Isolation of yeast strains
Samples were collected from naturally fermented nectar of toddy palm during mid day of hot summer (~40 °C) from different regions of Telangana State, India. Commercial probiotic yeast S. boulardii (Econorm, Dr. Reddy’s Laboratories, Hyderabad, India) was used as a referral organism.
Isolation of yeasts was carried out by using standard methodology described by Tikka et al. (2013) and Zaky et al. (2014). After incubation, colonies were selected based on morphology and sub-cultured on yeast extract peptone dextrose (YPD) agar slants for further observations.
Growth and characterization of yeast isolates
Isolates were characterized by their cultural and morphological characteristics following the protocols as described by Reis et al. (2013). After incubation, cultural and morphological characteristics were observed by visible growth pattern followed by simple staining and microscopic observation.
The sugar utilization ability of yeast isolates was achieved by their sugar assimilation profiling. The sugar assimilation property enables us to determine the ability of yeast to use a specific sugar/carbohydrate as a sole source of carbon. Yeast nitrogen base (YNB) medium with 2% (w/v) specific selected sugar (s) like glucose, maltose, ribose, fructose, xylose, cellobiose, dextrose, mannose, starch, sucrose, lactose and galactose were used for determination of yeast sugar assimilation property on the basis of biomass formation (Marinho et al. 2010).
Assessment for probiotic properties
Antimicrobial drug sensitivity
The yeast isolates were tested for antimicrobial drug sensitivity against 8 antifungal agents like Cefmetazole 30 µg (CMZ 30), Ceftazidime 10 µg (CTR 10), Clotrimazole 10 µg (CC 10), Fluconazole 10 µg (FLC 10), Itraconazole 10 µg (IT 10), Ketoconazole 50 µg (KT 50), Miconazole 50 µg (MIC 50) and Nystatin 100 µg (NS 100) and 20 antibacterial agents like Amoxicillin 10 µg (AMX 10), Ampicillin 10 µg (AMP 10), Ampicillin 25 µg (AMP 25), Bacitracin 10 µg (B 10), Ceftriaxone 10 µg (CT 10), Chloramphenicol 10 µg (C 10), Clindamycin 10 µg (CD 10), Cloxacillin 10 µg (COX 10), Enrofloxacin 10 µg (EX 10), Erythromycin 10 µg (E 10), Gentamicin 10 µg (GEN 10), Kanamycin 30 µg (K 30), Lincomycin 10 µg (L 10), Methicillin 10 µg (MET 10), Neomycin 10 µg (N 10), Norfloxacin 10 µg (NX 10), Penicillin G 10 µg (P 10), Streptomycin 10 µg (S 10), Streptomycin 25 µg (S 25), Teracycline 10 µg (TE 10), Teracycline 30 µg (TE 30), Trimethoprim 10 µg (TR 10) and Vancomycin 10 µg (VA 10) of Sigma Aldrich (Sigma Aldrich Pvt. Ltd., India) were placed on yeast inoculated YPD medium plates and incubated for 48 h at 30 °C. After incubation drug sensitivity of the yeast isolates was evaluated by measuring the zone of inhibition (Mashad et al. 2012).
Antagonistic activity against pathogens
Antagonistic activity of yeast isolates was evaluated by agar well plate method by measuring the zone of inhibition around the wells as per protocol described by Sibanda et al. (2010) against human intestinal pathogenic bacteria like Escherichia coli 0157:H7, Pseudomonas aeruginosa, Klebsiella pneumonia, Staphylococcus aureus, Salmonella typhi and Salmonella paratyphi.
Co-culture activity with healthy commensals
Co-culture activity assay is yet another method to determine the positive or negative impact and/or ability of candidate probiotic yeast isolates to coexist with normal bacterial flora of the human intestine. Yeast isolates and bacterial cultures like E. coli 0150:H5, Lactobacillus acidophilus were activated in their basal media and equal volumes (0.1 ml) of their suspensions were inoculated in a modified medium (1% yeast extract, 1% peptone, 0.25% NaCl and 1% dextrose) with pH 6.5 and incubated at 37 °C for 24 h. After incubation, culture suspensions were diluted to tenfold and 10 µl of 10−3 dilution was inoculated on YPD agar medium with Ampicillin (30 µg/ml), Kanamycin (30 µg/ml) for yeast growth and nutrient agar medium with Geneticin (200 µg/ml) for bacterial growth. The YPD agar medium plates were incubated at 30 °C for 48 h and nutrient agar plates were incubated at 37 °C for 24 h. After incubation, percent viability of yeast isolates and bacteria was determined on the basis of colony forming units (CFU) using modified methodology of Paschos et al. (2015).
