Pichia kudriavzevii UNJCC Y-137 and Candida tropicalis UNJCC Y-140 isolated from Durio kutejensis as potential probiotic agents

Abstract

Durio kutejensis, commonly known as Lai durian, has a unique characteristics of a creamy texture and a combination of sweet and bitter tastes. This study aimed to isolate and screen yeast from fruits as a potential probiotic agent. The tests consisted of tolerance to bile salt and gastric acid at pH 2, antibacterial activity against Listeria monocytogenes and Salmonella enteriditis, and hemolytic activity on blood agar medium. The results showed that 40 yeasts isolated from Lai durian fruit and 34 of these isolates grew on YMA medium. The two isolates showed high significance in the probiotic tests. These two isolates were able to grow on bile salt up to a concentration of 2% and gastric acid for up to 6 h, with survival rates of 99.06% and 100%, respectively. Two isolates were identified as Pichia kudriavzevii UNJCC Y-137 and Candida tropicalis UN-JCC Y-140 Therefore, these two yeast isolates can be used as potential probiotic agents.

Introduction

Probiotics are defined as living microorganisms that are beneficial to the health of the host when given in sufficient quantities (FAO/WHO, 2006). Probiotics can grow in the human digestive tract and stimulate the immune system by inhibiting the growth of pathogens in the body (Amorim et al., 2018; Marteau et al., 2002). Probiotics can adhere to and colonize the human digestive tract by competing with pathogenic bacteria (Amorim et al., 2018; Moradi et al., 2018; Oliveira et al., 2017). They produce killer toxins that can neutralize pathogenic bacterial toxins (Punder et al., 2015), and induce the formation of Immunoglobulin A (IgA), and stimulate the activation of macrophages (Collado et al., 2009). Therefore, consumption of probiotics in sufficient amounts is proven to give advantages for human health.

Until today, the exploration of probiotic agents has been limited to certain microorganisms. Most probiotics in current use are lactic acid bacteria (LAB), particularly from the genera Lactobacillus, Enterococcus, and Bifidobacterium, and non-pathogenic Saccharomyces boulardii (Alvarez and Oberhelman, 2001; Martins et al., 2005; Moradi et al., 2018; Sharma et al., 2021; Zullo et al., 2019). However, for microorganisms to be classified as probiotic groups, they should meet certain requirements and have some features. These requirements include the AA's high tolerance activity during stressful conditions in the human body (Wendel, 2022), indicated by the presence of enzymes in the oral cavity such as amylase and lysozyme, as well as the gastric juice which has a pH condition that ranges from 2.5 to 3.5 (Horáčková et al., 2018; Ogunremi et al., 2015). According to Kumar et al. (2012), microorganisms should also be resistant to intestinal bile salts to be able to grow and colonize the intestines. More importantly, probiotic microorganisms must have adhesion ability, antipathogenic activity, and safety for consumption, as well as pass the clinical trials and show no haemolytic activity (De Melo Pereira et al., 2018).

Currently, researchers have started to explore the broader types of microorganisms that have potential as probiotic agents (Sen and Mansell, 2020; Staniszewski and Kordowska-Wiater, 2021). Yeasts hold promise as probiotic agents for several compelling reasons, including their ability to grow in a wide pH range of 4.5 to 6.5, even under extreme conditions as low as pH 1.5, their antibiotic resistance, and their ability to colonize the digestive tract (Caselli et al., 2013; Czerucka et al., 2007; França et al., 2015). Yeast can be found in various substrates, including fruits (Sukmawati et al., 2021a, b; Wulandari et al., 2018), plant surfaces (Limtong et al., 2014; Sukmawati et al., 2015), flowers (Risandi et al., 2019), and fermented foods such as cheese, wine, and kefir (Amorim et al., 2018; Cho et al., 2018; Martins et al., 2005). Among various substrates, fruits serve as a significant habitat for yeast growth due to their abundant concentration of simple sugars, providing an ideal environment for yeast proliferation (Glushakova and Chernov, 2004; Sukmawati et al., 2020a, b).

The definition of probiotics has been widely understood, namely products (food) that contain live microorganisms with clear characteristics, sufficient quantities and capable of providing benefits to the body. Various products in the probiotic category have been circulating on the market. Even globally, in this pandemic season, the market for probiotic products, which are believed to improve the body's immune system, is increasing. Probiotics can be marketed in the form of fermented food or after being processed as powder, then integrated into food ingredients to produce functional food. Now, new terms have emerged, namely parabiotics and postbiotics. Until now, there is no clear definition for both of them. But we still want to write down the main differences between the two.

Paraprobiotics, tyndalized probiotic (heat inactivated probiotic), are heated first to make the cells non-viable. Dead probiotic cells (intact or lysed) still have health benefits, especially through their cell walls (peptidoglycan), which can stimulate the immune system. However, several researchers say this par probiotic is mixed with supernatant, which contains various important metabolites, such as SCFA, antibacterial, and enzymes.

Meanwhile, postbiotics are metabolites produced by probiotics that also benefit intestinal health, often called cell-free supernatant. Metabolites such as SCFA, especially butyrate, are vital in maintaining gut health. A healthy intestine can also support a strong immune system. Other metabolite products are antibacterials, such as bacteriocins, which can suppress the growth of pathogens in the intestine. However, several papers state that postbiotics are also mixed with cells that have been lysed. An important thing that needs to be considered in postbiotic production is the composition of the media so that important metabolites are obtained during fermentation.

