Isolation and screening of yeast isolates
For the isolation of yeast isolates from traditional mango pickle, sample of pickle was homogenized with 0.9% NaCl solution and serial dilutions were plated on Yeast Malt agar (peptone—0.5%, yeast extract—0.3%, malt extract—0.3%, dextrose—1%, and agar—2.5%) procured from HiMedia, Mumbai, India, which was enriched with ampicillin (0.05 g/L) and acidified with 1N HCl to pH 5.0 to prevent bacterial growth. Plates were incubated at 30 °C for 48 h. Isolates were screened for different parameters (i.e., morphological, biochemical tests, carbohydrate fermentation (API 20C AUX), tolerance to acidic conditions and antimicrobial activity). Acid tolerance of the isolates was studied according to the method of Yu et al. [51]. Overnight grown yeast isolates were harvested (10,000 × g, 5 min, 4 °C), washed twice with PBS (phosphate buffer saline) buffer (pH 7.0). The cell pellet was then resuspended into PBS adjusted to pH 2, 3, and 7 (control) and incubated at 30 °C for 3 h. Survival of yeast isolates was calculated in terms of log cfu/mL. Antimicrobial effect of yeast isolates against three gram positive bacteria (Bacillus cereus MTCC 1272, Staphylococcus aureus subsp. aureus MTCC 96, Listeria monocytogenes MTCC 657) and five gram negative bacteria (Pseudomonas aeruginosa MTCC 424, Escherichia coli MTCC 118, Shigella, Salmonella typhi Aeromonas hydrophilla) were examined by agar well diffusion method [31]. Zone of inhibition was measured as millimeters (mm). Finally, yeast isolate Y33 was selected on the basis of acid tolerance and best antimicrobial activity for further study.
Molecular identification of yeast isolate
The selected isolate was identified through isolation and amplification of the ITS DNA region at Biologia Research India Pvt. Ltd., New Delhi, India. Amplification of ITS region was done by using universal primers ITS1: TCCGTAGGTGAACCTGCGG and ITS4: TCCTCCGCTTATTGATATGC. The sequence was assembled, compared using BLAST, and submitted to GenBank database under accession number OM037458. A phylogenetic tree was constructed to determine the closest yeast species by the neighbor-joining approach, using MEGA 11.
Evaluation of probiotic properties
Probiotic potential of P. kudriavzevii Y33 was examined for various factors viz., bile tolerance, cholesterol assimilation, cell surface hydrophobicity, autoaggregation, antibiotic susceptibility, exopolysaccharide production, and hemolytic activity.
Bile tolerance
An overnight grown culture of selected yeast isolate was harvested (10,000 × g, 5 min, 4 °C) and washed twice with PBS buffer (pH 7.0). The cell pellet was resuspended into the bile solution containing 0.5%, 1%, and 2% ox bile (HiMedia) and incubated at 30 °C for 3 h [15]. The broth without bile solution was used as control. The results were expressed as log cfu/mL.
Cholesterol assimilation
Cholesterol assimilation by yeast isolate was done using o-phthalaldehyde method according to the method given by Liong and Shah [24]. Three different bile salts viz. cholic acid, sodium taurocholate, and ox bile were used in the study. Cholesterol solution (Sigma-Aldrich) 10 mg/ml in 96% ethyl alcohol was made and sterilized using a filter. To simulate approximate levels in the digestive system, 70 μl of cholesterol solution was added to 10 ml of yeast peptone dextrose (YPD) broth (final cholesterol concentration of 70 μg/ml) with 0.2% (w/v) bile salts (ox bile/cholic acid/sodium taurocholate). YPD broth was inoculated with selected yeast isolate (1%) and incubated at 30 °C for 20 h. An uninoculated sample was used as control. After the incubation, the cells were centrifuged, and the leftover cholesterol concentration in the broth was quantified using Rudel and Morris [42] modified colorimetric method. Cholesterol assimilation was calculated as percentage by using formula:
$$A=100-\left(B/C\right)\times 100$$
where A = % of cholesterol removed, B = absorbance of the sample containing the cells, and C = absorbance of the sample without cells at 550 nm.
Cell surface hydrophobicity
The ability of isolate to adhere to different hydrocarbons (cell surface hydrophobicity) was studied as per the method described by Rosenberg et al. [41]. The cell suspension was prepared as described in bile tolerance experiment and an optical density of 0.8 at 600 nm was adjusted. Then, equal volume of yeast suspension and hydrocarbons (n-hexadecane, xylene, and toluene) were mixed by vortexing for 5 min and allowed for phase separation by incubating at 37 °C for 1 h. The lower aqueous phase was withdrawn carefully, and the OD was determined. Hydrophobicity was calculated by using formula:
$$\mathrm{Hydrophobicity}\ \left(\%\right)=\left[\left(A0-A\right)/A0\right]\times 100$$
where A0 and A are absorbance before and after mixing with solvents at 600 nm.
