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Comparing the antifungal activity of edible essential oils against Saccharomyces cerevisiae
Comparing the antifungal activity of edible essential oils against Saccharomyces cerevisiae
Hooria Malik
Blacktown Girls High School
Abstract
Three edible essential oils - turmeric, thyme and cinnamon oil - were tested for their antifungal activity against the common food spoilage agent Saccharomyces Cerevisiae using the disc diffusion method to to assess for suitability as a natural preservative to prevent the spoilage of food. The zones of inhibition around discs soaked in each of these oils were compared to a control disc to determine the extent of inhibitory activity. Zones of inhibition were measured in millimetres, and a one-way ANOVA and post-hoc contrasts were used to interpret the results obtained. All of the tested oils revealed a statistically significant difference in zones of inhibition when compared to the control disc (M = 5.0000), with cinnamon oil showing the highest mean zone of inhibition (M = 6.9375), followed by turmeric oil (M = 6.4375) and then thyme oil (M= 6.1250). However, there was no statistically significant difference observed between the antifungal activity of the oils, suggesting that no oil was more effective than the other. However, the study had a wide range of associated assumptions and limitations, such as a small sample size (N = 16), which may have affected the viability of results. Hence, additional research should be implemented to thoroughly investigate the oils’ relative effectiveness against Saccharomyces Cerevisiae. Even further research should be conducted on other aspects of food preservatives, such as the effect of oils on the taste and appearance of food, to determine their suitability as natural, edible preservatives.
Keywords: Saccharomyces Cerevisiae, turmeric oil, cinnamon oil, thyme oil, zone of inhibition, antifungal properties, disc diffusion.
Literature review
As the world population continues to rise, food demand is expected to increase by 5070% in 2050; yet already, around 2.37 billion people around the world struggle to access adequate food (Food and Agriculture Organisation of the United Nations, 2021). While there is sufficient food produced to satisfy current demands, one third of this food is estimated to be wasted (Bond et.al., 2013). In a world where food insecurity impacts millions of people, the continuous wasting of food can catalyse further destructive consequences. According to the Food and Agriculture Organisation (FAO), food waste contributes up to 10% of global greenhouse gases, influencing catastrophic climate events which can detrimentally impact the growth and nutritional quality of crops, as well as food distribution. This could lead to economic disadvantage for growers and further food shortages, potentially forcing many more people to endure malnutrition and hunger (FAO, 2022).
Food spoilage is a major contributor to this wastage. When food is not promptly sold, not stored appropriately or mishandled, it becomes difficult for retailers to sell; consequently, such produce is rejected and thus wasted (Raak et.al., 2017). Food waste also occurs due to post-purchase deterioration. In the UK, where household wastage is the largest contributor to national food waste, two thirds of household food waste was due to spoilage (Quested et.al., 2011). Similar results were obtained in a Polish study, in which the majority of interviewed respondents agreed that spoilage was the main reason for their household food waste. (Bilska et.al., 2019). Therefore, as food spoilage is a major contributor to global food wastage, there is a significant need for the employment of suitable strategies to prevent it.
Microbial spoilage is a common and dangerous type of spoilage; not only does it significantly contribute to food waste, consumption of contaminated food can also make consumers very sick (Alpers et.al., 2021; World Health Organisation, 2022). Therefore, manufacturers are unable to sell contaminated produce, which may cause significant economic disadvantage (Smits & Brul, 2005). A large majority of fruit juice manufacturers agreed in a previous survey that microbial spoilage was a threat to the profits and reputation of their brands. (Snyder & Worobo, 2018). Therefore, microbial contamination of food is a significant issue for both consumers and manufacturers.
There are a myriad of microorganisms responsible for food spoilage, including bacteria and fungi like yeasts and moulds (Blackburn, 2006). However, fungi are especially prevalent, as they are able to survive a myriad of environmental conditions (Sevindik & Uysal, 2021), unlike bacteria, which are typically limited by low moisture and pH (Saranraj & Geetha, 2011). Saccharomyces Cerevisiae, commonly called bread yeast is one of many food spoilage agents, affecting foods with a high sugar content such as bread, canned fruits and vegetables, fruit juices, soft drinks and dairy (Viljoen & Heard, 1999; Smits & Brul, 2005).
