (Tian, 2011). There are various previous studies performed in order to establish a positive correlation of essential oils with inhibiting growth of fungi.

In the experiment relative inhibition of treated colony as % of control (also called mycelial growth inhibition or MGI = ([dc-dt]/dc) * 100.

Where dc = mean colony diameter for controls and dt = mean colony diameter for each treatment. 

For the experiment purpose essential oil used is Clove oil and fungal organism used is Botrytis sp. The following lab manual had been used for the purpose of materials and methods related to the study (Hyldgaard, 2012). No changes were made to the scope of following journal for the purpose of the study. Ciolfi, J. and Carpenter-Cleland, C. (2017). Lab 4 Effect of Essential Oils on Fungal Growth. In BIOL 2P96 D3 2017FW Biology of Fungi Lab Manual. St. Catharines; Brock University. 

The sampling mean for the sample was 79.64 where the standard error was 8.1332. Value of the F-statistic = 5387.63 along with P-value for the ANOVA was less than 0.0001. Mean MGI(s) for control plate was 0, 98.2 for 125 UL/L, 100 for 250 UL/L, 100 for 375 UL/L and 100 for 500 UL/L concentration of the oil. Standard errors for control plate were 0, 1.3565 for 125 UL/L, 0 for 250 UL/L, 0 for 375 UL/L and 0 for 500 UL/L concentration of the oil.

The control plate was the outlier and the data was skewed because of which large F value was observed. Tukey HSD values were 2.57 for 5% level of significance and 3.22 for 1% level of significance. Pair wise comparison between the mean MGI were insignificant for plates 2-5 whereas when compared with control plate the null hypothesis was rejected due to very small (less than 0.01) p-values.

Conclusion

The sampling mean for the sample was 75.88 where the standard error was 8.2864. Value of the F-statistic = 47.02 along with P-value for the ANOVA was less than 0.0001. Mean MGI(s) for control plate was 0, 81.2 for 125 UL/L, 98.2 for 250 UL/L, 100 for 375 UL/L and 100 for 500 UL/L concentration of the oil. Standard errors for control plate were 0, 13.9549 for 125 UL/L, 1.8 for 250 UL/L, 0 for 375 UL/L and 0 for 500 UL/L concentration of the oil.

The control plate was the outlier and the data was skewed. Tukey HSD values were 26.7 for 5% level of significance and 33.4 for 1% level of significance. Pair wise comparison between the mean MGI were insignificant for plates 2-5 whereas when compared with control plate the null hypothesis was rejected due to very small (less than 0.01) p-values.

The sampling mean for the sample was 75 where the standard error was 8.4459. Value of the F-statistic = 35.38 along with P-value for the ANOVA was less than 0.0001. Mean MGI(s) for control plate was 0, 75 for 125 UL/L, 100 for 250 UL/L, 100 for 375 UL/L and 100 for 500 UL/L concentration of the oil. Standard errors for control plate were 0, 15.2788 for 125 UL/L, 0 for 250 UL/L, 0 for 375 UL/L and 0 for 500 UL/L concentration of the oil.

Tukey HSD values were 30.89 for 5% level of significance and 38.65 for 1% level of significance. Pair wise comparison between the mean MGI were insignificant for plates 2-5 whereas when compared with control plate the null hypothesis was rejected due to very small (less than 0.01) p-values. 

The sampling mean for the sample was 59.96 where the standard error was 7.6791. Value of the F-statistic = 23.49 along with P-value for the ANOVA was less than 0.0001. Mean MGI(s) for control plate was 0, 48.2 for 125 UL/L, 70 for 250 UL/L, 83.8 for 375 UL/L and 97.8 for 500 UL/L concentration of the oil. Standard errors for control plate were 0, 13.7419 for 125 UL/L, 7.9937 for 250 UL/L, 2.2 for 375 UL/L and 0 for 500 UL/L concentration of the oil.

 Tukey HSD values were 33.44 for 5% level of significance and 41.83 for 1% level of significance. Pair wise comparison between the mean MGI of the control plate with other 4 concentrations ensured that the null hypothesis was rejected due to very small (less than 0.01) p-values. Pair wise comparison of mean MGI score for 125 uL/L with 375 uL/L and 500uL/L also gave p values less than 0.05 and 0.01 which showed that null hypothesis 4 was rejected.

References

Botrytis Sp. and Penicillium Notatum are two kinds of fungi which are very important in food and drug production.

The purpose of the laboratory experiment was to study the fungi static activity of two essential oils namely clove oil (Eugonel) and peppermint oil (Menthol) against two fungi Botrytis sp. and Penicillium Notatum (Sánchez-González, 2010).

Null hypothesis1:: The concentration level of clove oil has no effect on the control of Botrytis fungal growth.

Across the four different concentration levels and one control plate the overall average MGI was 79.64 which indicated that on an average inhibition along the samples were high. The concentration of 250uL/L, 375 uL/L and 500 uL/L completely (100%) inhibited the Botrytis fungal growth. Standard error of 8.1332 implies that for 5% level of significance the confidence interval for sampling mean is [71.5068, 87.7732].The F value of the ANOVA clearly shows that calculated F value of 5387.63 which is extremely large and also p has value less than 0.0001, which implies that the null hypothesis is rejected and it can be concluded that clove oil has significant antifungal effect on Botrytis fungus.

Null hypothesis2:: The concentration level of peppermint oil has no effect on the control of Botrytis fungal growth.