Earlier investigations have successfully delved into the capacity of yeast strains derived from specific fruits to act as probiotic agents. Nasir et al. (2017) found the probiotic characteristics of Saccharomyces cerevisiae isolated from citrus fruits based on its ability to grow at pH 2 and in bile salt concentrations of up to 1%. In addition, Meyerozyma caribbica originated from pineapple was found to tolerate up to 1% bile salt (Amorim et al., 2018), while S. boulardii taken from grape substrate exhibited gastric acid and antibiotic resistance (Dixit et al., 2009). In this context, conducting studies on potential probiotic yeasts isolated from fruit substrates is of paramount importance, as it not only expands our understanding of yeast ecology but also holds promise for identifying novel probiotic candidates with unique properties.

Lai fruit (Durio kutejensis) is one of the edible species of the genus Durio that has the potential to be explored for its yeast ecology and probiotic characteristics. This fruit is a type of Durian endemic to Kalimantan, Indonesia (Sukmawati et al., 2021a, b) that is notable for its nutritional content. Lai durian is rich in protein of 1.94–2.93%, carbohydrates of 31.65–42.05%, sugar content ranging from 11 to 19%, and an acid pH value of 4–5, hence constituting a good substrate for yeast growth. Despite its uniqueness and regional significance, Lai fruit is not widely recognized by the general public, especially in areas outside of Kalimantan (Hadi et al., 2013). Researching on Lai fruit is still limited to the aroma, taste, and nutritional values such as fat, carbohydrates, and protein (Belgis et al., 2017), while information about the existence of yeast originating from this fruit, especially its potential as a probiotic agent is still rare.

Therefore, it is necessary to research the yeast diversity living on fruit as Lai and examine their potential probiotic characteristics. In the broader context, these research findings not only enhance our understanding of probiotics but also highlight the potential of exploring diverse and unconventional natural sources, emphasizing that probiotic research extends beyond traditional avenues. It underscores the notion that nature may harbour numerous undiscovered beneficial microbes with implications for human health. Furthermore, this study offers a window into the intricate interactions between host organisms, such as humans, and their resident microbes, contributing to a deeper comprehension of gut health and the microbiome. This study aimed to isolate and identification from Lai durian, then determined the activity yeasts as probiotic by depending on some important tests resembling stress conditions in the human digestive tract such as bile salt tolerance, gastric acid tolerance, activity against pathogenic antibacterial activity, and haemolytic activity.

Materials and methods

Yeast isolation from Durio kutejensis

A total of six Lai fruit (D. kutejensis) were collected from four different locations in Bukit Sawit Village, Central Kalimantan, Indonesia. Isolation was done using washing method and direct method (Sukmawati et al., 2015; Sukmawati et al., 2020a, b). Every fruit sample was cut into small pieces with approximate dimension of 1 × 1 × 1 cm, of which 1 g was then added to a 9 mL of yeast–peptone–dextrose (YPD) broth containing 1% yeast extract, 2% peptone, and 2% dextrose to a total volume of 10 mL with pH 4.5. The mixture was subsequently homogenized using a 120 rpm shaker for 24 h, producing a suspension, 100 μL of which was then spotted onto the surface of YMA (yeast malt agar) containing 10% dextrose and chloramphenicol (50 µg/mL), and incubated at 30 °C for 2 days for observation. For the direct method, the pulp samples from the same homogenized suspension were taken and placed directly onto the YMA medium and incubated under the same conditions. All the resulting colonies with yeast-like morphology were stored at the Universitas Negeri Jakarta Culture Collection (UNJCC), preserved using the L-drying method and 10% glycerol at − 20 °C.

Initial screening of yeast isolates on YMA pH 2 medium

Ayyash et al. (2021), reported that the selection of potential probiotic strains can be determined by performing the physiological capacity of strains in the gastrointestinal tract through the acid tolerance test, bile metabolism and tolerance test, and adhesion capability test. Ayyash et al. (2021) reported that potential probiotic strains can be selected by evaluating their physiological capacity in the gastrointestinal tract through the acid tolerance test, bile metabolism and tolerance test, and adhesion capability test. In this study, the yeast isolates were screened for their ability to grow at pH 2 using a YMA medium that was added with HCl. The yeasts were inoculated on YMA medium containing peptone (5%), yeast extract (3%), malt extract (3%), dextrose (10%), and agar (20%), with a pH of 2 to obtain yeast isolates which have potentials as probiotic candidates. A total of 40 yeast isolates from Lai fruit were inoculated by streak method onto the YMA in duplicates. Finally, the colony morphology such as thickness, colony margin, and colony texture were observed during the 24 h incubation at 28 °C. The colony features such as texture, colour, surface, profile, the edge of the colony, their budding types, and cell shapes were also observed using a phase contrast microscope (Olympus, Olympus Corporation, Tokyo, Japan) at × 400 magnification.