Food manufacturers may prevent the spoilage of their products by adding artificial preservatives, however, the use of chemical and microbial preservatives is becoming more challenging as some of them are known to cause possible adverse health effects, and modern consumers are demanding alternatives from natural sources (Al-Maqtari et.al., 2021). Many yeasts are also becoming resistant to certain preservatives used to prevent spoilage of juices. (Aneja et.al., 2014). Therefore, it becomes necessary to find suitable natural preservatives to mitigate fungal growth on food.
While Saccharomyces Cerevisiae is responsible for the spoilage of many foods and beverages, including fruit juice, canned fruit, dairy, soft-drinks and bakery products (Viljoen & Heard, 1999) there is, apparently, limited research towards finding antifungal agents in the form of plant essential oils (Konuk & Ergüden, 2017).
The fungus has been dubbed the “most useful yeast” (Stewart, 2014) due to its contributions to research and industry. It is commonly used to manufacture goods such as bread and alcoholic beverages (Stewart, 2014; Viljoen & Heard, 1999), and even in microbiology to study infectious agents like prions and viruses (Wickner et.al., 2011).
Thus, despite its major role in food spoilage, there is a lot more research towards exploiting those favourable properties of Saccharomyces Cerevisiae for societal benefits rather than towards mitigating its growth on food products (Viljoen & Heard, 1999). This study therefore endeavoured to find essential oils with desirable attributes and compare their effectiveness towards inhibiting the growth of Saccharomyces Cerevisiae, in order to find the most suitable antifungal agent. This research may assist manufacturers in finding suitable alternatives to artificial preservatives which is especially important in satisfying the growing consumer demand for natural preservatives. Therefore, this research may also contribute towards the reduction of overall food waste, and assist in mitigating the detrimental impacts of food waste on the environment.
Essential Oils with proven antifungal properties
Thyme oil is used to add flavouring and spice to food products. The oil is a safe and natural food preservative also known to possess significant antifungal properties (Mandal & DebMandal, 2016). It has been used to treat mould on ceilings (Šegvić Klarić et al., 2007) and to mitigate the growth of a fungus causing decay in mango (Esquivel-Chávez et al., 2021). In one study conducted by KunickaStyczyńska (2011) thyme oil was the one of the most effective oils in mitigating the growth of nine yeasts (including Saccharomyces Cerevisiae) out of eight tested essential oils. Another study, conducted by Lis-Balchin et.al. (1998) inoculated quiche filling with Saccharomyces Ludwigii (another food spoilage agent) and then treated it with thyme oil. Again, out of eight oils, thyme oil showed the highest antifungal activity. Therefore, thyme oil shows promising antifungal qualities.
Furthermore, turmeric plant is widely used in Indian cooking and traditional medicine (Das, 2016). It has also been proved to possess antifungal properties. Curcumin is a component of turmeric that has shown activity against Saccharomyces Cerevisiae One study, conducted by Minear et.al. (2011) tested the effect of curcumin against wildtype Saccharomyces Cerevisiae, and revealed that exposing the yeast to curcumin slowed down its cell cycle and lowered its growth rate. However, all yeast samples were still completely viable; thus curcumin did not kill Saccharomyces Cerevisiae, rather only delaying its growth. Another study, conducted by Stępień et.al. (2019) similarly tested the impact of curcumin on the ageing rate of Saccharomyces Cerevisiae, also showing that the addition of curcumin slowed the growth of wild-type Saccharomyces Cerevisiae. However, the supplementation of curcumin to some mutant variants of the yeast had an insignificant effect on their growth rate. While turmeric oil showed inhibitory activity against 15 dermatophytes (Apisariyakul et al., 1995), it was proved ineffective against inhibiting the growth of Saccharomyces Cerevisiae in a study conducted by Kamble & Patil (2008). Regardless, due to its ability to slow its microbial growth, turmeric could still be a beneficial preservative.
