DPPH assay was conducted according to the suggested method by Hassan (3) with some modifications. DPPH solution (0.1 mM) was first prepared in absolute methanol and added into different tubes at 1 mL volumes. Then, 1.0 mL yeast extract was added into each tube at different concentrations (125-2000 μg/mL) and mixed well. A set of blanks was prepared by adding 1.0 mL methanol with 1.0 mL yeast extract at different concentrations. All the mixtures were incubated in the dark at room temperature for 30 min. Negative control was prepared by the same procedure without the yeast extract. Ascorbic acid solution was used as a positive control. The absorbance of the resulting mixtures was measured at 517 nm using a 10S UV-Vis spectrophotometer (Genesys, Daly City, CA, USA). The measurements were taken in triplicate and the mean values were calculated.

The ABTS assay was carried out based on the method proposed by Hameed et al. (13) with some modifications. The stock solution was prepared by reacting the 7.00 mM ABTS solution with 2.45 mM potassium persulfate at the ratio of 1:1 (V/V), and kept in the dark at room temperature for 12-16 h before use (to generate ABTS˙⁺). The ABTS˙⁺ solution was diluted with absolute methanol and adjusted to the absorbance from 0.7 to 0.75 at 734 nm. A volume of 200 µL of the yeast extract at different concentrations (15.63-2000 µg/mL) and 1.8 mL ABTS˙⁺ solution were mixed well in the tubes and incubated in the dark at room temperature for 30 min. Negative control was prepared according to the same procedure without the yeast extract. Ascorbic acid solution was used as a positive control. The absorbance was measured at 734 nm using Infinite 200 PRO microplate reader (Tecan, Männedorf, Switzerland). The measurements were taken in triplicate and the mean values were calculated.

The FRAP reagent was prepared by mixing 300 mM acetate buffer at pH=3.6, 10 mM TPTZ in 40 mM hydrochloric acid (Merck), and 20 mM FeCl₃·6H₂O at the ratio 10:1:1. The mixture was incubated for 15 min at 37 °C before use. Ascorbic acid was used in distilled water at different concentrations (200-1000 μg/mL) as the positive control to generate a standard curve. A volume of 150 μL of the yeast extract (in ethyl acetate) or standard was mixed thoroughly with 2.85 mL FRAP reagent in Falcon tubes. The formation of an intense blue colour complex suggests the reduction of Fe³⁺ to Fe²⁺ in TPTZ complex. The mixture was incubated in the dark for 30 min. The absorbance (A) of the solution was measured at 593 nm using the Infinite 200 PRO microplate reader (Tecan). The measurements were taken in triplicate and the mean values were calculated.

In this test, the yeast extract sample was used against Cutibacterium acnes (ATCC 6919), Staphylococcus aureus (ATCC 6538) and Staphylococcus epidermidis (ATCC 12228). The concentrations of the yeast secondary metabolites were 100, 200, 300 and 400 mg/mL. The extract was dissolved in DMSO. Gentamycin discs (Thermo Scientific™ Oxoid™) with 10 µg drug acted as the positive control, while the disc with DMSO solvent but without any extract acted as the negative control. A volume of 7 µL of the dissolved extract and solvent was impregnated into sterile discs and dried in the laminar flow hood (model AHC-4DI; ESCO, Selangor, Malaysia) to remove the solvent completely. The antimicrobial tests against S. aureus and S. epidermidis were carried out in MHA, while the disc diffusion test against C. acnes was carried out on TSA as C. acnes cannot grow on MHA. The three tested bacterial strains were cultured in broth and the concentration of the bacteria was adjusted to 0.5 McFarland standard. According to Clinical and Laboratory Standards Institute (CLSI), 0.5 McFarland standard is equivalent to an absorbance value range from 0.08 to 0.13 at 625 nm. The TSA plates with C. acnes were incubated in the anaerobic jar at 37 °C for 48-72 h, while the MHA plates were incubated in the presence of oxygen at 37 °C for 8-16 h. The formed inhibition zones were observed after every 6 h of incubation; the diameter of the zones was measured in mm. This test was performed in triplicate.