Molecular identification of yeast isolates

To determine the specific genetic identity and phylogenetic relationships of the yeast isolates, molecular identification was performed. In this study, yeast identification is conducted using the specific D1/D2 region of the 26S rDNA gene, which is the common region used to identify yeast isolates. D1/D2 26S rDNA region sequencing facilitates earlier and more reliable identification of yeast than phenotypic methods, and this region has been used by many researchers to identify yeasts from different resources (Walker, 2009). Molecular identification was performed for the two selected isolates using forward primer NL1 (5′-GCATATCAATAAGCGGAGAAAG-3′) and reverse primer NL4 (5′-GGTCCGTGTTTCAAGACGG-3′) based on Makene (2014) for DNA amplification. The procedure is conducted as follows. Each colony of the yeast isolates was inoculated into a YMA medium and incubated overnight at 37 °C. DNA was extracted as described previously by Sukmawati et al. (2015), and 3 µL of DNA template was added with nuclease-free water (8.5 µL), Go Taq Green Mastermix (12.5 µL), NL1 forward primer (0.5 µL), NL4 reverse primer (0.5 µL) for PCR reaction. PCR conditions were set for 35 cycles as follows: pre-denaturation (95 °C for 2 min), post-denaturation (95 °C for 30 s), annealing (58 °C for 30 s), elongation (72 °C for 1 min), final elongation (72 °C for 10 min), and extension (4 °C) for final condition. Visualization of PCR products was performed using electrophoresis with 1% agarose gel and 1 × TAE buffer (Tris acetate EDTA) based on Sambrok and Russel l (2001) and sent to First Base DNA sequencing services. The sequence data was then edited using the ChromasPro version 2.6.2 application program followed by analysis using the Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov). The phylogenetic tree was constructed using the MEGA 7 application program with 1000 times bootstrap with the neighbour-joining method (Tamura et al., 2013).

Tolerance activity test of yeast isolates to bile salts

From the initial screening, two potential yeast isolates were chosen to perform further tests. The tolerance activity of yeast isolates to bile salts was carried out based on the modification of the García-Hernández et al. (2012) method. In this study, the test was carried out by varying the bile salt concentrations (0, 0.5, 1.0, 1.5, and 2.0%) and S. cerevisiae UNJCC Y-117 was used as a positive control. The selection of S. cerevisiae UNJCC Y-117 as a control strain is based on its genetics and physiological capability as a probiotic agent (Abid et al., 2022). Saccharomyces cerevisiae is a widely recognized and extensively studied yeast species, often used as a model organism in exploring probiotic yeast from various substrates.

A total of 15 yeast streak isolates were inoculated on yeast peptone dextrose agar (YPDA) slant agar medium (3 g yeast extract; 5 g peptone; 10 g glucose; 20 g agar; and 200 mg chloramphenicol for every 1000 mL of the medium) followed by incubation at 27 °C for 48 h. Serial dilution method was performed using 5 mL of phosphate buffer saline (PBS) solution (8 g sodium chloride; 0.2 g potassium chloride; 1.44 g disodium phosphate; 0.24 g potassium phosphate and dissolved in 1000 mL) then homogenized using vortex for 1 min, until it reaches an absorption value of 1 at 600 nm using a spectrophotometer (≅ 10⁷ CFU/mL cell density) (Sherman, 2003). Yeast cell suspension (0.1 mL) was spread onto YPDA medium (3 g yeast extract; 5 g peptone; 10 g glucose; 20 g agar; and 200 mg chloramphenicol for every 1000 mL of medium) which contained bile salts with a concentration of 0 (control), 0.5, 1.0, 1.5, and 2.0%. The tolerance activity was estimated by the percentage of yeast isolates treated with bile salts compared to those not given the bile salts (Ogunremi et al., 2015). A survival rate of > 70% indicated that the yeast isolate has tolerance activity to bile salt and considered as a potential probiotic candidate (Pennacchia et al., 2008). The survival rate is calculated by the following equation:

$$\text{Survival rate (\%)=}\frac{\frac{\text{CFU}}{\text{mL}}\text{treatment}}{\frac{\text{CFU}}{\text{mL}}\text{control}}\times \text{100.}$$

Tolerance activity test of yeast isolates to gastric acid

A tolerance activity test for gastric acid was carried out based on Amorim et al. (2018) with modification of incubation time variations (0, 2, 4, and 6 h). A total of 15 yeast streaks were inoculated on YPDA medium for 48 h incubation at 28 °C. Serial dilution was subsequently performed in the same way as described for the bile salt tolerance activity test. The yeast cell suspension was then centrifuged at 4500 rpm, 28 °C for 10 min. The obtained biomass was washed twice using 10 mL of PBS solution (PBS g/L: sodium chloride 8.0; potassium chloride 0.2; disodium phosphate 1.44; potassium phosphate 0.24 with the addition of 600 µL 1 M HCl). A total of 1% (v/v) yeast suspension was put into 9 mL of PBS pH 2 followed by incubation with different duration at 37 °C. After incubation, 0.1 mL of the cell suspension was inoculated into YPDA medium using the spread plate method. The number of yeast colonies in each treatment and replication was counted after 24 h incubation at 37 °C. Finally, the survival rate is calculated based on Ogunremi et al. (2015). The survival rate of > 70% indicated the isolate has the potential to become a probiotic (Pennacchia et al., 2008).

Antibacterial activity test of yeast isolates against pathogenic bacteria

Antibacterial activity test was performed based on Amorim et al. (2018). The yeast isolates were tested for their antibacterial activity against Salmonella enteritidis ATCC 5190 and Listeria monocytogenes with 0.8–0.9 absorbance values at a spectrophotometric wavelength of 600 nm (≅ 10⁷ CFU/mL cell density) (Yuliani et al., 2018). Twenty microlitres yeast cell suspension with 10⁷ cells/mL density was inoculated into YPDA medium using a Drugalsky spatula. The antibacterial test was carried out by pouring 10 mL of nutrient agar (0.7% agar) which was added with 1 mL of pathogenic bacteria, into a Petri dish containing yeast isolates. After 24-h incubation at 37 °C, the antibacterial activity was observed for the presence of a clear zone around the yeast colony.

