Exploring the Antifungal Activity of Various Natural Extracts in a Sustainable Saccharomyces cerevisiae Model Using Cell Viability, Spot Assay, and Turbidometric Microbial Assays

Abstract

Saccharomyces cerevisiae is a sustainable yeast with many applications in the food industry. Here, we study the use of a Saccharomyces cerevisiae model composed of three different industrial strains (a wine, a beer and a baker’s strain) to assess the antifungal activity of three organic plant-based extracts (Hypericum perforatum 10% w/w, Pistacia lentiscus var. Chia 20% w/w and Rosmarinus officinalis 6% w/w). Three different methods were employed (agar disc diffusion, spot assay, and growth curve analysis). Only the Rosmarinus officinalis extract (6% w/w) exhibited inhibitory activity against all the tested yeast strains in the agar disc diffusion method. In the spot assay, all extracts and their carrier oils (sunflower oil and caprylic triglyceride) exerted similar mild antifungal activity. In the growth curve analysis, all extracts significantly lowered the growth rate of the yeasts, but this was not observed for the carrier oils. The results highlighted that it is important to consider more than one method for testing the antimicrobial activity of different compounds. The three yeast strains exhibited differences in their susceptibility to pharmaceutical antifungals, and the beer and baker’s yeasts were resistant to itraconazole. Moreover, polyphenols were detected in all natural extracts which may be linked to their antifungal activity. Our results suggest that we might consider multiple use of these natural extracts in the food industry as food additives or even preservatives to delay food spoilage.

1. Introduction

Natural plant-based extracts are currently garnering a lot of attention due to their multifunctional biological roles and their different industrial applications in foods, pharmaceuticals, and cosmetics. Indeed, plant-based extracts can be used as food additives, which is considered a sustainable development trend in the food industry [1]. Specifically, plant-based food additives can be used as preservatives to prevent food spoilage caused by microorganisms to prolong food shelf life, as antioxidants to stop or delay the autoxidation of foods, or as antimicrobials to protect from microbes and other predators [2]. In addition, plant-based food additives have gained a lot of attention because of their benefits over synthetic ones, including environmental protection, health benefits, and green safety and food legislation issues [2]. As a result, the development and application of plant-based food additives is an emerging and active field in the food industry [1].

In this study we focused on three natural extracts that are derive from three highly related medicinal plants within the Greek flora: Hypericum perforatum (H. perforatum), Pistacia lentiscus var. Chia (P. lentiscus var. Chia), and Rosmarinus officinalis (R. officinalis). H. perforatum is a medicinal herb that is traditionally used to treat various ailments, including depression, anxiety, and inflammation. It is known that it contains a variety of phytochemicals, including flavonoids and hypericin, which are believed to have antioxidant, anti-inflammatory, and antimicrobial properties [3]. Specifically for foods, H. perforatum can be found in a variety of food and beverage goods as a functional ingredient. According to previous studies, 0.5 g of H. perforatum mixed with 100 g of wheat flour enhances the bioactive compounds of wheat cookies [4], and H. perforatum water honey extract is used to extend the shelf life of bread [5]. In addition, it is reported that H. perforatum is a safe and non-cytotoxic ingredient as it is used in pharmaceuticals, cosmetics, and foods [6,7]. Mastic is a natural resin derived from the P. lentiscus var. Chia tree and has been used for centuries for medicinal purposes and studied for its antifungal properties. Mastic has demonstrated antimicrobial properties against various strains of bacteria. Research has shown that mastic extract can inhibit the growth of bacteria associated with stomach ulcers and gastroenteritis [8]. Specifically, mastic can serve as an extra natural preservative in a variety of susceptible products such as cosmetics or pharmaceutics. In addition, in combination with other food additives, it has been shown to enhance the preservation and the quality of different products [9]. On the other hand, R. officinalis L., either as extract or essential oil, is a rich source of phenolic compounds with anti-inflammatory [10], antidiabetic [11], hepatoprotective [12], and antimicrobial activity [13]. Both the extract and essential oil of R. officinalis L. are used for the treatment of illnesses and in food preservation [13]. This rationale has stimulated consumer and industry interest in replacing or decreasing synthetic antioxidants in foods. The European Food Safety Authority (EFSA) has reviewed the safety of rosemary extract for use as a food additive [14].