A crucial step towards the identification and selection of potential probiotic candidates is to evaluate their resistance to the extreme conditions of the gastrointestinal tract. The barriers that must be overcome are temperature, low pH, organic acids and digestive enzymes, i.e. pepsin and amylase in the stomach and trypsin and bile in the upper intestine (Corzo and Gilliland 1999). The probiotic organism should be able to tolerate high temperature, i.e. above 37 °C and the diverse conditions of the gastric juice. Therefore, to test the parameters of stress tolerance of yeast isolates the following experiments were performed.
Resilience to gastrointestinal parameters
Actively growing yeast suspensions (1 ml) were inoculated in 100 ml phosphate buffered saline (PBS) and incubated for 4 h at different temperatures (30, 37, 40 and 45 °C), with different pH (2.0, 2.5, 3.0 and 3.5), different percentages of ox bile (0.25, 0.5, 0.75 and 1.0) and different percent mixture of 3 organic acids like propionic, butyric and acetic acids in 7:2:1 ratio (0.25, 0.5, 0.75 and 1.0). After incubation, 100 µl of inoculum from each set was inoculated on YPD agar at 30 °C for 24 h and percent tolerance was calculated by using the formula (log N/log N
) × 100 where N is count after incubation and N0 is count before incubation (both expressed as cfu/ml) (Syal and Vohra 2013).
Aqueous solution (1 ml) of 5% NaCl was taken to create an in vitro gastric environment with different concentrations of enzymes like Pepsin (2.0, 4.0, and 6.0 g l−1), Trypsin (0.25, 0.5 and 0.75 mg ml−1) and Amylase (250, 300 and 350 IU ml−1) (Pennacchia et al. 2008). The pH of pepsin containing solution was adjusted to 2.0 and trypsin and amylase containing solution was adjusted to 7.5. All the sets were inoculated with 0.1% inoculum of yeast isolates and incubated at 30 °C in a shaking incubator at 150 rpm for 4 h. After incubation of all the sets, 100 µl of inoculum from each set was inoculated on YPD agar medium for the determination of percent tolerance of yeast isolates (Syal and Vohra 2013).
Pathogenicity of yeast isolates
Yeast such as S. boulardii are best studied probiotics being used successfully and well known to play a crucial role in the treatment and management of diarrhoea and inflammatory bowel disease and to reduce the duration of disease. Despite an excellent record of safe use, yeasts may still be the cause of localized infections in some patients.
Therefore, in order to determine whether the yeast isolates show pathogenicity, it was studied by the detection of enzymes like protease, phospholipase, the ability of haemolysis and by detection of specific pathogenic genes through polymerase chain reaction (PCR) amplification. Protease activity was tested using milk casein and gelatin as substrates by the method described by Lechuga et al. (2016) and was adapted to verify the phospholipase activity. Similarly, haemolytic activity was determined by a modified method of Manns et al. (1994). Protease and phospholipase production was detected by halos formation around the culture and haemolysin activity was detected by measurement of haemolytic index (Luo et al. 2001). Extracted genomic DNA of each isolate was amplified with universal primer SR6R (5′-AAGTAAAGT-CGTAACAAGG-3′) and LR1 (5′-GGTTGGTTTCTTTTCCT-3′) of human pathogenic Candida albicans. The reaction mixture for PCR and conditions are as per protocol described by Koehn (1982). The amplicons formed as PCR product were visualized on an agarose gel.
Caco-2 cells derived from human intestinal epithelial cells were purchased from National Centre for Cell Science (NCCS), Pune, India and maintained in Eagle’s minimal essential medium (MEM) with glutamine (0.584 g/l), sodium bicarbonate (3.7 g/l), penicillin (100 U/ml), streptomycin (100 µg/ml), and 5% serum and the final pH of medium was adjusted to 7.2 before sterilization. Culture flasks with medium were maintained in a CO2 incubator at 37 °C and 5% CO2 as per the protocol described in Nikolic et al. (2012). Inoculated monolayers were washed thrice with warm MEM medium without serum. Adhesion of yeast cells with monolayer was fixed with cold methanol, stained with Giemsa stain and observed under microscope for adherence. The mean number of yeast cells was determined and the adherence score was expressed for each yeast isolate.