Hemolytic activity of yeast isolates

The hemolytic activity of the yeasts isolated from Lai fruit was determined using the procedure described by Foulquié et al. (2003) with some modifications. The test was conducted using a blood agar medium containing 15 g/L agar, 10 g/L beef extract, 10 g/L peptone, 5 g/L NaCl and supplemented with 5% (v/v) sheep's blood. Yeast suspension (20 µL) with spectrophotometric absorbance of 1.0 at 600 nm (≅ 10⁷ cells/mL cell density) was inoculated into a well-drilled blood agar medium. They were then incubated at 28 °C and checked after 48 h to examine the hemolytic activity. If there was a clear zone tending toward the colour and transparency of the base medium in the area around the colony, the hemolysis was considered positive (Buxton, 2005). On the other hand, if a reaction was not observed in the surrounding medium, it indicated a lack of hemolysis.

Statistical analysis

The data obtained in the bile salt tolerance test and stomach acid tolerance were analyzed by using SPSS V17 software. Two-way analysis of variance (ANOVA) was employed. The significance in statistic analysis was set at P ≤ 0.05, means performed using Duncan Multiple Range Test (DMRT) 5%.

Results and discussion

Yeast isolates obtained from Lai fruit (D. kutejensis) and initial screening results in YMA medium pH 2

A total of forty yeast isolates were successfully obtained from three Lai fruits, with 14 isolated from the first fruit, 10 from the second fruit, and 16 from the third fruit. Following the initial screening, 34 yeast isolates demonstrated growth in the medium at pH 2, representing an ultra-acidic condition. Macroscopic and microscopic observations revealed that the colonies were predominantly white (100%), with the majority displaying a butyrous, rough surface (79.51%), while the remaining exhibited a mucoid, smooth surface (20.59%). Among these isolates, two strains that exhibited extensive growth at pH 2, coded DU 1.23 (with a butyrous, rough surface) and DU 3.2 (with a mucoid, smooth surface) were chosen for further testing. These isolates were subsequently identified as UNJCC Y-137 and UNJCC Y-140, respectively, afterwards. According to Alkalbani et al. (2022), yeast isolates that exhibited growth in ultra and extremely acidic environments are recognized as potential probiotics, given that the majority of yeast strains did not demonstrate growth under such highly acidic conditions.

Molecular identification result of yeast isolates

The D1/D2 26S rDNA sequencing using NL1 and NL4 primers on the two yeast isolates resulted in a PCR product of 500 bp for each strain. The alignment result showed that the two isolates, which were then classified as UNJCC Y-140 and UNJCC Y-137, had the closest homology to Candida tropicalis ATCC 750 (99.83%) and Pichia kudriavzevii NRRL Y 5396 (99.66%), respectively (Table 1). The phylogenetic tree showed that the two yeast isolates belonged to the order Saccharomycetales which is the only order from the class Saccharomycetes. The order Saccharomycetales has asci free during sexual reproduction and are not enclosed in ascomata (fruiting bodies). Isolate UNJCC Y-140 is in a monophyletic clade with C. tropicalis with a bootstrap value of 94 while isolate UNJCC Y-137 is in a monophyletic clade with P. kudriavzevii with a bootstrap value of 100 (Fig. 1). The bootstrap value in the phylogenetic tree shows the topology of the tree formed. The confidence in the resulting phylogenetic tree is substantiated by bootstrap values ranging from 70 to 100%, in line with the assessment provided by Simpson (2006).

Table 1.

BLAST analysis result of isolates UNJCC Y-140 and UNJCC Y-137 based on D1/D2 rDNA region

Isolate codes	BLAST result	Max score	Query cover (%)	E-value	Accession	Identity	Gaps (%)
UNJCC Y-140	Candida tropicalis ATCC 750	1090	96	0.0	NG_054834.1	99.83%	0/593 (0%)
UNJCC Y-137	Pichia kudriavzevii NRRL Y-5396	1064	95	0.0	NG_055104.1	99.66%	0/582 (0%)

Fig. 1.

Phylogenetic tree of isolate UNJCC Y-140 and isolate UNJCC Y-137 based on D1/D2 region using Neighbour Joining method (1000 × bootstrap)

According to its microscopic profile, isolate UNJCC Y-137 was found to be white, butyrous textured, with a rough colony surface. Its colony edge was of pseudohyphae with a mountainous colony profile. It had round, oval cell shapes and monopolar budding. This is in line with the typical P. kudriavzevii that is commonly found in fermented food and fruit resources (Lata et al., 2022). Chamnipa et al. (2018) also discovered a white-coloured colony of P. kudriavzevii with a butyrous surface, flat to lobulated edges, round to oval cells, and monopolar budding. This species, originally known as Issatchenkia orientalis, has different cell shapes (round, oval or elongated). In terms of its potential as a probiotic agent, this yeast species is also able to inhibit the growth of pathogens and resist various types of antibiotics. Current research conducted by Swaruparani and Bhima (2023) has successfully found the probiotic potential of this isolate using in vitro assessment. The research found that P. kudriavzevii has high acid tolerance at pH 2, shows strong antimicrobial activity against some pathogens, can withstand a concentration of 2% of bile salt, and exhibits resistance towards different antibiotics, especially for exopolysaccharides. The isolate was observed to produce several enzymes such as β-galactosidase, protease, amylase, phytase, and lipase (Swaruparani and Bhima, 2023).