In addition, Saccharomyces cerevisiae (S. cerevisiae) has been studied extensively, because it is a microorganism with a high genetic diversification due to adaptations after many years of industrial applications that have resulted in the evolution of many different strains with distinct phenotypic characteristics [15]. Therefore, to facilitate research and draw conclusions, S. cerevisiae is often chosen as a study model because it is harmless, easy to culture, and well-studied [16]. Yeasts, also, are more complex genetically and structurally than bacteria [17] and may be more resistant to various antimicrobial compounds. Therefore, it is important to evaluate not only the antibacterial activity of various extracts, but also their antifungal activity. Also, although there are many types of yeasts that cause food spoilage (such as Brettanomyces spp., Zygosaccharomyces spp., Rhodotorula spp.), in some cases, the presence of S.s cerevisiae also has the potential to cause spoilage (e.g., sweet wines, yogurts, mold-ripened soft cheeses) [18]. Moreover, it has been noted that S. cerevisiae is considered as a multifaced single-cell factory with many industrial applications with homology to other eukaryotic organisms that can provide information on the mechanism of action or scavenging potential under various conditions [19,20,21]. Here, we explore the use of an industrial and commercially available S. cerevisiae cellular-based model by applying three different strains—a wine, a beer and a baker’s strain—as used in three representative food industries, with the aim of evaluating the antifungal activity of three natural extracts. The extracts are H. perforatum (10% w/w), P. lentiscus var. Chia (20% w/w), and R. officinalis (6% w/w), which are commercially available and ready to be used in the food industry. For this purpose, three different methods have been employed: the classical agar disc diffusion method, spot assay, and turbidometric growth curve analysis. In addition, LC–MS was used complimentarily, to assess the presence of phenolic compounds in these natural extracts.

2. Materials and Methods

2.1. Selected Yeasts

Commercial dried yeasts were used as cell-based models to test the antifungal activity of various extracts. More specifically, three S. cerevisiae strains were used: (a) a wine yeast—fast-fermenting classic 8 Turbo Yeast (producer: Still Spirits, New Zealand), which is a high strength alcohol yeast for wine production; (b) a beer yeast—M15 Empire Ale yeast, a top-fermenting ale yeast suitable for a variety of full-bodied ales (producer: Mangrove Jack’s, New Zealand); and (c) a baker’s yeast—an instant dried yeast for bakeries (Alimentaria, Greece).

2.2. Natural Extracts Tested as Antimicrobial Agents

All extracts are organically certified from 100% Hellenic plants produced by local farmers. Specifically, the extracts are: (a) H. perforatum extract that contains the non-polar fraction of components from H. perforatum organic flowers and leaves (10% w/w) in a carrier of caprylic/capric triglyceride 90% and 0.2% sorbic acid, (b) mastic resin extract that contains the non-polar fraction of components from P. lentiscus var. Chia (20% w/w) in a carrier of caprylic/capric triglyceride 80% w/w, and (c) rosemary extract that contains R. officinalis leaves (6% w/w) in 94% sunflower (Helianthus annuus) seed oil as a carrier. The extracts were produced and kindly provided by the Natural Food Additives company, Athens, Greece.

2.3. Agar Disc Diffusion Method

The disc diffusion method was used to evaluate the effect of natural extracts against the yeasts [22]. A suspension of the tested yeast (in sterilized phosphate-buffered water APHA) containing about 10⁶ cfu/mL was prepared from an overnight culture in the stationary phase. A 100 μL volume from this suspension was spread on the surface of chloramphenicol glucose yeast extract agar plates (Biolife, Milan, Italy). Blank sterilized paper discs (HiMedia) 6 mm in diameter were placed on the surface of the inoculated agar plates and impregnated with 50 μL of each natural extract. The antifungal activity of 6 pharmaceutical compounds was also tested with the same method. Ready-to-use discs impregnated with the specific antifungal compound were used (HiMedia, Modautal, Germany): clotrimazole (10 μg), fluconazole (25 μg), itraconazole (10 μg), ketoconazole (10 μg), nystatin (100 units), amphotericin B (100 units). The plates were then incubated for 48 h at 37 °C. The incubation temperature of at 37 °C was chosen as a factor inducing possible pathogenic traits of S. cerevisiae strains, according to the review by Annop et al. (2015) [22]. The diameter of the inhibition zones (including the diameter of the paper disk) was measured using calipers and was expressed in millimeters (Supplementary Data Figures S1 and S2). The preparation of the petri dishes was carried out within the laboratory’s Biosafety Cabin Class II to ensure sterile conditions. All experiments were performed in triplicate.

