Determination of the Probiotic and Functional Properties of Yeasts Isolated from Different Dairy Products

Abstract

This study aimed to explore new probiotic yeast strains, considering that Saccharomyces cerevisiae is the only yeast used industrially. To this end, yeasts were isolated from various dairy products and their probiotic potential was investigated. All yeast strains demonstrated survival potential under simulated gastrointestinal conditions, mimicking the stomach and intestinal passage. Among the isolates, 13.04% were identified as moderate biofilm producers, 34.78% as weak biofilm producers, and 52.17% exhibited no biofilm-forming ability. Catalase enzyme production was detected in all isolates, and it was determined that their hydrophobicity varied depending on hydrocarbon interactions. In this study, the percentage of autoaggregation increased with prolonged incubation times across all strains. After 24 h, the percentage of autoaggregation exceeded 60% for all isolates. It was concluded that the coaggregation ability depended on incubation time and strain type. Most of the isolated yeasts exhibited antimicrobial activity against E. coli ATCC 25922, E. coli O157:H7, B. cereus, C. sakazakii ATCC 29544, L. monocytogenes ATCC 7644, S. aureus ATCC 25923, and S. typhimurium ATCC 140828. Furthermore, the yeast strains were resistant to all tested antibiotics. The 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity revealed that the antioxidant activity of the cells was higher than that of their intracellular extracts for all tested strains. The yeasts were initially identified using MALDI-TOF and further characterized through 18S-28S rRNA analysis. There are not many recent studies on the selection of probiotic yeasts for use in the dairy industry. Therefore, in this study we wanted to investigate some functional properties of different yeasts to be proposed as probiotic cultures in this specific sector. In particular, the antimicrobial and antioxidant properties and enzymatic activity highlighted by isolates have promising implications for food safety and health. These preliminary results provide a valuable basis for the future industrial application of probiotic yeasts in the dairy sector.

1. Introduction

Although numerous studies have focused on probiotic bacteria, relatively fewer efforts have been directed toward probiotic yeasts and their applications in fermented dairy products. Using fermented dairy products as a source of probiotic microorganisms with therapeutic effects is a promising concept in food science. Cheese, kefir, and yogurt are the most common fermented dairy products containing probiotic yeasts.

Yeasts are eukaryotic microorganisms with biotechnological potential in the food industry. Yeast produces a wide range of fermented products, including alcoholic beverages obtained using various substrates, fermented dairy products such as cheese, grain-based leavened products, and seasonings [1]. During food fermentation, yeast plays crucial roles in producing and utilizing organic acids, improving taste, aroma, and texture, enhancing nutritional properties, and reducing toxins [2].

Yeast strains isolated and characterized from naturally fermented foods have been successfully applied as starter or co-starter cultures for the industrial production of functional foods [3]. In fermented food products, yeasts act synergistically with other groups of microorganisms, increasing the production of bioactive compounds and improving functional properties through enzyme and metabolite production [4]. Additionally, yeasts reduce or eliminate undesirable spoilage caused by microorganisms responsible for low product quality by producing mycotoxins and antibiotic factors [1].

Probiotic microorganisms in supplements or fermented foods are used to improve health. Several properties are required for a microorganism to be considered probiotic, including the ability to grow in low pH and bile, surface hydrophobicity, and tolerance to auto-aggregation [5]. S. boulardii is a yeast species that possesses numerous probiotic properties and has been extensively studied. Furthermore, S. boulardii is utilized as an economical and effective probiotic yeast against intestinal diseases, and it is commonly used in the treatment of various types of diarrhea in both humans and animals [6,7]. Yeasts offer several advantages over bacteria, as they are generally resistant to antibiotics [8]. While most studies on probiotic microorganisms have focused on bacteria, yeasts have recently shown potential as probiotics.

Although Saccharomyces boulardii remains the only commercially accepted probiotic yeast, the list of promising yeast candidates continues to grow. Probiotic yeasts include various species such as S. cerevisiae and S. boulardii [9,10,11,12], Yarrowia lipolytica [13], Candida famata [14], C. tropicalis [15], Debaryomyces hansenii [16], Issatchenkia orientalis [17], Kluyveromyces lactis [18], Kluyveromyces marxianus [9,18,19], Metschnikowia gruessii [19], Pichia jadinii [20], Pichia kluyveri [17], Pichia kudriavzevii [17], Pichia pastoris [21], and Pichia guilliermondii [22].

The selection of probiotic strains involves a complex process comprising several steps [23]. When determining the properties of a probiotic yeast, resistance to gastric and pancreatic conditions, adherence to hydrocarbons, autoaggregation and coaggregation abilities, as well as antimicrobial and antioxidant properties are assessed. Due to the increasing demand for natural components in food preservation, new studies have been conducted to isolate new probiotic microorganisms [24]. Since probiotic properties are strain-specific, studies must be conducted to evaluate new probiotic candidates. There are not many recent studies on the selection of probiotic yeasts for use in the dairy industry. Therefore, in this study we wanted to investigate some functional properties of different yeasts to be proposed as probiotic cultures in the dairy sector.

2. Materials and Methods

The yeasts were isolated from the following dairy products: cottage cheese, karın kaymağı cheese, butter, kefir, yogurt, and string cheese.

2.1. Isolation of Yeasts

The oxytetracycline–glucose–yeast extract agar base (OGYEA) (Merck, Darmstadt, Germany) medium was used to isolate probiotic yeasts. Pure cultures obtained were cultered in Sabouraud 2% dextrose broth (SDB) at 25 °C for 48 h (active yeast), and to ensure long-term storage, they were transferred to Eppendorf tubes containing 20% sterile glycerol (800 µL active yeasts + 200 µL glycerol) and stored at −60 °C [25].

2.2. Tolerance of Yeasts to 37 °C and pH 2.0

The isolates were subjected to tolerance tests at pH 2.0 at 37 °C, according to the method described by Ramos et al. [26]. Yeast cells grown in SDB medium at 30 °C for 24 h were centrifuged (4 °C, 5000 rpm for 5 min) and resuspended in SDB medium. The pH was adjusted to 2.0 using 1 N HCl, and the cells were incubated at 37 °C for 3 h. Isolates that showed growth at pH 2.0 and 37 °C were selected for further experiments.

2.3. Determination of Hemolytic Activity in Yeasts

Hemolytic activity was determined by inoculating yeast strains onto blood agar plates containing 5% defibrinated sheep blood and incubating them at 37 °C for 48 h. Developing a clear hydrolysis zone around the colonies was considered a positive result. Strains that formed greenish zones (α-hemolysis) or showed no effect (γ-hemolysis) were considered non-hemolytic colonies, while strains showing a zone of hemolysis around the colony (β-hemolysis) were classified as hemolytic and were not selected for probiotic use [27].