While isolate UNJCC Y-137 has a butyrous textured and rough colony surface, isolate UNJCC Y-140 has a mucoid texture with a smooth colony surface, flat colony edge, and mountainous colony profile. It had round cells, monopolar budding, and no pseudohyphae (Table 2). Aspergillus niger was used as an outgroup in the reconstruction of the phylogenetic tree to contrast the diversity. In the context of phylogenetic analysis, the selection of outgroups can be made either randomly or based on a clear relationship between the ingroup and outgroup. Outgroups are taxa closely related to the group of species under study but do not belong to that specific group, as described by Luo et al. (2010) and Staton (2015). While the information on the probiotic potential of P. kudriavzevii is widely discovered, it is still limited for C. tropicalis. This strain is known as pathogen fungi and is a constituent of the human flora, a commensal of the skin, gastrointestinal and genitourinary tracts (Guerra et al., 2020).

Table 2.

Morphological observation of yeasts on YMA medium incubated at 28 °C for 48 h

Isolate codes	Colour	Texture	Surface	Edge	Profile	Cell shape	Budding type	Hyphae
Pichia kudriavzevii UNJCC Y-137	White	Butyrous	Rough	Pseudo-hyphae	Mountainous	Rounded-oval	Monopolar	No
Candida tropicalis UNJCC Y-140	White	Mucoid	Smooth	Flat	Mountainous	Round	Monopolar	No
Saccharomyces cerevisiae
UNJCC Y-117	White	Mucoid	Smooth	Flat	Mountainous	Rounded, oval	Monopolar	No

Probiotic profile of isolate UNJCC Y-140 and UNJCC Y-137 from Lai fruit: bile salt tolerance, gastric acid tolerance, antibacterial activity against pathogen, and haemolytic activity

The number of tested yeast colonies is directly proportional to the survival value, where a higher yeast colony count means a higher survival value, and vice versa. The two-way ANOVA test showed that there was an interaction between yeast isolates and survival percentage in the testing of bile salt tolerance and gastric acid at P =  < 0.05. Isolate S. cerevisiae UNJCC Y-117 has a significantly different interaction between the bile salt concentrations of 1.5 and 2% against the yeast colony count of 4.02 and 0.00 log CFU/mL (Fig. 2). In this study, C. tropicalis UNJCC Y-140 showed the highest colony count of 7.01 log CFU/mL at 0% bile salt concentration, with a survival percentage of up to 100% at 2% bile salt concentration (Fig. 2). This study showed P. kudriavzevii UNJCC Y-137 to have the highest number of colonies (7.16 log CFU/mL) at 0% bile salt concentration, with a survival rate of up to 99.06% at 2% bile salt concentration (Fig. 3). The highest colony count (6.71 log CFU/mL) of the control positive S. cerevisiae UNJCC Y-117 at 0% bile salt concentration dropped to 0% viability as the bile salt concentration was increased to 2% (Fig. 3).

Fig. 2.

Colony number (log CFU/mL) of yeast isolates in 0.5, 1, 1.5, and 2% bile salt incubated at 37 °C for 24 h

Fig. 3.

Effect of incubation time on the number of colonies (log CFU/mL) of Candida tropicalis UNJCC Y-140, Pichia kudriavzevii UNJCC Y-137, and Saccharomyces cerevisiae UNJCC Y-117 at incubation times of 0, 2, 4, and 6 h

Isolate P. kudriavzevii UNJCC Y-137 had the highest colony count of 6.55 log CFU/mL at 2 h of incubation and the count continued to decrease to 5.30 log CFU/mL at 6 h incubation with a survival rate of 78.45, S. cerevisiae UNJCC Y-117 had the highest colony count of 5.99 log CFU/mL at 0 h, 5.64 log CFU/mL at 2 h, and the count continued to decrease to 3.33 log CFU/mL at an incubation time of 6 h with a survival value of 55.64%. Overall, this study showed that the isolate C. tropicalis UNJCC Y-140 and P. kudriavzevii UNJCC Y-137 had a strong survival ability of > 70% that they can be considered having probiotic activity. Isolate C. tropicalis UNJCC Y-140 showed the best survival rate of 95.34 ± 0.59 at pH 2 with an incubation time of 6 h.

The result of the gastric acid tolerance test showed that all the yeast isolates had a slight decrease in growth with longer incubation time in a medium containing gastric acid solution (Fig. 4). Isolate C. tropicalis UNJCC Y-140 had the highest colony count of 6.93 log CFU/mL at 0 h of incubation, slightly decreased to 6.86 log CFU/mL at 2 h, and to 6.61 log CFU/mL at 6 h with a survival rate of 95.34% (Fig. 4).

Fig. 4.

Effect of incubation time on the survival rate (log CFU/mL) of Candida tropicalis UNJCC Y-140, Pichia kudriavzevii UNJCC Y-137, and Saccharomyces cerevisiae UNJCC Y-117 at incubation times of 0, 2, 4, and 6 h

Pichia kudriavzevii UNJCC Y-137 and S. cerevisiae UNJCC Y-117 showed a clear zone formation against S. enteritidis and L. monocytogenes, demonstrating the yeasts’ ability to inhibit the growth of both pathogenic bacteria. Hemolytic activity testing was carried out on the three yeast isolates to determine whether they could lyse erythrocytes on a blood agar medium. Hemolytic test results were grouped into three groups, namely α-hemolytic (a green zone was formed around the colony), β-hemolytic (a clear zone was formed around the colony), or γ-hemolytic (no clear zone was formed around the colony) on blood agar media.