Autoaggregation
Autoaggregation ability of yeast isolate was performed by the modified method of Collado et al. [9]. Overnight grown cells were resuspended in PBS buffer and optical density of suspension was adjusted to 0.5 at 600 nm. The suspension (4 ml) was vortexed for 10 s and incubated at 30 °C. The autoaggregation ability was checked at different time intervals (0 h, 3 h, and 24 h), and autoaggregation ability was expressed in percentage by using formula as:
$$\mathrm{Autoaggregation}\ \left(\%\right)=\left[1- At/A0\right]\times 100$$
where At represents the absorbance at time t and A0 the absorbance at t = 0.
Antibiotic susceptibility
For testing the antibiotic susceptibility of the yeast isolate, disk diffusion method was used [49]. Both antifungal and antibacterial antibiotics were used in the experiment. Antifungal agents used were ketoconazole (KT) (30 μg), clotrimazole (CC) (10 μg), itraconazole (IT) (30 μg), amphotericin-B (AP) (50 μg), nystatin (NS) (50 μg), and fluconazole (FLC) (10 μg), and antibacterial antibiotics used were vancomycin (VA) (30 μg), penicillin (P) (10 units), erythromycin (E) (15 μg), clindamycin (CD) (2 μg), and ampicillin (A) (10 μg).
Exopolysaccharide production
For the evaluation of exopolysaccharide production, overnight grown yeast isolate was streaked on the ruthenium red milk agar plate (10% w/v, skim milk powder, 1% w/v, sucrose and 0.08 g/l ruthenium red, 1.5% w/v agar) and incubated at 30 °C for 24 h [34].
Hemolytic activity
Yeast isolate (24 h grown culture) was streaked on the Columbia 5% Sheep Blood agar plate, and after 24 h of incubation at 30 °C, the plate was observed for the zone of clearance [25].
Enzymatic profiling
The activity of different enzymes viz. β-galactosidase, protease, amylase, phytase, and lipase was checked in yeast isolate. For β-galactosidase activity, yeast culture was exponentially grown in YPD (yeast peptone dextrose) broth. Cells (50 μl) were treated with SDS (sodium dodecyl sulfate, 100 μl, 0.1M) for permeabilization; o-nitrophenyl β-galactopyranoside (100 μl; 0.1 M) (ONPG, HiMedia Mumbai, India) was used as substrate and incubated at 30 °C for 15 min. After the incubation, 1 ml of sodium carbonate (0.1 M) was added to stop the reaction and absorbance was recorded at 420 nm [30].
Supernatant of 48 h grown yeast isolate was used for protease assay. In assay, 150 μl of substrate (0.5% casein), supernatant (100 μl), and 750 μl of 50 mM Tris HCl buffer (pH 7) were added and incubated at 30 °C for 30 min. The reaction was stopped by adding 1 ml of 5% TCA (Trichloroacetic acid), reaction mixture was centrifuged, and absorbance was recorded at 275 nm [26].
To check the amylase activity of yeast isolate, 100 μl of substrate (0.5% starch), 100 μl of 48 h supernatant (enzyme), and 800 μl of acetate buffer (50 mM, pH 5.5) were incubated at 30 °C for 30 min. To stop reaction, 1 ml of DNS reagent was added to reaction mixture and then kept in boiling water for 5–10 min. After cooling, the absorbance was recorded at 540 nm [29].
Phytase activity of yeast isolate was assayed by determining the amount of phosphate liberated from sodium phytate. The reaction mixture comprising of 200 μl of enzyme and 800 μl of buffered substrate (30 mM sodium phytate in 0.25 M sodium acetate buffer pH 5.5) was incubated at 30 °C for 30 min. The reaction was quenched by adding 1 ml of 10% TCA, followed by 2 ml of coloring reagent (FeSO4.7H2O, 7.32%; ammonium molybdate, 1.5%; H2SO4, 4.8%). Optical density of the reaction mixture was determined at 750 nm [2].
The ability of yeast isolate to produce lipase enzyme was determined by photometric assay. In assay, 60 μl of p-nitrophenyl palmitate (30 mg p-NPP in 100ml of iso-propanol) as substrate, 40 μl of 24 h supernatant (enzyme), and 2.9 ml of Tris-HCl buffer (pH 8.0) were added and incubated at 37 °C for 30 min. The reaction was stopped by chilling at − 20 °C for 2 min. Absorbance was recorded at 410 nm [21]. Reaction buffers were used as blank during enzymatic reactions.
Statistical analysis
The data was obtained from three separate experiments and provided as mean values. The statistical analysis was carried out with SPSS Inc. software (version 21.0). ANOVA and Tukey’s multiple comparison test (p < 0.05) were used to compare the results.