2.4. Survival Capacity of Yeasts in Gastrointestinal Conditions

2.4.1. Survival of Yeasts in Gastric Juice

The survival capacity of yeasts in gastric juice was determined [28]. Synthetic gastric juice was prepared in a buffer solution at pH 2.0 (adjusted with 1 M HCl), containing NaCl (2.05 g/L), KH₂PO₄ (0.60 g/L), CaCl₂ (0.11 g/L), and KCl (0.37 g/L). After sterilization, pepsin (0.0133 g/L) and lysozyme (0.01 g/L) were added to obtain the gastric solution. After growing yeast cultures on PDA (for 24 h at 25 °C) and developing them in tryptic soy broth (TSB) yeast broth, the cultures were centrifuged (5000 g for 10 min at 4 °C), and the pellets were washed twice with sterile 0.9% NaCl solution. The cells were then resuspended in synthetic gastric juice, adjusted to an optical density (OD) of 0.5 (λ = 600 nm), and incubated in an orbital shaker at 200 rpm at 37 °C for 2.5 h to simulate peristaltic movements. The survival percentages of the samples in gastric juice were calculated by colony counting on PDA.

2.4.2. Survival of Yeasts in Pancreatic Juice

The survival capacity of yeasts in pancreatic juice was determined [28] using a buffer solution formulated at pH 8.0 (adjusted with 1 M HCl) containing bile salts (3.0 g/L), pancreatin (0.1 g/L), Na₂HPO₄ (26.9 g/L), and NaCl (8.5 g/L). Cells collected from the previous gastric digestion step were centrifuged, washed twice in sterile 0.9% NaCl solution, and resuspended in the simulated pancreatic juice solution. After shaking the suspension (200 rpm at 37 °C for 3.5 h), the pellet was washed in 0.9% NaCl solution, and cell viability was assessed by incubation on PDA plates at 25 °C for two days. The survival percentages of the samples in pancreatic juice were calculated.

2.5. Determination of Yeast Biofilm Formation Properties

Biofilm formation ability was evaluated using the method described by Kopit et al. [29] with minor modifications. The strains were incubated in brain heart infusion (BHI) broth at 37 °C for 24 h. Then, 200 μL of the prepared culture was taken and added to 1800 μL of BHI broth. The negative control consisted of 2000 μL BHI broth. After growing the strains at 37 °C for 24 h, wells were washed with 2000 μL of phosphate-buffered saline (PBS) and the plates were inverted to dry at 55 °C for 30 min. The wells were stained with 2000 μL of 1% crystal violet. After washing the wells twice with PBS, they were inverted and dried at 55 °C for 30 min. The wells were then treated with 2000 μL of acetone and ethanol solution (20:80 v/v), and the OD was measured after 15 min. The mean value of the negative control was calculated, and three standard deviations were added to it to set an upper limit. This value is the cutoff point (ODc) at which the test result will be evaluated as either “positive” or “negative.” Biofilm-forming ability was evaluated according to the method of Pieniz et al. [30] with results classified as: OD ≤ ODc (non-biofilm producer), ODc < OD ≤ 2 ODc (weak biofilm producer), 2 ODc < OD ≤ 4 ODc (moderate biofilm producer), or OD ≥ 4 ODc (strong biofilm producer).

2.6. Determination of Enzymatic Activities in Yeasts

Yeast enzymatic activity was assessed using qualitative tests. Strains were initially grown on an OGYEA medium, and each test was conducted separately.

Catalase Activity: After growing all yeast isolates on an OGYEA medium, 30% hydrogen peroxide (H₂O₂) was dropped onto the colonies, and bubble formation was considered a positive result [31].

Protease Activity: For protease activity, a growth medium was prepared using 10 g skim milk, 1 g glucose, 5 g peptone, 25 g yeast extract, and 15 g agar in 1 L of water. The yeast isolates were grown on this agar at 30 °C for 48 h, and the formation of a clear zone around the colonies was considered a positive result [32].

Esterase Activity: To determine the ability of yeasts to hydrolyze esters, a growth medium was prepared using 10 g Tween 80 (polyoxyethylene-sorbitan monooleate), 5 g NaCl, 0.1 g CaCl₂. 2H₂O, 10 g peptone, and 20 g agar in 1 L of water, adjusted to pH 6. After inoculation, yeast isolates were grown on this agar at 30 °C for 48 h, and the formation of a clear zone around the colonies was considered a positive result [33].

Amylase Activity: For amylase activity, a medium was prepared with 5 g starch, 5 g peptone, 5 g yeast extract, 0.5 g MgSO₄.7H₂O, 0.01 g FeSO₄.7H₂O, 0.01 g NaCl, 20 g agar, and 1000 mL distilled water, and sterilized at 121 °C for 15 min. After incubation (5 days at 30 °C), the medium was flooded with iodine solution, and the appearance of a brown color around the colony was considered a positive result. The iodine solution for Gram’s iodine staining was prepared with 7 g iodine, 3 g KI, and 100 mL distilled water [34].

β-Glucosidase Activity: A mixture containing 5 g arbutin, 10 g yeast extract, 200 mg iron chloride, and 15 g agar was prepared, entirely dissolved in 1 L of distilled water, and sterilized at 121 °C for 15 min. This medium was used to determine the β-glucosidase enzyme activities of the isolates [35]. Pure yeast isolates were inoculated onto the β-glucosidase activity medium and incubated at 27 °C for 2 days. The presence of growth was considered a positive result [31].

2.7. Yeast Adhesion to Hydrocarbons

The ability of yeast isolates to adhere to hydrocarbons was determined according to the method of Rosenberg [36]. The isolates were activated by incubating them in TSB yeast broth at 37 °C for 48 h. Active cultures were centrifuged at 9000 rpm for 15 min, and the resulting pellets were washed with 0.9% NaCl solution. The pellets were resuspended in 0.1 M KNO₃ buffer (pH 6.2) and the optical density (OD) was adjusted to 0.6 ± 0.02 at 600 nm (OD₁) using a spectrophotometer. A 2 mL yeast suspension was layered onto 0.5 mL of p-xylene (a non-polar neutral solvent), chloroform (a monopolar acidic solvent), and ethyl acetate (a monopolar basic solvent) hydrocarbons and incubated at room temperature for 1 h. After a 1-h pre-incubation at room temperature, the two phases were vortexed for 2 min and left at room temperature for another 4 h. After incubation, the OD of the aqueous phase was measured at 600 nm (OD₂) using a spectrophotometer. The microbial adhesion of yeast isolates to hydrocarbons was calculated using the following formula:

% Hydrophobicity = (OD₁ − OD₂)/OD₁ × 100

OD₁ represents the optical density of the yeast cultures, while OD₂ represents the optical density of the mixture after 4 h.

2.8. Determination of Yeast Auto-Aggregation Properties

The auto-aggregation properties of yeasts were determined according to the method of Collado et al. [37]. After activation in SDB broth at 37 °C for 48 h, yeast isolates were centrifuged at 9000 rpm for 10 min. The yeast pellet was washed twice with phosphate buffer (PBS; 0.02% KCl, 1.44% K₂HPO₄, 8% NaCl, 0.24% KH₂PO₄, pH: 7.0) and resuspended in the same buffer. The optical density of the resulting solution was adjusted to 0.6 ± 0.02 at 600 nm. After 2, 4, and 24 h of incubation at room temperature, 1 mL of the upper phase was taken, and the OD was measured at 600 nm. The percentage of yeast auto-aggregation was calculated using the following formula:

% Auto-aggregation= (OD₁ − OD₂)/OD₁ × 100

OD₁ represents the initial optical density, while OD₂ represents the optical density after 2, 4, and 24 h.