The results showed that the isolates C. tropicalis UNJCC Y-140, P. kudriavzevii UNJCC Y-137 and S. cerevisiae UNJCC Y-117 were categorized as having γ-hemolytic or non-hemolytic activity, which was unable to lyse erythrocytes. Yeasts obtained from food substrates such as fruit, cheese, and kefir, have a very low frequency of hemolytic activity.

According to the sequential tests, it can be seen that both isolate C. tropicalis UNJCC Y-140 and P. kudriavzevii UNJCC Y-137 isolated form Lai fruit have probiotic profile that can be considered as probiotic agents. Yeast can be found from a variety of substrates, including fermented cocoa beans (Sukmawati et al. 2021a, b), Artocarpus heterophyllus (jackfruit) (Wulandari et al. 2018), Moringa oleifera leaves (Sukmawati et al., 2020b) and Cerbera manghas (sea mango) leaves (Sukmawati et al., 2020a). These yeasts could have antagonistic ability against pathogenic molds in postharvest fruit (Sukmawati et al., 2020a). Colony morphology of yeasts both of pigmented and non-pigmented can be found from various substrates (Risandi et al., 2019). Study conducted by Santos et al. (1996) found yeast from the internal tissues of ripe cashews, instead of the unripe fruits. According to Nasreen et al. (2014), fruits contain high concentrations of sugars such as glucose and fructose and therefore yeast species can be easily isolated from fruits. All the yeasts obtained in this study had white-colored colonies, except some of them which showed no pigmentation. The mucoid texture of yeasts is due to the presence of polysaccharide substances contained in their extracellular components (Marham et al., 2017). Goldhawke et al. (2016) added that some yeast species are covered by extracellular components in the form of slimy polysaccharides and heteropolysaccharides. In the research conducted by Sukmawati et al. (2015), it was reported that the colonies of phylloplane yeasts had predominantly peach (Astaxanthin) and cream pigments. Yeasts obtained from the fruit of the pindo palm (Butia capitata), loquat (Eriobotrya japonica), blackberry (Rubus sp.), and hackberry (Celtis sp.) had white- to cream-coloured colonies (Lentz et al., 2014).

It is known that the pigment in yeast is produced as a self-protection function against extreme environmental conditions, such as temperature and UV light exposure (Moline et al., 2010). Yeasts can produce various kinds of pigments such as carotenoids (red to orange) and melanin (black color) (Dufossé, 2016). Research by Moline et al. (2010) reported that the yeast Rhodotorula mucilaginosa could withstand exposure to UV-B rays due to the presence of carotenoid pigments produced by the yeast.

Based on the results of the study 34 yeast isolates showed growth in the medium at pH 2 (ultra-acidic condition). Research by Dixit et al. (2009) obtained yeast from grapes and found 57 isolates of them could grow at pH 2. Amorim et al. (2018) also found 13 yeast isolates from pineapple that could grow at pH 2. The pH value is an environmental factor that can affect the growth of yeast cells. Inappropriate pH prevents yeasts from performing metabolism properly, hence limiting their growth. Lai fruit has an acidic pH ranging from 4 to 5 which can be tolerated by yeasts. In a lowered pH medium, yeast cells can activate an adaptive response to acid conditions and continue to grow after the lag phase. The most common yeast adaptation mechanisms involve plasma membrane transporters and pro-ton translocating ATPases (Dorighetto et al., 2020). The plasma membrane transporter Pdr12p is inducible by acidic compounds. The accumulation of Pdr12p in the plasma membrane is dependent on the transcription factor War1p. In earlier research, Martínez-Muñoz and Kane (2008) found that the presence of Pdr12p will balance the pH inside and outside of yeast cells, enabling yeasts to grow in low pH environments.

Crucially, the appropriate selection of probiotic microorganisms is contingent upon their tolerance to bile salt concentrations ranging from 0.5 to 2% (Czerucka et al., 2007). These concentrations closely mimic those found in the human intestine, which typically ranges from 1 to 40 mmol/L or 0.05 to 2% under normal physiological conditions (Islam et al., 2011), and can reach 0.5 to 2% when in contact with food (Cho et al., 2018). Bile fluid, originating from hepatocyte cells, constitutes a yellow–green liquid composed of cholesterol, phospholipids, biliverdin pigment, and bile acids (Horáčková et al., 2018; Kumar et al., 2012). Notably, some studies have indicated that concentrations of 1.5–2% are present only in the initial hour of digestion, gradually decreasing to 0.3% over time (Cho et al., 2018).

This study revealed that both C. tropicalis UNJCC Y-140 and P. kudriavzevii UNJCC Y-137 exhibited tolerance to bile salt concentrations, with a survival rate exceeding 70%. These findings suggest their potential as probiotic agents, as previously noted by Pennacchia et al. (2008). Furthermore, distinct ultrastructural characteristics set this yeast apart from the majority of other Ascomycetes, as highlighted by Suh et al. (2006). Additionally, P. kudriavzevii demonstrated the ability to remain metabolically active even under extreme conditions, withstanding temperatures as high as 45 °C and maintaining viability at pH levels as low as 2, 3, or 4, as documented by Kurtzman and Fell (1998). This yeast species is commonly found in fruits such as apples and pears, as reported by Vadkertiova et al. (2012). Previous research, including the work of Ramachandran et al. (2008), has explored this yeast potential as a probiotic agent, showcasing its resistance to bile salts, pepsin, and pancreatic enzymes.