2.4. Spot Assay

Spot assays were used to quantitatively evaluate the growth and survival of yeast cells. Spot assay were performed according to the protocol of., Petropavloskiy et al. 2020 with some modifications [23]. This method detects and quantifies the growth of yeasts in agar plates by taking images of the colonies (spots) of yeasts as they grow and applying image analysis to measure the gray value of the spots. Yeasts were grown at 37 °C overnight in Brain Heart Infusion (BHI, Condalab, Madrid, Spain) broth supplemented with 20 g/L dextrose, and the inoculum 200 μL of OD₆₀₀ 0.5 was placed in row A of a 96-well plate. Two serial 1:5 dilutions were performed in rows B and C by adding 30 μL from row A to 120 μL of ddH₂O in row B and then adding 30 μL from row B to 120 μL of ddH₂O in row C. Then 50 μL of the natural extracts and carrier oils were added to all rows. All samples were added in triplicate. Equal volumes (10 μL) from each well were transferred to the surface of an agar plate (Brain Heart Infusion (BHI, Condalab) agar supplemented with 20 g/L dextrose) at equal distances to form visible spots after incubation. The plates were incubated for 48 h at 37 °C, and images were taken with a mobile phone. The third dilution (25×) after incubation at 24 h was selected as the most appropriate for image analysis. Preprocessing of the images was implemented using GIMP 2.99.16 software to remove any elements that could confuse the analysis, such as various light reflections in the background, and to isolate each yeast spot. MATLAB software (Matlab R2022b) was used for image analysis, first by separating the spots (yeast cultured regions) from the background using the threshold algorithm to focus only on the yeast spots to be used for analysis and to select regions of interest of equal diameter. Then, image analysis of yeast spots was performed by measuring the distribution of gray level values (values of gray level intensity ranging from 0, which corresponds to black, to 255, which corresponds to white) of the spots and calculating the mean gray level values.

2.5. Growth Curve Analysis

Growth curves of yeasts were generated by automatic turbidometry using a Bioscreen C (Lab systems Helsinki, Vantaa, Finland) instrument in 100-well honeycomb plates [24]. Yeast strains were incubated at 37 °C overnight in Brain Heart Infusion (BHI, Condalab) broth supplemented with 20 g/L dextrose; after vortexing, 100 μL aliquots of inoculum were added to 9 mL sterilized phosphate-buffered water APHA to obtain a turbidity of 0.5 McFarland standard. Samples were loaded into the plates by adding 50 μL of the microorganism, 50 μL of tested natural extract, and 200 μL broth (Brain Heart Infusion, BHI + 20 g/L dextrose) to a total volume of 300 μL. The preparation of the plates was carried out within a Biosafety Cabin Class II. The optical density (OD) of the wells was measured at 600 nm every 30 min for 48 h at 37 °C and at 30 °C (Supplementary Data Figure S3). Before each measurement, the wells were automatically shaken for 10 s. All measurements were performed in triplicate. After incubation, data were extracted in Excel, and corresponding graphs of optical density versus time were constructed for each sample. These graphs represent the growth curves of microorganisms under the specified conditions. Results were processed with the ComBase (https://www.combase.cc/index.php/en/, accessed on 3 December 2023) tool DMFit for Excel, which is an Excel add-on that fits a primary curve to log CFU or OD counts versus time data and estimates microbial kinetic parameters such as the maximum specific growth rate (μ_max) and lag time of the growth curve of microorganisms [25]. Antimicrobial activity is observed if in the presence of the antimicrobial agent, an extended lag phase and a lower growth rate during the logarithmic phase are observed.