2.9. Determination of Yeast Co-Aggregation Properties

The co-aggregation abilities of yeast isolates with Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028, Staphylococcus aureus ATCC 25923, and Listeria monocytogenes ATCC 7644 strains were determined according to the method of Rinkinen et al. [38]. The isolates were activated by incubating them in SDB broth at 37 °C for 48 h. Active cultures were centrifuged at 9000 rpm for 10 min, and the yeast pellets were washed twice with PBS (pH 7.0) and resuspended in the same buffer. The OD of each sample was adjusted to 0.6 ± 0.02 at 600 nm. Equal amounts of yeast culture (2 mL) and bacterial test culture (2 mL) were mixed in the same tube, vortexed for 10–15 s, and incubated at room temperature for 24 h. After incubation, 1 mL of the upper phase was taken, and the OD values were measured at 600 nm. The co-aggregation percentages were calculated using the following formula:

%Co-aggregation = (ODA + ODB − 2ODC)/(ODA + ODB) × 100

ODA represents the yeast culture’s optical density, ODB represents the optical density of the test bacteria, and ODC represents the optical density of the mixed culture after 24 h.

2.10. Determination of Antimicrobial Properties of Yeasts

The antagonistic activity of yeast strains against E. coli ATCC 25922, E. coli O157:H7, B. cereus, C. sakazakii ATCC 29544, L. monocytogenes ATCC 7644, S. aureus ATCC 25923, and S. typhimurium ATCC 140828 was determined using the well diffusion assay [39]. The isolated probiotic yeast strains were grown in TSB yeast broth at 25 °C for 48 h to test antimicrobial activity. The yeast cultures were centrifuged at 9000 rpm for 20 min at 4 °C. Supernatants were filtered through a 0.22 µm filter (Merck, Darmstadt, Germany) to remove cellular debris. The pH of the yeast supernatants was adjusted to 7 using a 1.0 N NaOH solution. To analyze whether the antimicrobial activity was due to secondary metabolites, proteinase K (100 µg/mL) was added to denature peptides and incubated at 37 °C for 1 h. The supernatants were tested for antagonistic activity against the mentioned pathogens. Pathogens were spread onto Mueller–Hinton agar (Becton, Dickinson & Co., Franklin Lakes, NJ, USA). Wells with a diameter of 3 mm were created in the agar, and 30 µL aliquots of the sterile-filtered supernatant were pipetted into each well. Plates were incubated at 4 °C for 2 h and then at 37 °C for 12 h, after which the diameters of the inhibition zones were measured.

2.11. Determination of Yeast Antibiotic Resistance

The yeast strains selected for antibiotic tolerance testing were activated in TSB yeast broth at 25 °C for 24 h. Active cultures were adjusted to a density equivalent to 2 McFarland (6 × 10⁸ CFU/mL) and plated onto OGYEA plates using the spread plate method by inoculating 100 µL onto each plate. Antibiotic susceptibility testing was conducted using erythromycin (15 µg), gentamicin (10 µg), chloramphenicol (30 µg), amoxicillin (20 µg), ampicillin (10 µg), trimethoprim (5 µg), and tetracycline (30 µg) antibiotic disks (Oxoid) via the disk diffusion method. Plates were incubated at 25 °C for 24 h. At the end of the incubation period, the diameters of the zones around the disks were measured in millimeters. The resistance of yeast isolates to antibiotics was categorized as Resistant (R), Intermediate (I), or Sensitive (S) based on the Clinical and Laboratory Standards Institute criteria (son I, 2015) [40].

2.12. Determination of Yeast Antioxidant Activity

Antioxidant Activity of Intact Cells and Intracellular Extracts

The antioxidant activity of yeasts was assessed in two ways: using intact cells and intracellular free extracts. For intact cells, yeasts incubated in TSB yeast broth at 25 °C for 24 h were centrifuged (5000 g, 10 min, 4 °C). The resulting pellets were washed twice with PBS buffer (pH 7.4, OD600 nm 0.400 ± 0.05) and resuspended. For intracellular free extracts, intact cell suspensions were disrupted via ultrasonication (Sonics, VCX750, Newtown, Pennsylvania, USA). (37 kHz, 30 min). The cells were separated by centrifugation (5000 g, 10 min, 4 °C), and the resulting supernatants were considered intracellular free extracts [41].

The antioxidant activity of yeasts was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method [42]. Briefly, 2 mL of intact cells or intracellular extracts were mixed with 2 mL of freshly prepared DPPH (0.2 mM in methanol), incubated in the dark for 30 min, and centrifuged (5000 g, 10 min). The absorbance of the supernatant was measured at 517 nm. Samples prepared without yeast served as controls. The DPPH radical scavenging ability was calculated using the following formula:

% DPPH =1 − OD(Sample)/(OD (Contol)) × 100

2.13. Yeast Identification via MALDI-TOF MS

Yeast identification was conducted using a MALDI-TOF Bruker Microflex system (Bruker Daltonics, Inc., Bremen, Germany) through the direct colony method. A single colony grown on YGC agar was transferred onto a 96-well plate in a spiral motion. A 1 µL aliquot of 70% formic acid stock solution was added to each spot and allowed to dry at room temperature. Subsequently, 1 µL of the matrix solution (α-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 1.5% trifluoroacetic acid) was applied and dried again. The prepared spots were analyzed using a MicroFlex LT mass spectrometer, and the results were interpreted with MALDI Biotyper 3.0 software. The analysis was repeated if the identification score was <1.7 [43].

2.14. Identification of Yeasts via 18S-28S rRNA

DNA isolation from cultured yeast isolates was performed using the EZ1 DNA Tissue Kit with the BioRobot EZ1 DNA isolation device (Qiagen, Hilden, Germany). The extracted DNA was amplified using ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) primers [44]. The amplified PCR products covered the ITS1-5.8S rRNA gene and partial regions of the ITS2-18S and 28S rRNA genes. Amplicons were sequenced using the BigDye Terminator v3.1 cycle sequencing kit on an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The sequences obtained were compared with other isolates in the NCBI database using the BLAST (Basic Local Alignment Tool) server (http://www.ncbi.nlm.nih.gov (assessed on 1 November 2024))

2.15. Statistical Analysis

All analyses were performed in duplicate with at least two replicates. The results’ mean values and standard deviations were calculated and presented in tables. One-way analysis of variance (ANOVA) and Student’s t-test were conducted using the SPSS statistical program to determine significant differences (p ≤ 0.05).

3. Results

3.1. Isolation of Yeasts from Samples

Twenty-three strains with different colony characteristics were isolated from six homemade natural cheeses, butter, kefir, yogurt, and string cheese samples. The probiotic properties of these isolates were investigated. The source of the isolates, their numbering, and colony morphologies are shown in

Table 1. Sources, isolate numbering, and colony morphologies of isolated yeasts.