The minimum inhibitory concentration (MIC) of the extracts was determined in a 96-round bottom well microplate. The initial concentration of the secondary metabolites of S. cerevisiae used for the MIC determination was the lowest concentration that formed an inhibition zone in the disc diffusion method for the three bacterial strains. Two-fold serial dilution was carried out to make a total of eight different concentrations of the extract. Sterile MHB was added to each well containing S. aureus or S. epidermidis, while sterile TSB was added for C. acnes. The extract was prepared in 100% DMSO at the concentration of 300 mg/µL, then 100 µL of the prepared extract were transferred to the first column of the well, mixed, and 100 µL of the mixture were also transferred to the next well in each row. Gentamicin was used as the positive control at the concentration of 1 mg/µL, while the negative control was the broth and solvent without any extract. The suspension of the three tested bacterial strains was adjusted to the concentration equivalent to 0.5 McFarland standard using a UV-Vis spectrophotometer (Genesys). A volume of 100 µL bacterial suspension was added into each well, then the microplate was covered, sealed, and incubated at 37 °C. The microplates containing S. aureus and S. epidermidis were then incubated for 6 h and those containing C. acnes 12 h. After the incubation period, 50 µL of 0.4 mg/mL p-iodonitrophenyltetrazolium violet indicator (Sigma-Aldrich, Merck) was added into each well and further incubated for 30 min. The colour change of each well was observed. The colour of the indicator turns pink when there is active bacterial growth in any well, while no colour change indicates no active bacterial growth. The lowest concentration of the extract that inhibited bacterial growth was recorded as the MIC. This assay was performed in triplicate.

The DPPH radical scavenging activity of the S. cerevisiae extract was evaluated based on its ability to reduce DPPH free radicals, which decolourises purple DPPH solution (16). Fig.1a shows the radical scavenging ability of the S. cerevisiae extract compared to ascorbic acid. The highest DPPH radical scavenging activity of ascorbic acid and the yeast extract was 95.91 and 90.96% at the concentrations of 250 and 2000 μg/mL, respectively. Ascorbic acid exhibited a significantly higher DPPH radical scavenging activity than the S. cerevisiae extract. Ascorbic acid is one of the active reducing agents and scavengers of free radicals in biological systems, acting as a scavenger of free oxidizing radicals and harmful oxygen species (17). Hassan (3) documented that 25 mg/mL baker’s yeast autolysate showed (69.06±1.22) % DPPH radical scavenging activity due to the reduction of the molarity of the DPPH solution from 0.2 to 0.1 mM. Moreover, the extraction method might affect the antioxidant capacity of an extract ( 18). The study by Sugiyama et al. (19) evaluated the antioxidant activity of indole derivatives from a marine sponge-derived yeast. All compounds showed weak activities in the DPPH assay. The extract of S. cerevisiae has been reported to exhibit the highest DPPH radical scavenging activity due to its high tryptophol content from alcoholic fermentation (20). Ethanolic extracts of Hibiscus sabdariffa and Croton caudatus leaves have been evaluated for free radical scavenging activity in the model system of S. cerevisiae. H. sabdariffa and C. caudatus at a concentration of 500 μg/mL demonstrated an immense free radical scavenging capacity of DPPH with an IC₅₀ value of 184.88 and 305.39 μg/mL, respectively ( 21). Furthermore, it has been demonstrated that soybean with Tricholoma matsutake and S. cerevisiae showed considerable DPPH radical scavenging activity (22).

The ABTS radical scavenging activity of the S. cerevisiae extract was assessed based on its ability to reduce ABTS radical cation. The ABTS˙⁺ was produced from the oxidation of ABTS with potassium persulfate, which involves the loss of an electron from the nitrogen atom of ABTS (23). The ABTS˙⁺ solution is bluish-green in methanol. Its absorbance was determined at 734 nm to minimize the interference from other absorbing components and sample turbidity. Fig. 1b shows the relative ability of the S. cerevisiae extract to scavenge ABTS˙⁺ compared to that of ascorbic acid. The standard ascorbic acid and yeast extract at the concentration of 2000 μg/mL recorded the highest inhibition values of 99.17 and 92.20% respectively. This result indicated that both ascorbic acid and yeast extract effectively inhibited the formation of ABTS˙ ⁺. It has been reported that endophytic yeasts isolated from the lower stem and roots of Phragmites australis Cav showed 88% radical scavenging activity in the ABTS assay (24). Furthermore, Aspergillus awamori DT11 exhibited ABTS˙⁺ scavenging activity of 34.07% compared to ascorbic acid (44.5%), which was used as a positive control (25).