2.6. Determination of Total Phenolics, Phenolic Acids and Flavonoids

The presence of polyphenols in the extracts was determined using a LC-ESI single quadrupole MS (Shimadzu, Kyoto, Japan) according to a previous work of our group [26]. Briefly, the mobile phase of the system was 95% ddH₂O, 5% MeOH, 0.2% acetic acid (solvent A) and 50% acetonitrile (ACN)/50% ddH₂O (solvent B) in gradient mode. For the detection of different m/z values, we used both positive and negative mode. The findings represent the mean value ± SD of three measurements.

2.7. Statistical Analysis

For all analyses, three independent experiments were conducted followed by triplicate measurements to calculate average values and standard deviation (SD). The statistical analysis of all the results was based on one-way ANOVA (ANOVA) followed by Duncan post hoc tests (p < 0.05). The statistical analyses were performed using SPPSS 18.0 (SPSS Inc., Chicago, IL, USA).

3. Results and Discussion

The antifungal activity of the various plant extracts at concentrations that are available commercially was assessed by different methods: (a) agar disk diffusion method, (b) spot assay, and (c) growth curve analysis.

3.1. Agar Disc Diffusion Method

This method is a classical microbiological technique to test pharmaceutical antimicrobial agents such as antibiotics and antifungals in many clinical laboratories [27]. In this method, the antimicrobial agent, which is placed on a disk in the surface of an inoculated agar, diffuses into the agar and inhibits the growth of the test microorganism. The inhibition creates a clear zone around the impregnated discs, which represents “no growth” areas, and the diameter of inhibition growth zones is an indication of the susceptibility of the microorganism to the specific antimicrobial agent (the greater the inhibition zone, the greater the susceptibility).

It was observed (Figure 1) that the natural extracts showed negligible inhibition zones (<10 mm) or no inhibition zones (6 mm), except for the rosemary extract, which showed an inhibition zone (>10 mm) higher than that of fluconazole in the case of beer yeast. Indeed, rosemary has been extensively studied, and there is significant evidence of its antimicrobial activity against both Gram-negative and Gram-positive bacteria [28]. These properties make rosemary of particular interest as a natural food preservative [29]. The EU approved rosemary extract as a food preservative after extensive toxicity studies and determining that the observed adverse effect level (NOAEL) range was wide enough to not pose any safety concerns [30]. However, when an extract exerts low fungal growth inhibition, there may be no results in the disc diffusion method, and it would be worth-while assessing the extract’s antimicrobial activity using alternative methods. Moreover, it should be noted that the carrier oils of the natural extracts, caprylic triglyceride and sunflower oil, were also tested for antifungal activity with the agar disc diffusion method and showed negligible inhibition activity (<10 mm) (Figure 1), although there are studies indicating antifungal activity of caprylic acid [31].

Pharmaceutical antifungals are categorized as fungicidal if they kill the fungus (such as amphotericin B) or fungistatic if they inhibit the growth of fungi [32]. Many known antifungals that are used for medical treatments such as azoles (e.g., fluconazole, clotrimazole, itraconazole, ketoconazole) can have either fungistatic or fungicidal activity depending on the dose and the species of microorganism [33,34]. In this study, six pharmaceutical antifungal compounds were tested against the three S. cerevisiae strains (Figure 1). It was observed that the three strains of the same species S. cerevisiae demonstrated different susceptibilities to the various pharmaceuticals. All three strains exhibited susceptibility to amphotericin B, nystatin, ketoconazole, and clotrimazole, which clearly exerted fungicidal activity (diameter of inhibition zone > 20 mm). However, the beer and baker S. cerevisiae strains showed resistance to itraconazole (no inhibition zone), and the beer strain had low susceptibility to fluconazole (diameter of inhibition zone < 14 mm), which are known pharmaceutical agents used to treat fungal diseases. It is known that industrial yeasts have adapted to tolerate various environmental stresses such as high osmotic pressure, oxidative stress, temperature shifts, as well as chemical compounds; for example, wine yeasts have adapted to sulfite compounds and copper-based pesticides used in wineries [35,36]. It is possible that the well-adapted industrial yeasts employed in this work have resistance mechanisms that allow them to thrive in the presence of specific pharmaceutical azoles. The low susceptibility of S. cerevisiae to some azoles has already been documented, and although the yeast is considered GRAS (generally recognized as safe), there have been cases in which it has caused invasive infection [37].