Source	Isolate	Colony Morphology
Cottage Cheese	1	Large, white, round
Cottage Cheese	2	Medium, white
Cottage Cheese	3	Medium, white, round
Cottage Cheese	4	Large, white, round
Cottage Cheese	5	Small, white
Karın Kaymağı Cheese	6	Large, white, round
Karın Kaymağı Cheese	7	Large, white
Karın Kaymağı Cheese	8	Small, white, round
Karın Kaymağı Cheese	9	Small, white, rough, round
Karın Kaymağı Cheese	10	Large, white
Butter	11	Large, white, rough, round
Butter	12	Medium, white, rough
Butter	13	Medium, white, round
Kefir	14	Large, yellow, rough, round
Kefir	15	Medium, white, rough
Kefir	16	Small, white, round
Kefir	17	Medium, white, round
Yogurt	18	Medium, yellow, round, black center
Yogurt	19	Large, white, rough, round
Yogurt	20	Large, white, rough, round
String Cheese	21	Large, white, round
String Cheese	22	Large, white
String Cheese	23	Small, yellow, round

None of the isolates exhibited β-hemolytic activity; all showed γ-hemolytic activity, indicating that their use is safe as they do not exhibit hemolytic activity. Small colonies were less than 1 mm in diameter, medium colonies were 1 mm in diameter, and large colonies were greater than 1 mm in diameter.

.

3.2. Survival Capacity of Yeasts Under Gastrointestinal Conditions

Under simulated gastrointestinal conditions, the yeasts showed good survival. The survival rates under intestinal conditions simulated with the addition of bile salts and pancreatin are provided in

Table 2. Survival rates of yeast isolates in gastric and pancreatic conditions.

Isolate	Gastric (%)	Pancreatic (%)
1	50.8 ± 0.1	28.1 ± 0.2
2	47.2 ± 0.0	21.6 ± 0.1
3	00.0 ± 0.0	00.0 ± 0.0
4	80.0 ± 0.2	13.2 ± 0.0
5	80.1 ± 0.1	16.2 ± 0.0
6	79.0 ± 0.0	19.1 ± 0.1
7	65.2 ± 0.1	29.2 ± 0.0
8	57.4 ± 0.1	32.2 ± 0.0
9	77.8 ± 0.0	16.1 ± 0.1
10	84.5 ± 0.1	15.2 ± 0.0
11	64.0 ± 0.1	32.1 ± 0.2
12	39.0 ± 0.0	30.7 ± 0.1
13	84.8 ± 0.1	17.8 ± 0.1
14	79.4 ± 0.0	14.8 ± 0.2
15	36.0 ± 0.1	33.9 ± 0.0
16	49.9 ± 0.1	34.1 ± 0.0
17	90.0 ± 0.2	43.2 ± 0.0
18	38.1 ± 0.0	31.4 ± 0.0
19	53.9 ± 0.1	22.6 ± 0.2
20	48.6 ± 0.0	25.3 ± 0.6
21	50.6 ± 0.4	26.0 ± 0.1
22	72.8 ± 0.1	18.8 ± 0.4
23	65.4 ± 0.4	26.4 ± 0.1

. The strains tested in this study exhibited survival potential under gastrointestinal conditions after simulated gastric and intestinal passage exposure. It was concluded that the yeasts we obtained were more resistant to gastric conditions than pancreatic fluids. Strain 3 showed no ability to survive in gastric and pancreatic conditions. When all strains were examined, 30.4% showed survival in gastric conditions below 50%.

For probiotic strains, it is necessary to pass through the stomach and intestines. The pH of the stomach ranges from 2.5 to 3.5, and yeasts can survive across a wide range of pH levels.

3.3. Biofilm Formation Properties of Yeasts

None of the isolates were classified as strong biofilm producers. Of the isolates, 13.04% were classified as moderate biofilm producers, 34.78% as weak biofilm producers, and 52.17% showed no biofilm formation ability (Table 3).

3.4. Enzymatic Activities of Yeasts

The enzymatic activity results of our isolates are provided in Table 3.

Table 3. Results of biofilm formation and enzymatic activities of isolates.

Isolate	Biofilm Formation ¹	Protease 2	Esterase 2	Amylase 2	β-Glucosidase 2
1	0.066 (−)	1 (+)	1 (+)	3 (+)	0 (−)
2	1.088 (+ +)	1 (+)	1 (+)	1 (+)	0 (−)
3	0.697 (+)	1 (+)	1 (+)	1 (+)	0 (−)
4	0.891 (+ +)	1 (+)	1 (+)	1 (+)	7 (+ +)
5	0.767 (+ +)	1 (+)	1 (+)	1 (+)	0 (−)
6	0.225 (−)	3 (+)	1 (+)	1 (+)	0 (−)
7	0.368 (+)	2 (+)	1 (+)	1 (+)	10 (+ +)
8	0.184 (−)	1 (+)	2 (+)	1 (+)	0 (−)
9	0.707 (+)	1 (+)	1 (+)	2 (+)	12 (+ +)
10	0.460 (+)	4 (+ +)	1 (+)	1 (+)	15 (+ +)
11	0.154 (−)	1 (+)	1 (+)	1 (+)	9 (+ +)
12	0.249 (−)	1 (+)	1 (+)	1 (+)	0 (−)
13	0.156 (−)	2 (+)	1 (+)	2 (+)	14 (+ +)
14	0.444 (+)	1 (+)	1 (+)	1 (+)	15 (+ +)
15	0.260 (−)	2 (+)	5 (+ +)	1 (+)	0 (−)
16	0.090 (−)	1 (+)	3 (+)	1 (+)	12 (+ +)
17	0.024 (−)	3 +	1 +	1 +	18 ++
18	0.376 (+)	2 (+)	1 (+)	1 (+)	17 (+ +)
19	0.566 (+)	4 (+ +)	3 (+)	2 (+)	0 (−)
20	0.261 (−)	5 (+ +)	1 (+)	1 (+)	0 (−)
21	0.349 (−)	2 (+)	1 (+)	1 (+)	0 (−)
22	0.520 (+)	2 (+)	1 (+)	5 (+ +)	9 (+ +)
23	0.084 (−)	1 (+)	1 (+)	1 (+)	12 (+ +)

¹ Biofilm formation: non-producer (−), weak producer (+), moderate producer (+ +), strong producer (+ ++). ² Zone formation: no zone (−), zone/colony diameter < 3 mm (+), zone/colony diameter > 3 mm (+ +).

Table 3. Results of biofilm formation and enzymatic activities of isolates.