While information regarding P. kudriavzevii UNJCC Y-137 is widely spread, research on the probiotic potential of C. tropicalis UNJCC Y-140 is still limited. This yeast grows optimally at 25–35 °C (Kurtzman and Fell, 1998). However, current research conducted by Ogunremi et al. (2015) and Saber et al. (2019) considered yeast C. tropicalis as a potential probiotic agent because of its resistance to concentrations of gastric acid and bile salts, and its ability to produce digestive enzymes such as lipase, esterase, and phytase. The observed characteristics of Candida, including its white colonies, round shape, smooth surface, and vegetative reproduction through budding, align with the descriptions provided by Talaro and Chess (2012). Typically, Candida colonies on solid media exhibit a round, convex, smooth surface and display white to cream colours. They reproduce vegetatively through budding without engaging in sexual reproduction, consistent with findings reported by Deorukhkar (2018) and Zuza-Alves et al. (2017).

The ability of yeast to survive in bile salt is closely related to the bile salt hydrolase (BSH) enzyme which is produced by microorganisms such as S. cerevisiae. This enzyme will deconjugate bile salts into secondary bile salts (litholic acid and deoxycholic acid) which are poorly absorbed by the intestine and, thus, are excreted through feces. Ogunremi et al. (2015) reported that only 10.41% C. tropicalis was only able to survive 0.5% bile salt concentrations and lost its viability at 1–2% bile salt concentrations. Ogunremi et al. (2015) revealed that the survival of yeast P. kudriavzevii declined from 92.99 to 66.66% as the bile salt concentration changed from 0.3 to 2%. A similar study conducted by Syal and Vohra (2013) showed that the yeast type S. cerevisiae was only able to survive up to 1% bile salt concentration with 93% survival value. According to Chen et al. (2010), the ability of probiotic microorganisms to survive through the digestive tract varies and depends on the type of microbial strain. Amorim et al. (2018) reported that yeast isolates from pineapples showed different resistance when grown in environments containing 0.1 and 1% bile salts. This study showed C. tropicalis UNJCC Y-140 and P. kudriavzevii UNJCC Y-137 having tolerance up to a concentration of 2% bile salts with a survival rate above 70%, suggesting they can be considered as potential probiotic agents (Pennacchia et al., 2008).

This phenomenon contributes to the reduction of serum cholesterol levels because the replacement of excreted bile acids necessitates the synthesis of new bile salts, a process that relies on cholesterol as its precursor, as elucidated by Hofmann and Mysels (1992). Notably, Tanaka et al. (2000) noted that the molecular weight and structure of the BSH enzyme are strain-dependent, highlighting variability in its characteristics. Furthermore, according to Ortiz et al. (1997), the resilience of S. cerevisiae to bile salt conditions can be attributed to the presence of an ATP-binding cassette (ABC protein) encoded by the bile acid transporter (bat1) gene within the yeast. This protein plays a pivotal role in the translocation of bile salts and exhibits efficient transport capabilities for conjugated bile salts. In the context of this study, the relatively low survival rate of S. cerevisiae UNJCC Y-117 may be attributed to the lower production or activity levels of the BSH enzyme and potential structural differences in the enzyme itself.

The modest reduction in both colony numbers and the survival rate of yeast isolates observed in this study can likely be attributed to their exposure to extreme acidic conditions. This exposure may have compromised the yeasts' ability to maintain pH stability within their cells, consequently diminishing cell viability. It is worth noting that the capacity of probiotic microorganisms to withstand the challenges of the digestive tract, including low pH, varies and is contingent on the specific yeast isolates, as highlighted by Moline et al. (2010). Furthermore, Brandão et al. (2014) conducted a comprehensive investigation involving various Saccharomyces strains, revealing that the composition and levels of fatty acids in cell membranes played a role in determining their tolerance to low pH. In certain yeast strains, there was an observed increase in the synthesis of oleic acid and arachidic acid following exposure to an acidic environment (pH 2) for 30 min.

The survival of yeasts at pH 2 can be attributed to their intracellular pH regulatory system, as elucidated by Brandão et al. (2014). Yeasts effectively maintain an intracellular pH higher than that of their extracellular environment through the action of the ATPase enzyme, which translocates protons (H⁺ ions) from the interior to the exterior of the cell. This process relies on the energy generated from ATP hydrolysis, as described by De Oliveira Coelho et al. (2019). In a study conducted by Saber et al. (2019), it was observed that C. tropicalis could endure a pH of 2 for up to 3 h, resulting in a colony count of 6.86 log CFU/mL and a survival rate of 89%. Similarly, research on P. kudriavzevii by Ramachandran et al. (2008) revealed that this yeast strain could survive at pH 2 for an extended period of up to 24 h, with a colony count of 8.41 log CFU/mL and a survival rate of 92%. This resilience enables yeasts to thrive in environments characterized by extremely low pH levels.