3.2. Spot Assay Analysis

In spot assays, yeast colonies are grown as spots on agar plates in the absence or in the presence of the tested antimicrobial compound at different concentrations. The sensitivity of the yeast towards the antimicrobial compound is analyzed based on the density of the cells grown in each spot. Comparison of growth inhibition in a specific spot with and without the antimicrobial compound allows assessment of the sensitivity of a strain to that particular agent [38]. For the spot analysis, the mean gray values of the yeast colonies visualized as spots were measured [23], and results are shown in Figure 2. Spots with high yeast growth yield a higher mean gray level value than spots in which the yeast has not grown due to the presence of an antimicrobial agent. Therefore, this assay can quantitatively assess the growth capacity and survival of yeasts based on the density of the cells in spots of the same size [39].

As can be observed in Figure 2, the “yeast only” samples for wine yeast (Figure 2a) and beer yeast (Figure 2b) in the absence of the natural extracts demonstrated higher growth capacity compared to all other samples in which the yeast was co-inoculated with a plant extract or carrier oils (p < 0.05). A similar trend was observed for the baker’s yeast, but only for Hypericum perforatum and the carrier oils. This implies that the natural extracts negatively influence the growth of the yeast. However, in this method, the carrier oils (sunflower oil and caprylic triglyceride) also exhibited the same antifungal activity as the plant extracts, and therefore there is no evidence to suggest that the ingredients of the natural extracts are the causative agents of antifungal activity. The co-inoculation of the carrier oils with yeast cells onto the surface of the agar resulted in mild inhibition of yeast growth, in contrast to the agar disc diffusion method, in which the carrier oil exhibited a negligible antifungal activity (Figure 1). This difference may be due to the fact that the oils had not adequately diffused from the disc to the agar in order to inhibit the growth of the yeast, whereas in the spot assay method the carrier oils were directly influencing the growth of the yeast cells due to the co-inoculation. Fatty acids may exert antifungal activity mainly by interfering with the lipid bilayers of fungal membranes, and it has been shown that caprylic acid ester derivative has potent membrane disruptive actions against C. albicans and M. furfur [40,41].