Based on the probiotic characteristics, 2 yeast isolates OBS1 and OBS2 were selected and characterized by sequencing of 5.8S rRNA and internal transcribed spacer (ITS) 1 and 2 (Fujita et al. 2001). Genomic DNA was extracted using an Insta Gene Matrix (BIO RAD, California, USA). The universal primers used for amplification of 5.8S rRNA, ITS1and ITS2 were 5′-TCCGTAGGTGAACCTGCGG-3′ and 5′-TCCTCCGCTTATTGATATGC-3′. The PCR conditions were set to 1 min each for denaturation at 95 °C, annealing at 55 °C, 2 min for an extension at 72 °C and finally finishing with a 10 min step at 72 °C. The amplicons were purified with a multiscreen filter plate (Millipore Corp., Bedford, MA, USA) and sequencing was performed using PRISM BigDye Terminator v3.1 Cycle sequencing Kit (Applied Biosystems, California, USA). The amplicons were added to Hi-Di formamide (Applied Biosystems, Foster City, CA) and the mixture was incubated at 95 °C for 5 min, followed by incubation on ice for 5 min and then analyzed by ABI Prism 3730XL DNA analyzer (Applied Biosystems). Complete sequence of 5.8S rRNA and partial sequences of internal transcribed spacer (ITS) 1 and 2 were BLAST searched in NCBI database (www.ncbi.nlm.nih.gov) for similarity. Based on maximum similarity of BLAST results, a phylogenetic dendrogram was constructed using Phylip 3.69.
Three test samples were prepared to assess the cytotoxic effect of yeast isolates on the chosen cancer cell lines MCF7 (Breast cancer) and IMR32 (Neuroblastoma). Among three samples, two were prepared by cultivating yeast isolates OBS1 and OBS2 separately overnight and one without yeast in YPD broth medium. After overnight incubation, all the three samples were centrifuged to separate yeast cells and supernatants were filtered using membrane filters to obtain clear sterile test samples. Sulforhodamine B (SRB) assay was performed to assess the anticancer property of yeast isolates (Szajewska et al. 2006). Briefly, 10 × 103 cells in 100 μl Dulbecco’s modified Eagle medium (DMEM) per well were seeded in triplicates in 96-well flat-bottom plates and are allowed to adhere overnight. Following this incubation period, the filtrate obtained from cultured broth of yeast isolates OBS1 and OBS2 were added to each of the wells in a volume dependent manner (2–40 µl) and incubated for 48 h. After 48 h, the media was replaced with 100 µl of 10% Trichloroacetic acid for fixation of cells for 1 h at 4 °C and stained with 0.4% sulforhodamine B dissolved in 1% acetic acid. Cells were then washed with 1% acetic acid to remove unbound dye. The protein-bound dye was extracted with 10 mM Tris base to determine the optical density at 510 nm wavelength.
The yeast autolysate for determination of antioxidant activity was prepared as per the protocol described by Chana et al. (2005). Cell pellets of two active yeast isolates, OBS1 and OBS2 were suspended in 20 ml sterile distilled water and incubated at 25 °C for 72 h. After incubation, the content was centrifuged and the volume of separated supernatant autolysate solution was made up to 500 ml with distilled water. The antioxidant activity of yeast isolates was assayed using phosphomolybdenum method (Kumaran and Karunakaran 2007). The increased absorbance of reaction mixture indicates the increased antioxidant property.
Statistical analysis was carried out for the data using the Statistical Package for the Social Sciences (SPSS), version 12.0 for Windows, for the determination of average and standard deviations. In this analysis, independent variables are temperature, pH, ox-bile, organic acid mixture, gastric enzymes like amylase, trypsin and pepsin and the dependent variables are the percent tolerance of yeast isolates. Significance was determined to be p < 0.01 using two-way analysis of variance (ANOVA), followed by Duncan’s multiple range test.
Sequence and culture deposition
Sequences were submitted to the NCBI GenBank and acquired accession numbers for both the strains. OBS1: KP998095 and OBS2: KP998094. Based on probiotic and therapeutic properties, yeast OBS2 was selected and deposited into DSMZ culture collection as S. cerevisiae DSM 103642.