Sampling average MGI was 75.88 and the concentrations of 375 uL/L and 500 uL/L completely (100%) inhibited the Botrytis fungal growth whereas 125 uL/L and 250 uL/L consistencies showed great positive result. Standard error of 8.2864 implies that for 5% level of significance the confidence interval for sampling mean is [67.5936, 84.1664].The calculated F value of 47.02 is sufficiently large and also p has value less than 0.0001, which implies that the null hypothesis is rejected and it can be concluded that peppermint oil has significantly positive antifungal effect on Botrytis fungus.

Null hypothesis3:: The concentration level of clove oil has no effect on the control of penicillium fungal growth.

Mean sampling MGI was 75 and the concentrations of 250 uL/L, 375 uL/L and 500 uL/L completely (100%) inhibited the Penicillium fungal growth. Standard error of 8.4459 implies that for 5% level of significance the accepting region for sampling mean is [66.5541, 83.4459].The ANOVA clearly shows that calculated F value of 35.38, which is sufficiently large and also p has value less than 0.0001, hence the null hypothesis is rejected and it can be concluded that clove oil has significantly positive antifungal effect on Penicillium fungus.

Null hypothesis4:: The concentration level of peppermint oil has no effect on the control of penicillium fungal growth.

Average MGI was 59.96 which indicate the fact that inhibition across the different samples was satisfactory. In this case the mean MGI was the lowest. Here the result was very interesting as increment in oil consistency of menthol extract from peppermint oil showed gradual improvement in control of Penicillium fungus (Soylu, 2010). Standard error of 7.6791implies that for 5% level of significance the accepting region for sampling mean is [52.2809, 67.6391].The ANOVA clearly shows that calculated F value of 23.49 which falls in the critical region and also p has value less than 0.0001 which is less than 0.05, implies that the null hypothesis is rejected and it can be concluded that peppermint oil has significantly positive antifungal effect on Penicillium fungus.

From table 6 it was evident from mean MGI values of the sampling distribution that clove oil with mean MGI scores 79.64 when inhibiting Botrytis fungus and 75.88 when inhibiting Penicillium fungus was much more effective in controlling fungal growth than peppermint oil with mean MGI scores 75 when inhibiting Botrytis fungus and 59.66 when inhibiting Penicillium fungus under the current scope of experiment.

In addition, it is possible to conclude from mean MGI values that growth of Botrytis fungus can be controlled more effectively than Penicillium fungus under the current scope of experiment.

It was earlier observed that by using antimicrobial reagents on edible films microbiological stability can be obtained. It also extended age of the food products and reduced the probability of fungal growth on food outer surface as obtained p value was less than 0.05 in analysis of variance (Avila-Sosa,2012). The outcomes of this experiment also resembled the very same fact.

Considering the limited number of sample data and restricted number of fungus variety it can be conclude that experiment outputs were still very productive in nature. It is also a matter of fact that instead of 125 uL/L increases in magnitude of essential oil concentration gradual increase may increase the probability of obtaining highly accurate and decisive statistical data. Gradual increase in oil concentration will be highly recommendable, as it will create a less skewed mean MGI graph for interpretation. Bigger diameter PDA plates can also be considered given that the samples were studied for 7 days.  The pooled data can also be collected after every 2 days of incubation to distinguish the antifungal effect of both oils used under varying concentration. Otherwise at higher concentration level the data becomes indistinguishable due to 100% per cent inhibition of the fungus.  

Reference Lists (Literature cited)

Avila-Sosa, Raúl, et al. “Antifungal activity by vapor contact of essential oils added to amaranth, chitosan, or starch edible films.” International journal of food microbiology 153.1-2 (2012): 66-72.

Balouiri, Mounyr, Moulay Sadiki, and Saad Koraichi Ibnsouda. “Methods for in vitro evaluating antimicrobial activity: A review.” Journal of Pharmaceutical Analysis 6.2 (2016): 71-79.

Baratta, M. Tiziana,Dorman, H. J. Damien,Deans, Stanley G.,Figueiredo, A. Cristina,Barroso, José G.,Ruberto, Giuseppe. “Antimicrobial and antioxidant properties of some commercial essential oils.” Flavour and Fragrance (1998).

Hocking, A.D. Hocking, A.D. Food preservation by moisture control. London: Elselvier Applied Science, 1998. 57-72.

Hua, Huijuan, et al. “Inhibitory effect of essential oils on Aspergillus ochraceus growth and ochratoxin A production.” PloS one 9.9 (2014): e108285.

Hyldgaard, Morten, Tina Mygind, and Rikke Louise Meyer. “Essential oils in food preservation: mode of action, synergies, and interactions with food matrix components.” Frontiers in microbiology 3 (2012): 12.

Perdones, A., et al. “Effect of chitosan–lemon essential oil coatings on storage-keeping quality of strawberry.” Postharvest Biology and Technology 70 (2012): 32-41.

Sánchez-González, Laura, et al. “Physical and antimicrobial properties of chitosan–tea tree essential oil composite films.” Journal of Food Engineering 98.4 (2010): 443-452.

Sokovi?, Marina, et al. “Antibacterial effects of the essential oils of commonly consumed medicinal herbs using an in vitro model.” Molecules 15.11 (2010): 7532-7546.

Soylu, Emine Mine, ?ener Kurt, and Soner Soylu. “In vitro and in vivo antifungal activities of the essential oils of various plants against tomato grey mould disease agent Botrytis cinerea.” International Journal of Food Microbiology 143.3 (2010): 183-189.

Tian, Jun, et al. “Chemical composition and antifungal activity of essential oil from Cicuta virosa L. var. latisecta Celak.” International Journal of Food Microbiology 145.2-3 (2011): 464-470.

Tolouee, Marziyeh, et al. “Effect of Matricaria chamomilla L. flower essential oil on the growth and ultrastructure of Aspergillus niger van Tieghem.” International journal of food microbiology 139.3 (2010): 127-133.