3.3. Growth Curve Analysis

Microbial population growth is usually graphically represented by a sigmoid growth curve that has three characteristic phases: the lag phase (λ) during which no growth occurs, the logarithmic (log) phase which is a phase of rapid growth in which the maximum growth rate (μ_max) of the microorganism is observed, and the stationary phase that is reached when nutrients are exhausted [40,41]. During optimum microbial growth, a short lag phase and a high maximum growth rate are observed. However, any factor that may stress the microorganism and compromise its viability (e.g., temperature variation, osmotic pressure, reactive oxygen species (ROS), antimicrobial agents, etc.) creates a shift away from optimum growth conditions [40]. This is usually observed by a longer lag phase, a slower maximum growth rate, and a lower level of maximum population in the stationary phase. One common method to obtain the growth curve of a microorganism is by measuring optical density (OD at 600 nm) at regular time intervals (e.g., every 30 min) in an inoculated microbial broth at a specific temperature throughout the incubation of the microorganism (usually 24–48 h) and constructing a curve of optical density versus time [42]. The growth curves of the three yeasts were measured by automatic turbidometry using a Bioscreen C instrument (Figure 3). The lag phase is the time in which the yeast cells are biochemically active but still not dividing since they are trying to adjust to the new growth medium [36]. After the lag phase, the cells start dividing and enter the logarithmic phase in which the maximum growth rate is observed, and the slope of the curve grows sharply. Figure 3A–C shows the sigmoid curves of the yeasts when they are grown in nutrient broth at 37 °C in the absence of the natural extract (yeast only) and the growth curves of the yeasts in the presence of a natural extract. According to our results, the three different S. cerevisiae strain-related models had different responses in growth rate in the presence of the three extracts. Moreover, the growth rate of the curves was calculated with the ComBase tool. At 37 °C, the rates of the yeasts grown in the presence of natural extracts were found to be significantly lower than that of the control (yeast only) (p < 0.05), which is indicative of antimicrobial activity (Figure 3D). The same trend was observed at 30 °C (Supplementary Data Figure S3). Furthermore, in this method, the carrier oils of the natural extracts, caprylic triglyceride and sunflower oil did not exhibit statistically significant antifungal activity. This may be due to fact that the assessment of growth was performed in broth and not in static agar conditions [43]. According to previous reports, H. perforatum extract may have antioxidant and antimicrobial activity toward microbial cells, which could be attributed to the presence of phytochemicals such as flavonoids and hypericin [44,45,46]. Moreover, H. perforatum could protect food from diseases caused by spore-forming bacteria such as B. mesentericus and B. subtilis along with improved organoleptic and rheological properties [5]. In addition, mastic extract has strong antioxidant properties due to the presence of polyphenolic compounds such as flavonoids and phenolic acids [47]. The essential oil of the resin of P. lentiscus var. Chia (mastic oil) was studied in vitro against a wide range of foodborne pathogenic and spoilage microorganisms; the results showed that Gram-positive were found to be more susceptible to the essential oil than Gram-negative microorganisms, and all fungi appeared very resistant to mastic oil [48]. In addition, several studies have suggested that mastic extract has antimicrobial activity against periodontal pathogens and the yeast Candida albicans [49]. Moreover, rosemary extracts showed biological bioactivities such as hepatoprotective, antifungal, insecticide, antioxidant, and antibacterial [13]. Rosemary has been extensively studied, and there is significant evidence of its anti-microbial activity against both Gram-negative and Gram-positive bacteria [28]. However, only a few studies reported the impact of R. officinalis L. in yeasts cells [50].

3.4. Determination of Total Phenolics, Phenolic Acids and Flavonoids

The LC-MS analysis revealed that the natural extracts of the study have a rich antioxidant phytochemical content in phenolic compounds including phenolic acids, flavonoids, and conjugated tannins. Specifically, the three natural extracts contain pyrogallic acid (m/z 125^-), catechol (m/z 109^-), 3,5-dimethoxy-4-hydroxy tannic acid (m/z 224.05^-), quercetin (m/z 300.5^-), curcumin (m/z 366.8^-), catechin (m/z 289^-), deosmin (m/z 607^-), kampferol (m/z 285), coumaric acid (m/z 163^-), and gallic acid (m/z 169.17^-) (

Table 1. Detection and Retention time (min) of phenolic compounds in hypericum (H. perforatum), liquid mastic (P. lentiscus var. Chia), and rosemary (R. officinalis) extracts.

		Hypericum Extract	Rosemary Extract	Liquid Mastic Extract
1	Pyrogallic acid	5.4 ± 0.01	5.4 ± 0.01	5.4 ± 0.01
2	Na-salicylate	17.5 ± 0.01	n.d *	17.5± 0.01
3	Catechol	32.0 ± 0.01	32.0 ± 0.01	32.0 ± 0.01
4	Tartaric acid	n.d	31.1 ± 0.02	31.1 ± 0.02
5	3,5-dimethoxy-4-hydroxy tannic acid	29.7 ± 0.02	29.7 ± 0.02	29.8 ± 0.02
6	Quercetin	22.2 ± 0.02	22.2± 0.01	22.2 ± 0.02
7	Rutin	33.2 ± 0.02	n.d	33.2 ± 0.02
8	Curcumin	62.5 ± 0.01	62.5 ± 0.01	62.5 ± 0.01
9	Tannic acid	n.d	26.5 ± 0.01	n.d
10	Catechin	12.4 ± 0.01	12.4 ± 0.01	12.4 ± 0.01
11	Silymarin	n.d	n.d	47.4 ± 0.06
12	Deosmin	37.5 ± 0.03	37.5 ± 0.03	37.5 ± 0.03
13	Kampferol	49.9 ± 0.03	49.9 ± 0.03	49.9 ± 0.03
14	Coumaric acid	24.8 ± 0.01	24.8 ± 0.01	24.8 ± 0.01
15	Ascorbic acid	n.d	n.d	2.8 ± 0.01
16	Cinnamic acid	n.d	n.d	2.9 ± 0.01
17	Gallic acid	4.4 ± 0.001	4.4 ± 0.001	4.4 ± 0.001

* n.d: non detected.

