Probiotic Potential of Ethanol-Producing Yeasts

The selected ethanol producing yeasts were investigated for their probiotic characteristics, which included the ability to grow at 37 °C, the resistance to grow in bile salt and low pH conditions, hemolytic activity and the adhesion capacity of cells [20 (link)]. Briefly, yeast cells were incubated in a simulated gastric juice containing 0.35% (w/v) pepsin at pH 2.0 and 3.0, and 0.3% (w/v) bile salt in phosphate buffered saline (PBS buffer) at pH 7 (PBS buffer pH 7 as control). The treated suspension was incubated at 37 °C for 3 h. Viable cell counts were determined by plating on yeast malt (YM) agar at 37 °C for 24 h, and the survival percentage was calculated as follows: survival (%) = [final (logCFU/mL)/control (logCFU/mL)] × 100. Hemolytic activity was determined by inoculating the yeast strain on a blood agar plate containing 5% defibrinated sheep blood and incubated at 37 °C for 72 h. The development of a clear zone of hydrolysis surrounding the colonies was observed and classified [21 (link)]. The cell surface hydrophobicity was tested in toluene (a nonpolar solvent). The yeast suspension was prepared in PBS to an optical density of 600 nm (OD600) = 1 (A0). Then, the volume of toluene was added into the yeast cell suspension at a ratio of 1:1 and the two-phase system was mixed for 5 min. Following 1 h of incubation at 37 °C, OD600 of the cell suspension was measured (A1) and the microbial adhesion to solvents (MATS) percentage was calculated as follows: MATS (%) = [(A0 − A1)/A0] × 100. Isolates with MATS above 50% were considered as hydrophobic [22 (link)]. The tannin-tolerance of the selected yeast isolates was also investigated on YM agar containing tannin, according to the method described by Kanpiengjai et al. [2 (link)]. A single colony of yeast isolate was picked up and spiked on YM agar with 10, 30 and 50 g/L tannin. The growth of the yeast isolates was observed after incubation at 30 °C for 3 days. The characteristics of low alcoholic producing properties, probiotic properties and tannin tolerance were used as the criteria for the selection of the proper yeast strain used in the healthy beverage fermentation using Miang fermentation broth residual byproducts.