Isolate	Biofilm Formation ¹	Protease 2	Esterase 2	Amylase 2	β-Glucosidase 2
1	0.066 (−)	1 (+)	1 (+)	3 (+)	0 (−)
2	1.088 (+ +)	1 (+)	1 (+)	1 (+)	0 (−)
3	0.697 (+)	1 (+)	1 (+)	1 (+)	0 (−)
4	0.891 (+ +)	1 (+)	1 (+)	1 (+)	7 (+ +)
5	0.767 (+ +)	1 (+)	1 (+)	1 (+)	0 (−)
6	0.225 (−)	3 (+)	1 (+)	1 (+)	0 (−)
7	0.368 (+)	2 (+)	1 (+)	1 (+)	10 (+ +)
8	0.184 (−)	1 (+)	2 (+)	1 (+)	0 (−)
9	0.707 (+)	1 (+)	1 (+)	2 (+)	12 (+ +)
10	0.460 (+)	4 (+ +)	1 (+)	1 (+)	15 (+ +)
11	0.154 (−)	1 (+)	1 (+)	1 (+)	9 (+ +)
12	0.249 (−)	1 (+)	1 (+)	1 (+)	0 (−)
13	0.156 (−)	2 (+)	1 (+)	2 (+)	14 (+ +)
14	0.444 (+)	1 (+)	1 (+)	1 (+)	15 (+ +)
15	0.260 (−)	2 (+)	5 (+ +)	1 (+)	0 (−)
16	0.090 (−)	1 (+)	3 (+)	1 (+)	12 (+ +)
17	0.024 (−)	3 +	1 +	1 +	18 ++
18	0.376 (+)	2 (+)	1 (+)	1 (+)	17 (+ +)
19	0.566 (+)	4 (+ +)	3 (+)	2 (+)	0 (−)
20	0.261 (−)	5 (+ +)	1 (+)	1 (+)	0 (−)
21	0.349 (−)	2 (+)	1 (+)	1 (+)	0 (−)
22	0.520 (+)	2 (+)	1 (+)	5 (+ +)	9 (+ +)
23	0.084 (−)	1 (+)	1 (+)	1 (+)	12 (+ +)

¹ Biofilm formation: non-producer (−), weak producer (+), moderate producer (+ +), strong producer (+ ++). ² Zone formation: no zone (−), zone/colony diameter < 3 mm (+), zone/colony diameter > 3 mm (+ +).

3.5. Adhesion of Yeasts to Hydrocarbons

The hydrocarbon adhesion values of the yeasts are provided in

Table 4. Adhesion properties of yeasts to hydrocarbons.

Isolate	Xylene (%)	Chloroform (%)	Ethyl Acetate (%)
1	35.7 ± 0.0	28.0 ± 0.0	33.2 ± 0.1
2	14.0 ± 0.4	46.2 ± 0.3	50.7 ± 0.2
3	13.5 ± 0.4	44.7 ± 0.1	52.6 ± 0.1
4	31.7 ± 0.8	56.1 ± 0.4	45.3 ± 1.3
5	39.6 ± 0.1	47.3 ± 0.0	43.3 ± 0.2
6	27.4 ± 5.5	33.1 ± 1.0	54.6 ± 1.3
7	20.9 ± 0.4	32.6 ± 0.4	49.2 ± 0.1
8	23.5 ± 1.3	32.0 ± 0.4	65.1 ± 0.1
9	19.2 ± 0.0	6.0 ± 0.0	0.0 ± 0.0
10	32.7 ± 0.6	21.2 ± 0.9	41.9 ± 0.2
11	26.7 ± 4.7	7.3 ± 0.4	65.9 ± 1.2
12	20.2 ± 0.0	5.6 ± 0.0	0.0 ± 0.0
13	36.0 ± 0.4	25.0 ± 0.3	45.5 ± 0.2
14	69.1 ± 0.2	24.1 ± 0.4	71.8 ± 0.1
15	34.0 ± 0.0	38.5 ± 0.0	49.4 ± 0.0
16	71.8 ± 0.3	24.9 ± 1.1	70.2 ± 0.4
17	36.6 ± 0.4	37.3 ± 0.2	34.2 ± 3.9
18	15.5 ± 0.0	12.2 ± 0.0	0.30 ± 0.4
19	25.8 ± 0.6	28.1 ± 0.3	46.3 ± 1.0
20	17.2 ± 1.0	26.6 ± 1.2	40.7 ± 0.3
21	24.6 ± 0.7	16.1 ± 2.6	17.2 ± 0.6
22	22.9 ± 0.1	23.7 ± 0.6	25.2 ± 0.2
23	00.3 ± 0.0	00.0 ± 0.0	15.6 ± 0.0

.

3.6. Auto-Aggregation Values of Yeasts

The auto-aggregation values of the yeast isolates are provided in

Table 5. Auto-aggregation results of yeast strains (%).

Isolate	2 h	4 h	24 h
1	19.9 ± 0.2	41.8 ± 0.1	79.6 ± 2.2
2	17.3 ± 0.0	42.2 ± 0.0	90.0 ± 0.0
3	14.0 ± 0.0	33.6 ± 0.2	82.2 ± 0.0
4	16.9 ± 0.0	32.1 ± 0.1	77.7 ± 0.0
5	10.8 ± 0.1	30.9 ± 0.0	86.1 ± 0.0
6	15.2 ± 0.0	34.1 ± 0.0	79.5 ± 0.0
7	12.6 ± 0.0	37.9 ± 0.0	82.5 ± 0.0
8	19.5 ± 0.0	56.9 ± 0.0	88.2 ± 0.0
9	10.1 ± 0.1	30.1 ± 0.0	88.6 ± 0.0
10	9.1 ± 0.0	27.4 ± 0.2	87.4 ± 0.0
11	31.2 ± 0.1	70.1 ± 0.0	94.1 ± 0.0
12	11.7 ± 0.0	32.0 ± 0.1	75.1 ± 1.2
13	28.5 ± 0.2	62.1 ± 0.0	93.7 ± 0.0
14	43.1 ± 0.2	71.9 ± 0.1	91.5 ± 0.0
15	9.1 ± 0.2	13.8 ± 0.0	64.5 ± 0.8
16	26.2 ± 0.2	56.6 ± 0.0	88.6 ± 0.0
17	25.3 ± 0.5	47.0 ± 0.0	100.0 ± 0.0
18	18.1 ± 0.3	35.7 ± 0.0	75.9 ± 0.0
19	9.1 ± 0.0	28.5 ± 0.0	85.7 ± 0.0
20	10.8 ± 0.0	33.8 ± 0.0	82.4 ± 0.1
21	7.7 ± 0.0	29.4 ± 0.0	88.9 ± 0.0
22	9.3 ± 0.1	29.6 ± 0.0	91.6 ± 0.0
23	5.2 ± 0.0	21.9 ± 0.1	73.8 ± 0.0

. In our study, it was observed that auto-aggregation percentages increased with longer incubation times for all strains. The auto-aggregation value for a probiotic yeast should be at least 30–60% [11,18]. After 24 h, the auto-aggregation value exceeded 60% for all isolates. As seen in Table 5, the highest auto-aggregation percentage in the isolates was observed after 24 h, with values approaching 100%. This is desirable for probiotic microorganisms.

3.7. Co-Aggregation Values of Yeasts

The co-aggregation abilities of yeast strains with E. coli ATCC 25922 were found to range from 0% to 95.7%, with S. typhimurium ATCC 14028 ranging from 0% to 91.9%, S. aureus ATCC 25923 from 0% to 95.1%, and L. monocytogenes ATCC 7644 from 0% to 88.7% (

Table 6. Co-aggregation results of yeast strains (%).