Additionally, cinnamon oil has been proven to possess significant antifungal properties. One study, conducted by Gairhe et.al. (2021), tested the effect of various essential oils on Colletotrichum sp., a fungus responsible for post-harvest banana spoilage, whereas another study, conducted by Burgute et.al. (2019) tested the effectiveness of essential oils against Colletotrichum Gloeosporioides, (a fungus responsible for decay in pomegranates). In both these studies, cinnamon oil showed the highest antifungal activity as compared to the other oils, eliminating 98.15% of Colletotrichum sp and 50.58% of Colletotrichum Gloeosporioides However, both studies focus on a specific genus of fruit-spoilage fungi; as Saccharomyces Cerevisiae does not typically cause the spoilage of fresh fruit (Viljoen & Heard, 1999). However, Niu et.al. (2022) used the broth microdilution method, showing a directly proportional relationship between the concentration of cinnamon oil and the inhibition of Saccharomyces Cerevisiae, suggesting that it is also effective against bread yeast. However, as cinnamon oil was the only plant-based extract investigated, the study does not provide any information on the oil's efficacy in comparison to other plant-based extracts. Yet, one study, undertaken by Çoşkun et.al. (2016), used the disc diffusion method comparing cinnamon oil to eight other plant-based oils, in which cinnamon oil proved to be the most effective against many foodrelated yeasts, including Saccharomyces Cerevisiae. Cinnamon oil also showed the greatest antifungal activity against Saccharomyces Cerevisiae in Dinkova-Kostova et.al. (2020), out of 3 other essential oils. Hence, cinnamon oil could be a highly beneficial inhibitory agent towards preventing the growth of Saccharomyces Cerevisae
Current use of Essential Oils as food Preservatives
The current commercial use of natural substances as food preservatives or coatings, however, is restricted mostly due to their impact on the sensory attributes of the actual product, including appearance and taste (Raybaudi-Massilia et.al., 2009). However, finding a way to utilise natural products as an alternative to artificial preservatives may help manufacturers cope with the demand for nature-based preservatives, allowing them to, theoretically, sell more of their foods, and produce less food waste (Novais et.al., 2022). Furthermore, emerging nano-emulsion technologies can make it possible to incorporate such substances into food packaging, without affecting the appearance or taste of the product inside (Espitia et.al., 2018).
All of the aforementioned essential oils have been widely reported to show antifungal activity, however there is minimal literature that compares their antifungal properties, creating the need for a comparative study to determine, between those oils, the most suitable antifungal agent. Therefore, this study aimed to compare the antifungal activity of those oils against Saccharomyces Cerevisiae in order to identify the substance that best inhibits its growth.
The results from this study may be used to assess between the edible oils for suitability as a natural preservative to prevent the spoilage of food. This may be beneficial to manufacturers, as there is a lot more pressure on them from consumers to use natural, environmentally-friendly preservatives as an alternative to synthetic ones. Finding a suitable plant-based antifungal agent against the common food spoilage agent Saccharomyces Cerevisiae can help those manufacturers sell more food and give consumers more time to use those products, assisting in the reduction of overall food waste and thus limiting the negative environmental impacts of food waste.
Scientific research question
Research Question: Which edible essential oil shows the greatest antifungal activity against Saccharomyces Cerevisiae?
Methodology
The disc diffusion method was used, similarly to (Gairhe et.al, 2021), (Kumar Tyagi et al., 2014) and (Burgute et.al, 2019) to compare the efficacy of each medicinal plant extract/oil. The preparation of agar plates and yeast solution was adapted from this method published on the North Carolina State University website (Stroud, 2010).
Procedure
The potato dextrose agar (PDA) plates were prepared by boiling together yeast extract with anhydrous dextrose, agar and one litre of water. 20 ml of PDA medium was poured into each sterilised plate, where the lids were opened and closed as necessary, and allowed to cool at room temperature. Once solidified, the plates were sealed with a rubber band, and then placed upside-down in a plastic sleeve and refrigerated until needed.