Notably, exposure to acidic conditions can lead to the entry of protons into yeast cells, causing cytoplasmic acidification and subsequently inhibiting the yeast's metabolic processes, as demonstrated in a study by Martínez-Muñoz and Kane (2008). To counteract this acidification, yeast activates the Pma1p proton pump in the plasma membrane, effectively expelling protons to maintain a balanced pH within the cell. Furthermore, gastric acid research conducted by Pennacchia et al. (2008) indicated that S. cerevisiae exhibited a colony count of 7.41 log CFU/mL at the start of incubation, and after 2 h of incubation in the presence of gastric acid, the growth slightly decreased to 7.00 log CFU/mL, with a notable survival rate of 94.4%. This underscores the yeast's capacity to withstand the challenges posed by gastric acid conditions.

In this study, C. tropicalis showed no clear zone between the colonies. Research conducted by Saber et al. (2019) showed that C. tropicalis inhibited the growth of L. monocytogenes with an inhibitory diameter of 21.9 mm. Perricone et al. (2014) reported that Candida sp. showed no inhibition against the pathogenic bacteria L. monocytogenes, Yersinia enterocolitica, Escherichia coli O157:H7, and Staphylococcus aureus. Similarly, Amorim et al. (2018) tested Candida sp. and found no inhibitory activity against the pathogenic bacteria E. coli ATCC 055, S. enteritidis ATCC 5190, S. aureus ATCC 8702, Bacillus cereus ATCC 14579, L. monocytogenes ATCC 11778 and Pseudomonas aeruginosa ATCC 27853.

One of the important characteristics of probiotic yeasts is their antibacterial activity against pathogenic bacteria that can penetrate the mucosal lining of the digestive tract. These results were similar to those by Syal and Vohra (2013) in which S. cerevisiae inhibited the growth of Salmonella sp., S. aureus, Pseudomonas sp., and Vibrio sp. The study by Saber et al. (2019) also reported that the yeast P. kudriavzevii could inhibit the pathogenic bacteria L. monocytogenes with an inhibition zone of 23.5 mm. Yeast can inhibit the growth of pathogenic bacteria because it can produce killer toxin, an extracellular toxin produced by yeast to kill other cells (Santos et al., 2009). The maximum toxin production ability of yeasts depends on the type of strain, genetic makeup, and the environmental conditions used during growth (Ratnaningtyas et al., 2019). Bajaj et al. (2013) revealed that the yeast P. kudriavzevii RY55 had a killer toxin in the form of a 39.8 kDa protein which inhibited the growth of Escherichia coli, Enterococcus faecalis, Klebsiella sp., S. aureus, P. aeruginosa, and Pseudomonas alcaligenes.

El-Banna et al. (2011) reported S. cerevisiae possesses a killer toxin in the form of a 19 kDa protein that destroys the target cell wall by attaching to the receptor and translocating to the cytoplasmic membrane. As a result, the cytoplasmic membrane function of the target cell was disrupted. S. cerevisiae is also known to inhibit the growth of pathogenic bacteria such as P. aeruginosa, S. aureus, and Salmonella sp. (Lentz et al., 2014). Yeast from the genus Saccharomyces was shown to have a 54-kDa serine protease that degraded toxins from the bacterium Clostridium difficile that causes colitis (Czerucka and Rampal, 2002).

Assessing hemolytic activity is a critical safety consideration in the selection of probiotic microorganisms. γ-Hemolytic yeasts, which cannot partially or completely lyse blood agar media, do not produce a zone on the media. To fully evaluate the probiotic potential of yeast isolates P. kudriavzevii UNJCC Y-137 and C. tropicalis UNJCC Y-140, further analyses such as adhesion testing and clinical testing are necessary to meet the criteria for probiotic agents. Pathogenic yeasts typically exhibit a reaction with blood agar media, resulting in the formation of a distinct zone around growing yeast colonies. In contrast, non-pathogenic yeast tests reveal no clear zone around the colonies, as demonstrated by Estifanos (2014). Additionally, a hemolytic activity test on P. kudriavzevii was conducted by De Oliveira Coelho (2019), who reported that this yeast strain was unable to form a clear zone on the blood agar medium.

Conclusion

In this study, we have successfully isolated some yeasts from Lai durian originating from Kalimantan, Indonesia. Two isolates showed efficacy in living and growing in conditions similar to the human intestine and exhibited a tolerance activity to gastric acid pH 2 and bile salt. In addition, one yeast isolate UNJCC Y-137 showed inhibition activity against two pathogenic bacteria but both isolates demonstrated no hemolytic activity as shown by no clear zone in the blood agar medium. Based on the profile of bile salt tolerance, gastric acid tolerance, activity against pathogenic antibacterial activity, and haemolytic activity, it can be seen that both isolates have potential as probiotic agents. This study holds significant importance in advancing our understanding of probiotic microorganisms, especially yeast that is isolated from indigenous fruit. This research is a contribution towards the knowledge of the potential role of microorganisms in enhancing the immune system. However, further investigation and substantiation are potential for supporting our assumption these strains to be considered as probiotic.

Author contributions

All authors had equal contributions as the main contributors to this manuscript paper. Conceptualization, DS and AS; methodology, DS, SS, SMA; software, CS, LHT; validation, HAE, and ZZ; formal analysis, DS, RHBS; investigation, SNA, ZNA; resources, SS; data curation, CS; writing—original draft preparation, DS, HAE, RHBS, SNA, MN; writing—review and editing, RHBS, SNA; visualization; supervision, LHT; project administration, DS; funding acquisition, RAR. All authors have read and agreed to the published version of the manuscript.

Declarations

Conflict of interest

The authors declare no conflict of interest.