Isolate	E. coli ATCC 25922	S. typhimurium ATCC 14028	S. aureus ATCC 25923	L. monocytogenes ATCC 7644
1	81.9 ± 0.1	50.0 ± 0.1	87.0 ± 0.2	57.8 ± 0.0
2	89.9 ± 0.0	66.6 ± 0.0	88.1 ± 0.0	71.9 ± 0.0
3	89.4 ± 0.0	63.8 ± 0.1	86.5 ± 0.0	71.1 ± 0.0
4	88.7 ± 0.0	69.1 ± 0.0	87.8 ± 0.0	72.7 ± 0.0
5	82.6 ± 0.3	60.8 ± 0.0	85.6 ± 0.0	69.7 ± 0.0
6	94.3 ± 0.0	71.9 ± 0.0	90.5 ± 0.0	85.0 ± 0.1
7	84.4 ± 0.0	79.0 ± 0.1	87.3 ± 0.0	74.4 ± 0.0
8	90.6 ± 0.0	72.5 ± 0.1	90.5 ± 0.0	77.4 ± 0.0
9	85.9 ± 0.0	72.2 ± 0.0	89.4 ± 0.0	75.8 ± 0.0
10	85.1 ± 0.0	73.6 ± 0.0	89.2 ± 0.0	74.0 ± 0.0
11	87.1 ± 0.0	74.3 ± 0.0	91.5 ± 0.0	84.5 ± 0.0
12	74.7 ± 0.1	53.8 ± 0.0	78.8 ± 0.0	66.2 ± 0.0
13	87.4 ± 0.0	74.3 ± 0.0	94.0 ± 0.0	87.4 ± 0.0
14	91.3 ± 0.2	74.6 ± 0.2	93.5 ± 0.0	83.9 ± 0.0
15	56.1 ± 0.0	56.5 ± 0.0	80.5 ± 0.1	59.6 ± 0.1
16	89.8 ± 0.0	72.6 ± 0.0	93.8 ± 0.0	85.3 ± 0.0
17	86.6 ± 0.0	69.9 ± 0.0	80.7 ± 0.0	76.8 ± 0.0
18	72.7 ± 0.2	61.3 ± 0.0	87.0 ± 0.1	69.8 ± 0.0
19	87.0 ± 0.0	75.0 ± 0.0	95.1 ± 0.0	78.7 ± 0.0
20	88.7 ± 0.0	71.4 ± 0.0	88.4 ± 0.1	79.3 ± 0.1
21	87.4 ± 0.0	73.1 ± 0.0	89.3 ± 0.0	73.0 ± 0.1
22	86.1 ± 0.0	66.6 ± 0.0	85.4 ± 0.0	71.7 ± 0.0
23	82.5 ± 0.0	49.5 ± 0.0	80.5 ± 0.0	54.0 ± 0.1

).

This study concluded that the co-aggregation ability is strain- and incubation-time dependent. All tested strains exhibited co-aggregation ability with E. coli ATCC 25922, S. typhimurium ATCC 14028, S. aureus ATCC 25923, and L. monocytogenes ATCC 7644.

3.8. Determination of Antimicrobial Properties of Yeasts

The antimicrobial properties of yeasts are presented in

Table 7. Antimicrobial zone diameters (mm) of yeasts against pathogenic bacteria.

	E. coli	E. coli	B.cereus	C. sakazakii	L. mono.	S. aureus	S. typhimurium
İsolate	ATCC 25922	O157H7		ATCC 29544	ATCC 7644	ATCC 25923	ATCC 140828
1	15.5 ± 1.5	12.7 ± 2.2	00.0 ± 0.0	12.0 ± 0.0	10.5 ± 2.5	00.0 ± 0.0	12.0 ± 1.0
2	16.5 ± 1.5	00.0 ± 0.0	00.0 ± 0.0	00.0 ± 0.0	12.5 ± 2.5	00.0 ± 0.0	13.0 ± 2.0
3	10.0 ± 2.0	00.0 ± 0.0	12.0 ± 2.0	20.0 ± 0.0	14.0 ± 3.0	00.0 ± 0.0	00.0 ± 0.0
4	15.0 ± 1.0	00.0 ± 0.0	11.0 ± 1.0	00.0 ± 0.0	14.5 ± 2.5	00.0 ± 0.0	00.0 ± 0.0
5	13.0 ± 4.0	00.0 ± 0.0	00.0 ± 0.0	03.5 ± 3.5	14.2 ± 4.2	12.0 ± 2.0	15.0 ± 2.0
6	15.0 ± 5.0	00.0 ± 0.0	00.0 ± 0.0	13.7 ± 1.7	17.5 ± 7.5	00.0 ± 0.0	0.0 ± 0.0
7	17.5 ± 3.5	00.0 ± 0.0	00.0 ± 0.0	10.5 ± 0.5	15.0 ± 6.0	00.0 ± 0.0	07.2 ± 5.2
8	20.5 ± 6.5	00.0 ± 0.0	00.0 ± 0.0	11.0 ± 0.0	14.0 ± 0.0	00.0 ± 0.0	10.5 ± 4.5
9	18.5 ± 0.5	00.0 ± 0.0	00.0 ± 0.0	10.5 ± 2.5	13.0 ± 6.0	00.0 ± 0.0	12.5 ± 4.5
10	12.7 ± 1.2	12.0 ± 4.0	00.0 ± 0.0	22.5 ± 0.5	15.5 ± 0.5	10.0 ± 1.0	11.5 ± 1.5
11	13.0 ± 1.0	15.5 ± 4.5	00.0 ± 0.0	10.0 ± 6.0	15.0 ± 3.0	12.0 ± 1.0	00.0 ± 0.0
12	14.0 ± 5.0	13.2 ± 1.2	00.0 ± 0.0	00.0 ± 0.0	15.0 ± 1.0	00.0 ± 0.0	12.5 ± 6.5
13	12.5 ± 0.5	17.2 ± 8.2	00.0 ± 0.0	25.0 ± 0.0	14.5 ± 0.5	00.0 ± 0.0	11.2 ± 0.7
14	12.7 ± 0.2	18.2 ± 1.7	00.0 ± 0.0	00.0 ± 0.0	14.5 ± 0.5	00.0 ± 0.0	14.0 ± 6.0
15	20.5 ± 1.5	12.0 ± 2.0	00.0 ± 0.0	00.0 ± 0.0	13.0 ± 1.0	00.0 ± 0.0	14.5 ± 2.5
16	12.5 ± 0.5	16.7 ± 0.2	00.0 ± 0.0	11.0 ± 5.0	15.5 ± 3.5	11.0 ± 1.0	14.5 ± 1.5
17	12.0 ± 0.0	00.0 ± 0.0	00.0 ± 0.0	14.0 ± 0.0	14.0 ± 0.0	15.0 ± 0.0	00.0 ± 0.0
18	14.0 ± 1.0	12.7 ± 3.2	00.0 ± 0.0	11.0 ± 1.0	14.0 ± 6.0	00.0 ± 0.0	11.2 ± 2.7
19	14.0 ± 0.0	14.7 ± 0.7	00.0 ± 0.0	11.0 ± 3.0	15.0 ± 0.0	00.0 ± 0.0	00.0 ± 0.0
20	20.7 ± 1.2	00.0 ± 0.0	00.0 ± 0.0	10.0 ± 0.0	15.5 ± 5.5	10.0 ± 0.0	17.0 ± 0.0
21	20.0 ± 6.0	15.7 ± 3.2	00.0 ± 0.0	11.0 ± 0.0	15.0 ± 0.0	11.0 ± 1.0	12.7 ± 0.2
22	15.0 ± 0.0	20.0 ± 6.0	00.0 ± 0.0	17.5 ± 1.5	11.5 ± 3.5	12.0 ± 2.0	15.7 ± 0.7
23	15.5 ± 4.5	15.0 ± 1.0	00.0 ± 0.0	10.0 ± 1.0	14.0 ± 2.0	00.0 ± 0.0	00.0 ± 0.0