The workbench was sanitised with 70% alcohol and paper towel before sterile agar plates were obtained. Three agar plates were sealed with rubber bands immediately, without opening the plates, and labelled “control” with a marker. Each unlabelled agar plate was divided into four sections with a marker and ruler; each section was then labelled. 5 millilitres of each oil was poured into different sterilised measuring cylinders, and then the oils in the cylinders were transferred into separate sterilised petri dishes. Filter paper discs (5 millimetres in size) were placed into each petri dish and allowed to soak.
The yeast solution was prepared in a large beaker by dissolving the dry yeast in 50mL of warm (37 to 43 degrees Celsius) water for each gram of dry yeast. The dissolution process was hastened by using a stirring rod.
A sterile syringe was used to transfer 1mL of the yeast solution onto the agar plates, which were opened at a 45 degree angle as needed. Sterile inoculating loops were used to gently rub the surface of the agar. While using the loops, plates were rotated 90 degrees clockwise until a full rotation was completed to allow uniform distribution of the yeast solution. Sterile tweezers were used to lift one piece of filter paper soaked in cinnamon oil, allowing any residue to drip back into the petri dish. Then the soaked filter paper disc was placed into the middle of the “cin” section of an agar plate. The remaining oil-soaked discs were then similarly placed into the middle of their respective sections on the agar plate. Unimpregnated filter paper discs were placed in the middle of the “con” section of each agar plate. Plates were sealed with rubber bands and inverted so the side with the gel was on top, and within 15 minutes of this process, the inoculated agar plates were stored in the incubator. All plates, including the "control" plates, were eventually stored in the incubator at 30 degrees for 24 hours (or a safe location at room temperature for 48 hours).
This process was repeated for 5 plates at a time, to ensure that the discs were timely placed on the agar, and that dishes were in the incubator within 15 minutes of the discs being placed. Plates were removed from the incubator after 48 hours and placed on the workbench. The zone of inhibition was measured using a ruler marked in millimetres. Results were recorded in an appropriate table.
Ethical Considerations
The use of living yeast microbes could cause illness or contamination, as it is possible for them to enter the body and cause infection. To mitigate this risk, plates will not be opened unless as indicated in the method, and will only be opened to a 45 degree angle. Containers will remain closed and sealed with tape after filter paper discs are added. Plates will be autoclaved and disposed of in biohazard bags and given to biological waste disposal facilities.
While essential oils may be generally safe to consume, they may be dangerous in their undiluted form. As concentrated oils are used in this experiment, there is a chance of spills, which may cause irritation to the skin. Therefore, syringes will be used and protective clothing such as leather shoes, gloves and safety goggles will be worn. Remaining oil will be diluted and given to the appropriate waste disposal facility.
All secondary resources used for the literature review or other parts of the project will be credited appropriately in the bibliography. The data collected from those sources will not be manipulated.
Results
Statistical analysis was performed using the Excel software. A total of 16 data points were collected for each treatment as the zones of inhibition around each disc were recorded up to the nearest millimetre.
These zones of inhibition were averaged as seen in Table 1, and these averages were compared in Table 1, with cinnamon oil showing the greatest mean zone of inhibition (M = 6.9375), followed by turmeric oil (M = 6.4375) and then thyme oil (M = 6.1250) (also see Figure 1). These means are all greater than the control value (M = 5.0000) as seen in both Table 1 and Figure 1. It should also be noted that, as seen in Table 1, zones of inhibition around cinnamon oil discs had the greatest standard deviation (SD = 1.6919), whereas the standard deviation of the zones of inhibition around discs treated with other oils was considerably lower (SD = 1.4083 for thyme oil, SD = 1.0935 for turmeric oil).
A one-way ANOVA was performed to compare the effect of essential oils on the size of these inhibition zones. The one-way ANOVA revealed that there was a statistically significant difference in the zones of inhibition of at least two groups, as seen in Table 2 (F(3, 60)= 7.144, p < 0.05).