). Moreover, we observed that the liquid mastic extract was richer in phenolic acids such as tartaric acid, ascorbic acid, and cinnamic acid (Table 1). This antioxidant activity is quite important in the food industry, where antioxidants can be used to prevent or stop the autoxidation of food items or to shield food from nutritional degradation during storage [51,52,53]. Plant-based food ingredients exhibit antioxidant protection through their content of phenolic compounds (carotenoids, phenolic acids, anthocyanins, flavonoids, tannins, hydroxytyrosol, and their derivatives) due to their ability to scavenge free radicals, donate free electrons, and chelate metals [27,28].

It has been suggested that the mechanism of antimicrobial action of various flavonoids varies depending on the group [54]. In a recent work, Kakouri et al. (2023) [55] studied the composition of nine Hypericum species from Greece and found high concentration of flavonoids. Regarding the non-polar extract of R. officinalis plants from the Greek territory, Lagouri and Alexandri (2013) found high concentrations of flavonoids [56]. Therefore, it may be assumed that the mode of antimicrobial action of the extracts is linked to the mechanisms of antimicrobial action of the flavonoids. Regarding the antifungal activity of flavonoids, in a review paper, Al Aboody and Mickymaray (2020) summarized the main inhibitory mechanisms that include plasma membrane disruption, induction of mitochondrial dysfunction, inhibition of cell wall formation, cell division, RNA and protein synthesis, as well as dysfunction of the cell membrane pump system [54].

4. Conclusions

This study evaluated the antifungal activity of three commercial natural plant extracts against three industrial S. cerevisiae strains: a wine, a beer, and a baker’s strain. S. cerevisiae is widely used as model eukaryotic microorganism but comprises numerous strains with significant genetic and phenotypic differences that are not well investigated [15,16]. In this study, S. cerevisiae strains were used as yeast models that are sustainable and well adapted to environmental pressures to assess the potential application of the natural plant extracts against yeast food spoilage. The antifungal activity of the natural plant extracts was assessed using three different methods, and it can be concluded that the degree of antimicrobial activity detected varied according to the applied method. In the agar disc diffusion method, only the rosemary extract appeared to be fungistatic against all the tested yeasts compared to the other two extracts. The spot assay method revealed mild antifungal activity against the wine and beer yeast by all of the natural extracts; the carrier oils showed similar antifungal activity, thus the antimicrobial activity of the extracts was not demonstrated. However, a more comprehensive analysis of the fungistatic activity was observed in the growth curve analysis, which revealed statistically significant slower growth rates of all yeasts in the presence of the natural plant extracts. Therefore, it was shown that the natural plant extracts delayed the growth of the S. cerevisiae strains studied. Also, the three yeast strains in this study exhibited differences in their susceptibility to pharmaceutical antifungals, and two of them were found to have acquired resistance to itraconazole (beer yeast and baker’s yeast).

A common characteristic of the samples was the carrier oils, which most likely hindered the inhibitory effect of the natural extracts due to low diffusivity in the agar disc method [43]. Yet, it is important to underline that mild antifungal activity of the oil carriers was only detected in the spot assay method. It is therefore advisable that the method of choice for the study of antimicrobial activity depends on many factors, and it would be better to apply more than one technique when investigating the antimicrobial activity of various components as reviewed by Tan et al. (2015) [43]. In addition, we confirmed the presence of several polyphenols in all the natural extracts that could be linked to their antifungal activity. Even though there is limited data concerning the antifungal activity of natural extracts on different species of yeasts, it should be noted that our results suggest that we could consider different use of these extracts in the food industry as, for example, natural preservatives in combination with lower than usual concentrations of chemical preservatives to inhibit or delay yeast food spoilage. Last but not least, we would like to underline that this is a preliminary investigation and that further research into other yeasts such as Brettanomyces spp., Zygosaccharomyces spp., Rhodotorula spp. should be conducted.