. Most of the yeasts we obtained showed antimicrobial effects against E. coli ATCC 25922, E. coli O157H7, B. cereus, C. sakazakii ATCC 29544, L. monocytogenes ATCC 7644, S. aureus ATCC 25923, and S. typhimurium ATCC 140828.

3.9. Determination of Antibiotic Resistance of Yeasts

The results obtained in this study showed that all isolated yeast strains demonstrated resistance to all tested antibiotics. This property enables probiotic yeasts to prevent diarrhea by preserving the intestinal microbiota without being affected by antibiotics prescribed during illness. The evaluation was based on breakpoints established by the NCCLS (National Committee for Clinical Laboratory Standards, 2000).

3.10. Determination of Antioxidant Activity of Yeasts

The antioxidant activity values of the obtained yeast strains are presented in

Table 8. Antioxidant activity of yeasts (%).

Isolate	Intact Cells (%)	Intracellular Extracts (%)
1	15.8 ± 0.5	88.4 ± 0.1
2	133.8 ± 0.1	15.3 ± 0.0
3	145.4 ± 0.0	28.3 ± 0.0
4	45.4 ± 0.0	47.9 ± 0.0
5	36.35 ± 0.0	47.9 ± 0.0
6	42.33 ± 0.0	59.5 ± 0.0
7	47.84 ± 0.0	19.1 ± 0.0
8	54.06 ± 0.1	ND
9	52.87 ± 0.0	79.2 ± 0.0
10	42.74 ± 0.1	23.7 ± 0.0
11	36.62 ± 0.0	23.3 ± 0.1
12	50.37 ± 0.0	63.2 ± 0.0
13	37.20 ± 0.0	ND
14	37.61 ± 0.0	69.0 ± 0.0
15	39.96 ± 0.0	73.1 ± 0.0
16	36.76 ± 0.0	ND
17	4.90 ± 2.0	2.17 ± 0.0
18	13.72 ± 0.1	64.5 ± 0.0
19	36.76 ± 0.1	0.0 ± 1
20	13.72 ± 0.2	6.5 ± 0.2
21	21.69 ± 0.1	36.0 ± 0.0
22	24.98 ± 0.0	1.5 ± 0.1
23	22.61 ± 0.1	4.8 ± 0.2

ND: Not determined.

. In all tested yeast strains, the antioxidant activity of intact cells was higher than that of intracellular extracts.

3.11. Identification of Yeasts Using MALDI-TOF and 18S-28S rRNA

The results obtained from the identification of yeasts using MALDI-TOF and 18S-28S rRNA are provided in

Table 9. Results of yeasts identified by MALDI-TOF.

Isolate	MALDI-TOF MS	MALDI-TOF MS Score	Accession No	Identify (%) with GenBank Database
1	Torulaspora delbrueckii	1.954	NR_111257.1	98
2	Candida parapsilosis	1.987	NR_130673.1	99
3	Candida parapsilosis	1.965	NR_130673.1	97
4	Metschnikowia pulcherrima	1.987	NR_164379.1	97
5	Candida parapsilosis	1.964	NR_130673.1	96
6	Pichia kudriavzevii	1.974	NR_131315.1	97
7	Pichia kudriavzevii	1.963	NR_131315.1	96
8	Saccharomyces cerevisiae	1.973	NR_111007.1	96
9	Pichia membranifaciens	1.985	NR_111195.1	97
10	Pichia kudriavzevii	2.054	NR_131315.1	97
11	Saccharomyces cerevisiae	2.147	NR_111007.1	98
12	Pichia membranaefaciens	1.994	NR_111195.1	95
13	Saccharomyces cerevisiae	2.057	NR_111007.1	96
14	Saccharomyces cerevisiae	2.231	HM191645.1	97
15	Pichia membranaefaciens	2.124	NR_111195.1	98
16	Saccharomyces cerevisiae	1.963	HM191658.1	99
17	Saccharomyces boulardii	1.987	MT449154.1	98
18	Kloeckera apiculata	2.140	NR_130660.1	97
19	Pichia kudriavzevii	2.120	NR_131315.1	96
20	Pichia kudriavzevii	1.963	NR_131315.1	95
21	Pichia kudriavzevii	1.987	NR_131315.1	96
22	Pichia kudriavzevii	1.974	NR_131315.1	97
23	Kloeckera apiculata	1.985	NR_130660.1	98

.

4. Discussion

This study evaluated yeasts isolated from various dairy products for their probiotic and functional characteristics. The results indicate that these yeasts have significant potential as probiotics, demonstrating survival capabilities under gastrointestinal conditions, biofilm formation abilities, and antimicrobial activities.

Few yeast species have survived in highly acidic environments, such as pH 1.5 [45]. Bonatsou et al. [28] found that 42 of 49 yeast strains showed over 50% survival during simulated gastric and pancreatic digestion. Similarly, in our study, 15 of 22 yeast isolates survived at a rate of 50% or more in gastric juice. Tolerance to in vitro gastric and pancreatic digestion is critical for potential probiotic candidates.

The study showed that most isolated yeasts could survive simulated gastric and intestinal conditions, with many isolates exhibiting survival rates exceeding 50%. This highlights their potential use as probiotic cultures in fermented foods. The ability to endure low pH levels and bile salts is crucial for probiotics, as they must survive through the gastrointestinal tract to confer health benefits [45].

In addition to survival, the ability of yeast to form biofilms is vital for probiotic effectiveness. Biofilms enhance yeast adhesion to the intestinal mucosa, facilitating colonization and improving gut health. The isolates in this study exhibited varying degrees of biofilm production, with some strains classified as moderate to weak biofilm producers. The capacity for biofilm formation can be influenced by the production of exopolysaccharides, which are essential for adhesion to the intestinal epithelium [15].