[The Bonferroni procedure was used to control the experiment-wise error rate at 0.05, with the critical F value being 6.63. Contrasts were performed to identify which of the results were statistically significant (see Table 3)]
As seen in Table 1 and Table 3, there was a significant difference in growth when the oil was present (M= 6.5) than no oil (M = 5) (F(1, 60)= 17.8759, p = 0.00008).
As seen in Table 3, there was no statistically significant difference between the zones of inhibition observed for turmeric oil (M = 6.4375) and thyme oil (M= 6.1250) (F(1, 60) = 1.9332, p = 0.1695).
No statistically significant difference was observed between the zones of inhibition of discs treated with turmeric oil (M = 6.4375) and cinnamon oil (M= 6.9375) (F(1, 60) = 0.7552, p = 0.1695)
There was also no statistically significant difference observed between the zones of inhibition for thyme oil (M= 6.1250) and cinnamon oil (M= 6.9375) (F(1, 60) = 1.9332, p = 0.1695).
Hence, despite there being observed differences in the means of the zones of inhibition, these differences are not statistically significant.
Discussion
Contrary to the hypothesis, while the oils all showed antimicrobial activity against Saccharomyces Cerevisiae, it was found that there was no statistically significant difference between the zones of inhibition of the oils.
Discs treated with turmeric oil revealed a statistically significant difference when compared with the control discs, indicating that turmeric oil was effective in inhibiting the growth of Saccharomyces Cerevisiae. The results obtained are consistent with those obtained by Minear et. al. (2011), which revealed that curcumin slows the growth cycle of Saccharomyces Cerevisiae. Hence, it may be that the curcumin present within the turmeric oil used in this study may have been what prevented Saccharomyces Cerevisiae from growing around the disc. However, the results obtained in this study do not align with those obtained by Kamble & Patil (2008), who used a similar disc diffusion method to show that turmeric oil was ineffective against inhibiting the growth of Saccharomyces Cerevisiae. This contradiction may be explained by the results obtained by Stępień et.al. (2019), that curcumin only slows the growth cycle of some variants of the fungus, and it may not significantly affect the growth of other variants. Therefore, it may have been that the variant of Saccharomyces Cerevisiae used in this study was susceptible to turmeric oil, whereas the variant used by Kamble & Patil (2008) may have been more resistant.
Discs treated with thyme oil also revealed a statistically significant difference when compared with the control discs, implying that thyme oil was effective in preventing the growth of Saccharomyces Cerevisiae. These results are consistent with those found by Kunicka-Styczyńska (2011), which discovered that thyme oil was highly effective in inhibiting the growth of Saccharomyces Cerevisiae. Yet, the results in this study only partially align with those in Kamble & Patil (2008), which revealed that thyme oil was more effective in inhibiting growth than turmeric oil. In this study, however, thyme oil had the lowest mean zone of inhibition out of the oils tested, including turmeric oil. Again, this may be due to the variant of Saccharomyces Cerevisiae used, or the variation of individual properties between oils from different brands.
Discs treated with cinnamon oil further revealed a statistically significant difference when compared with the control discs, proving that cinnamon oil was also effective in inhibiting the growth of Saccharomyces Cerevisiae. This aligns with the results obtained by Çoşkun et.al. (2016) and DinkovaKostova et.al. (2020) in which cinnamon oil proved to be more effective against Saccharomyces Cerevisiae than all the other oils tested. While there is very minimal literature comparing the inhibitory nature of cinnamon oil against that of thyme and turmeric oils, cinnamon oil has shown the highest inhibitory activity against yeast in the aforementioned studies. Thus, it is not surprising that discs treated with cinnamon oil had the largest average zone of inhibition in this study.
Concerningly, the zones of inhibition observed in the study were smaller compared to those in other investigations, for example cinnamon oil had an average of 6.9375mm when soaked in 5ml of oil, whereas Çoşkun et.al. (2016) observed a 38mm average when discs absorbed 20 microlitres of cinnamon oil. This significant difference may be a result of using smaller discs, more sterile agar plates or a different variant of Saccharomyces Cerevisiae in the study.
