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.
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.