Biofilm formation is an indicator of cell adhesion potential. The limited biofilm-forming capacity of the yeasts is likely due to their low ability to produce exopolysaccharides. The potential for intestinal colonization can also be evaluated through biofilm formation. Biofilms play an important role in the close relationship between the host and resident microorganisms in the intestines, and biofilm formation is a key prerequisite for intestinal colonization and adhesion of some probiotics to the intestinal mucosa. The co-aggregation and biofilm formation of probiotic yeasts have been reported to provide significant benefits. Okamoto et al. [46] investigated the biofilm-forming ability of the S. cerevisiae BY4741 strain, explaining that the negatively charged mannan compound in the yeast cell wall is responsible for co-aggregation. There is a correlation between biofilm formation and adhesion to Caco-2 cells, and high adhesion may suggest a role in modulating the immune system [47].

Enzymes produced by yeasts used in food play a crucial role in metabolizing complex compounds in foods, enhancing fermented foods’ nutritional value and organoleptic properties [48]. Esterase and lipase improve the aromatic profile of fermented foods by increasing the content of free fatty acids, which are precursors of various aromatic compounds [49]. Lipase and protease contribute to the digestion of lipids and proteins in the gastrointestinal system, supporting probiotic potential [40]. Amylase is responsible for the hydrolysis of polysaccharides, facilitating the breakdown of starch and glycogen in the human diet, and thus contributing to digestion [50]. The production of proteolytic enzymes by yeasts can benefit the host, as these enzymes have been reported to break down toxins [51]. Therefore, the production of proteolytic enzymes may protect the host from infections caused by enteropathogenic bacteria such as Salmonella, Vibrio, Clostridium, E. coli, and Bacillus species [52]. Lipolytic enzymes are precursors for forming various aromatic compounds, such as free fatty acids, ethanol, glycerol, higher alcohols, esters, and other volatile compounds. Another important enzyme is β-glucosidase, which significantly produces secondary metabolites and phenolic compounds and significantly impacts flavor [53]. For these reasons, high enzymatic activity is desirable in probiotic yeasts. Ogunremi et al. [15] identified Pichia kluyveri, Issatchenkia orientalis, Pichia kudriavzevii, and Candida tropicalis as yeasts with high protease, lipase, and phytase activity.

The capacity to adhere to hydrocarbons is related to cell surface hydrophobicity and determines the ability to adhere to the intestinal epithelium. While hydrophobicity alone is insufficient for epithelial adhesion, it should be evaluated alongside aggregation properties. Increased hydrophobicity is associated with more significant health benefits, as probiotic adhesion to the intestinal surface prevents pathogens from colonizing [54]. Probiotics that adhere to the intestinal surface compete for nutrients and inhibit pathogen growth by producing organic acids and antimicrobial compounds [55]. Additionally, it was found that hydrophobicity varies depending on the type of hydrocarbon; 36.36% of the yeasts adhered to xylene and chloroform, while 72.72% adhered to ethyl acetate, with more than 30% adhesion observed to hydrocarbons.

Ogunremi et al. [15] observed that the auto-aggregation rate increased with incubation time in all isolates. These results are similar to our findings. When the auto-aggregation activities of yeast isolates obtained from pineapple peel and pulp were examined, it was found that the auto-aggregation capacity of all samples increased from less than 16% after 2 h of incubation to 96% after 24 h [56]. Similar to our study, Gil-Rodriguez et al. [11] reported high levels of variability in yeast cultures after 2 h of incubation, with significant increases in aggregation percentages after 4 h of incubation. Auto-aggregation is influenced by several factors, such as the physicochemical properties of the cell surface, the presence of Ca + 2 ions in the environment, the presence of mannose, the incubation time of the inoculated culture, or differences in applied methods [57].

Ogunremi et al. [15] reported co-aggregation activities ranging from 57% to 71% in yeast strains isolated from traditional fermented grain-based food products in Nigeria.

The antimicrobial activity of the isolated yeasts against various pathogenic bacteria indicates their potential role in food preservation and health promotion. The isolates displayed significant inhibitory effects against pathogens such as E. coli, B. cereus, and S. aureus, supporting their use in functional foods. Previous studies have similarly reported the antimicrobial properties of yeasts, reinforcing the idea that they can enhance food safety and extend shelf life [40,58].

The antimicrobial activities of yeasts extend the shelf life of foods. Additionally, the competitive advantage of probiotic strains in preventing pathogen colonization in the intestines is crucial. Syal and Vohra [40] determined the antimicrobial activity of yeasts isolated from traditional fermented foods. Their study found that S. cerevisiae and C. tropicalis strains exhibited antimicrobial activity against Salmonella sp. and S. aureus. Ragavan and Das [58] reported that five of twenty yeast isolates from various food sources showed antimicrobial activity against Bacillus sp., E. coli sp., and Staphylococcus sp. bacteria.

The antibiotic resistance exhibited by the isolated yeasts is particularly noteworthy, as it suggests that these probiotics may help maintain a healthy gut microbiota even in the presence of antibiotics. This resistance is advantageous, allowing probiotic yeasts to flourish in environments where antibiotics are used, thus preventing dysbiosis and associated gastrointestinal issues.

Ragavan and Das [58] reported that among the twenty yeast isolates they obtained from various food sources, five exhibited resistance to antibiotics including ampicillin (20 μg/mL), gentamicin (10 μg/mL), ketoconazole (10 μg/mL), levofloxacin (10 μg/mL), tetracycline (30 μg/mL), tigecycline (20 μg/mL), and trimethoprim (20 μg/mL).

Furthermore, the antioxidant activity of the yeasts indicates their potential health benefits, particularly in mitigating oxidative stress-related diseases. The intact yeast cells demonstrated higher antioxidant capacities than intracellular extracts, suggesting that whole yeast cultures may be more effective for health promotion. This aligns with previous research indicating that yeasts can produce a variety of bioactive compounds with antioxidant properties [59].

Recently, studies have been initiated on the antioxidant activities of yeasts due to the wide variety of metabolites they produce. For instance, superoxide dismutase, catalase, carotenoids, resveratrol, exopolysaccharides, and lipids—particularly (1→3)-D-β-glucans—are believed to reduce the risk of pathological processes in the host by scavenging free radicals. The results indicate that intact cells possess a higher antioxidant capacity than extracts. This is likely attributed to the high content of (1/3)-β-D-glucans and other β-glucans present in the cell walls, which are believed to be the main contributors to the antioxidant activity of yeast [59]. Probiotic microorganisms may protect against gastric ulcers, obesity, cardiovascular diseases, and chronic diseases due to their antioxidant effects [60].

The identification of yeast strains using MALDI-TOF and 18S-28S rRNA analysis confirmed the presence of species known for their probiotic potential. This identification is crucial for the future application of these yeasts in food products, as specific strains may exhibit different health benefits.

5. Conclusions

In conclusion, the results of this study demonstrate the promising probiotic potential of yeasts isolated from dairy products. Their ability to survive gastrointestinal conditions, form biofilms, exhibit antimicrobial activities, show antibiotic resistance, and provide antioxidant benefits position them as valuable candidates for use in functional foods. Further research is necessary to elucidate the mechanisms underlying these properties and assess these yeasts’ in vivo effects on human health.