When the oils were individually compared with one another through post-hoc contrasts, neither of the lots returned a statistically significant difference. While these results suggest that no oil is more effective than the other in inhibiting the growth of Saccharomyces Cerevisiae, there are a variety of other possible explanations, as this study had many assumptions and limitations.
Firstly, it was assumed that all of the oils had a uniform composition and that the antifungal agents present in each oil (eg. curcumin in turmeric oil) were evenly soaked up by the discs. This may not have been the case, though, as discs may have absorbed varying levels of antifungal agents, therefore showing inconsistent antifungal activity between trials. It was further assumed that the oils were absorbed at the same rate between discs, regardless of varying levels of viscosity between oils, and that the yeast solution was evenly spread across the agar plates. Again, these assumptions may be incorrect and some discs may have shown higher antifungal activity than others because of more of the oil being absorbed, or because of their section on the plate containing a relatively lower amount of yeast solution. The small sample size of the study (16 discs per test) may have also contributed to the high p-values revealed after the contrast tests, as there is a greater likelihood of error and inconsistency when there are minimal trials. Some plates were removed due to a zone of inhibition forming around the control disc, or due to the discs falling out of place due to supersaturation, which contributed to the smaller sample size.
not be assumed that the oil that showed the greatest antifungal activity against Saccharomyces Cerevisiae would be a better edible compound in preventing food spoilage caused by the fungus. While all the oils are edible, some may need to be diluted before consumption. This method tests the oils at 100% concentration, thus the antifungal activity of those oils may vary if diluted to an edible concentration. Furthermore, this study only takes into account the zones of inhibition after 48 hours, whereas an oil may take a longer time to show activity, but may ultimately inhibit more fungal growth. There are also a variety of other variables to consider before use in food packaging and preservation, including their effect on the food’s appearance and taste. (RaybaudiMassilia et.al., 2009) and their ability to be used via nano-emulsion technology (all of which are ignored in this study.
Overall, despite its limitations, this study employed a timely, cost-efficient method that successfully measured the effect of edible essential oils on the growth of Saccharomyces Cerevisiae. However, there are a variety of future directions that should be implemented to improve and add more depth to this investigation. Researchers can use serial dilutions to determine the minimum inhibitory concentration of the oils as another measure of effectiveness against Saccharomyces Cerevisiae. It would also be beneficial for researchers to test the effectiveness of a greater variety of edible essential oils against Saccharomyces Cerevisiae to gain more knowledge on the relative inhibitory effectiveness of certain oils against it. A larger sample size also should be implemented to enhance the reliability of the experiment and reduce the effect of random errors.
Conclusion
As there was a significant difference between the zones of inhibition of the discs treated with oil and the control discs, it can be concluded that all of the oils exhibited antifungal activity against Saccharomyces Cerevisiae, hence they may potentially be incorporated in food packaging to prevent fungal spoilage. However, there was no statistically significant difference between the antifungal activity of those oils, hence the null hypothesis was accepted. However, this does not mean that there is no difference between the anti-inhibitory activity of these oils, as the myriad of limitations presented in this study (such as the small sample size) may have impacted the viability of the results obtained. Therefore, additional research is needed to investigate the differences between the antifungal activity of these oils more thoroughly. As the study only accounts for differences in inhibiting the growth of Saccharomyces Cerevisiae, further research on other characteristics of the oils, such as their effect on the appearance and taste of food, is needed to determine their suitability for use in food packaging and preservation.
Acknowledgements
I would like to acknowledge my supervising teacher, Joelle Rodrigues, and mentor, Dr Taylor Szyszka, for guiding me throughout the creation of this report. I would like to further acknowledge Aliyah Asad for assisting in the preparation of agar plates and arrangements of the experiment, and Therese Kanaan for guidance in statistical analysis and interpretation of results. I would like to additionally thank my peers and teachers who provided me with the ideas and moral support to persevere through this otherwise difficult journey.
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