The FRAP of S. cerevisiae extract was evaluated based on the ability to reduce iron(III) to iron(II)) via electron donation to the sample. This reduction was monitored by measuring the change in the absorbance at 593 nm. The assay was conducted at low pH values (pH=3.6) to maintain iron solubility and electron transfer (26). Table 2 shows the FRAP values of each yeast extract at different concentrations. In general, the absorbance readings increased when the concentration of ascorbic acid and yeast extract increased from 200 to 1000 μg/mL. This was due to the reduction of the Fe³⁺ in TPTZ complex to the intensely blue-coloured Fe²⁺ (26). The standard solution of ascorbic acid and yeast extract recorded the highest absorbance of 3.6291 and 0.1611 respectively, at the concentration of 1000 μg/mL. The yeast extract exhibited the highest FRAP value expressed as ascorbic acid equivalents of 44.40 μg/mL, indicating that S. cerevisiae can act as a potent antioxidant to reduce Fe³⁺ ion. Trolox, a water-soluble vitamin E analogue, was used as a reference standard to determine the antioxidant capacity of yeast microcarriers. The Trolox equivalent values for yeast cell wall particles and native yeast were reported to be 0.20 and 7.48 μM, respectively (27). Chen et al. (28) reported that Lactobacillus rhamnosus and Saccharomyces cerevisiae significantly improved FRAP in comparison to their non-fermented counterparts, and both activities were attributed to the released phenolics during the solid-state fermentation.

Benzeneethanol, 4-hydroxy, known as tyrosol, showed antioxidant activity in vitro and was able to inhibit or slow down the oxidation reactions brought by dioxygen or peroxides in animal tissues (29). However, tyrosol with its derivatives appeared to be less active than hydroxytyrosol and its analogues due to the influence of catechol structure (29). Antenucci et al. (30) reported that oligotyrosol exhibited higher antioxidant activity than tyrosol using DPPH, FRAP and hydroxyl radical scavenging assays. 1H-indole-3-ethanol is indolyl alcohol substituted with a 1H-indol-3-yl group. 1H-indole-3-ethanol plays a role as a metabolite of both plants and S. cerevisiae. Hexadecanoic acid is a fatty acid ester of plant and animal origins that acts as an insect repellent. Extracted from wild-growing mushrooms, it exhibited antioxidant activity at the concentration of 0.10 mg/mL ( 31). It is one of the twenty bioactive compounds isolated from Thesium humile Vahl with reported antioxidant activity (32). Gondwal et al. (33) reported that the water extract from the seeds and fruit pulp of Skimmia anquetilia can be used as a natural antioxidant due to its hexadecanoic acid content. Octadecanoic acid is a fatty acid ester and an algal metabolite that acts as a defoaming agent in processing beet sugar and yeast. Hexane extract of Sinapis alba exhibited slightly higher antioxidant activity than the standard ascorbic acid in phosphomolybdenum assay due to the existence of octadecanoic acid and other phytocomponents (34). Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro isolated from a marine bacterium Bacillus tequilensis MSI45 exhibited high antioxidant activity (35). The antioxidant activity of different pyrrolizidines, including pyrrolo[1,2-a]pyrazine-1,4-dione, and hexahydro-3-(phenylmethyl) in the ethyl acetate extract of Streptomyces omiyaensis has been reported by Tangjitjaroenkun (36). Also, Streptomyces strain MUSC 149(T) showed a strong antioxidant activity due to its pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- content (37).

The MIC of S. cerevisiae extract was determined using a colorimetric method in which the clear-cut end-points were determined via a colour change. Visual detection of bacterial growth via turbidity or pellet formation in the wells may be difficult and could lead to inaccurate results (50). To detect the presence of bacterial growth, p-iodonitrophenyltetrazolium violet was used as an indicator. When there is active bacterial growth, colour changes from light yellow to pink or violet (Fig. S2). The MIC of the extracted metabolites was 18.75, 31.25 and 75 mg/mL against S. aureus, S. epidermidis and C. acnes, respectively. This showed the potential of developing these metabolites into compounds with promising bioactivities against pathogenic microorganisms (Fig. 2). The obtained MIC of the extract against S. aureus showed consistency at 18.75 mg/mL in the triplicate tests. The mean MIC for S. aureus was the lowest among the three bacterial isolates, which indicates that the secondary metabolites of S. cerevisiae extract showed better antibacterial activity against S. aureus than the other two bacteria. C. acnes showed the highest level of resistance against both gentamycin and the tested secondary metabolites of S. cerevisiae.