Sampling site and sample collection
A total of fifty-six fruit samples, of which eight each from avocado (Persea americana), banana (Musa acuminate), grape (Vitis vinifera), mango (Mangifera indica), orange (Citrus sinensis L.), papaya (Carica papaya) and pineapple (Ananascomosus). All samples were collected from major local markets (Atikilit Tera and Merkato) in Addis Ababa City.
Fruit samples preparation for yeast isolation was done following the method indicated by . 100g of each fruit sample was separately cut, crushed with mortar in sterile plastic bags. The homogenate of each fruit sample was transferred into sterile beakers along with 50 ml of sterile distilled water. The covered beaker with each fruit homogenate was kept at room temperature for three days to allow fermentation to take place. In this study, active dry baker’s yeast (Saf instant) from DSM bakery Ingredients, Holland was used as a standard strain.
Isolation of yeasts
From each separate fermented fruit sample, 1ml was serially diluted in 9 ml sterile peptone water in a test tube. From appropriate dilutions, 0.1 ml of aliquots were spread plated on pre-dried potato dextrose agar (PDA, HIMEDIA) plates that contained chloramphenicol (0.1 g/L). All the plates were incubated at 30oC for 48 hrs.
From each countable plate, ten to twenty colonies were taken and purified on the similar freshly prepared medium. The purified isolates were transferred to PDA slants and preserved at 4oC for further study.
Test of CO2 and H2S production of yeast isolates
The level of CO2 production by each yeast isolate recovered in this study was tested first by inoculating each pure isolate into a test tube that contained 5 ml of Yeast Extract Peptone Dextrose (YEPD) broth with a Durham tube. All the tubes were incubated at 30oC for 24 hrs and those isolates that were producing more amount of CO2 by displacing the medium in the Durham tube were selected. H2S production by each purified yeast isolate was verified after having those potential isolates by streaking each isolate on Bismuth Sulfite Agar (BSA) and Kligler Iron Agar (KIA) plates. All plates were incubated at 30oC for 3 days following the procedures of . Isolates that exhibited black color on BSA plates and any blackening of the KIA along the line of inoculation or throughout the butt indicate hydrogen sulfide production.
Characterization of selected yeast isolates
Identification of yeast isolates to genus level was carried out on the basis of standard cultural, morphological and biochemical tests as described by . Induction of ascospore formation and its observation was done following the protocol given by  and .
The ability of each of selected yeast isolates in utilizing D-glucose, fructose, maltose, galactose, lactose and sucrose as a sole carbon source and production of gas was determined in Durham tubes following the standard methods as indicated in . Briefly, the medium was prepared by adding 10 g of yeast extract, and 10 g of peptone in 1000 ml of distilled water and thoroughly mixing. The pH was adjusted to 5 and the medium was boiled. Bromocresol purple carbohydrate (2%, w/v) fermentation indicator was added to an already boiled broth and dispensed in 5 ml amount into screw capped test tubes containing inversely placed Durham tubes. Both KNO3 (10 g/l), and (NH4)2SO4 (10 g/l) were used as nitrogen sources. The solutions were sterilized at 121oC for 15 minutes in separate flasks. The carbohydrate fermentation test was performed by inoculating yeast cells (6.6 log cfu/ml) of each isolate into separate tubes of 5 ml of yeast extract peptone broth with different sugar and nitrogen sources. The inoculated tubes were incubated at 30oC for 48 hrs. Change of color from violet to yellow due to acid production and accumulation of gas bubbles in inverted Durham tube (CO2 gas production) was taken as a positive result of sugar fermentation. No color change was taken as a negative result.
Effect of temperature, pH, glucose, and sodium chloride concentrations on growth of yeasts
The growth of each selected yeast isolates at different temperature values (25, 30, 35 and 42oC) was carried out by inoculating each actively growing yeast isolate (log 6.6 cfu/ml) into 50 ml of sterile YEPD broth (pH adjusted to 5 before autoclaving) in separate flasks.
Flasks were incubated at respective temperature values and the growth of each isolate was determined by reading the optical density at 550 nm using a spectrophotometer (6405 UV/Vis, JENWAY, United Kingdom) at intervals of 24, 48, 72, 96 and 120 hrs. To examine the optimum pH for the growth of the three selected yeast isolates, the pH of YEPD broth in separate flasks was adjusted to 4, 5 and 6 using 1N HCl and NaOH. Into each separate flask with a given pH value, each of an actively growing selected yeast isolate (log6.6 cfu/ml) was inoculated. The flasks were incubated at 30oC. The growth of each yeast isolates in a given pH value was followed at 24, 48, 72, 96 and 120 hrs by measuring the optical densities at 550 nm using a spectrophotometer (6405 UV/Vis, JENWAY, United Kingdom).
The ability of each yeast isolates to grow at different D-glucose concentrations (30, 40 and 50%) was conducted in YEPD medium. The pH of the broth medium was adjusted to 5. Similarly, log6.6 cfu/ml of each of an actively growing isolate was inoculated into 50 ml flasks. The flasks were incubated at 30oC. The growth of each isolate was determined after 96 hrs by measuring the optical densities at 550 nm using a spectrophotometer (6405 UV/Vis, JENWAY, United Kingdom).
The growth of the yeast isolates at different NaCl concentrations (5, 10 and 15%) (w/v) in YEPD medium was prepared and the pH of the broth was adjusted to 5. Each actively growing isolate (log6.6 cfu/ml) was inoculated into 50 ml flask and incubated at 30oC. The growth of each isolate was determined after 96 hrs by measuring the optical densities at 550 nm using a spectrophotometer (6405 UV/Vis, JENWAY, United Kingdom).
All data were presented as the average of triplicate experiments with standard deviation. Results were statistically interpreted with one-way analysis of variance (ANOVA) followed by post hoc analysis (Tukey’s test) to locate the significant differences. The data for ANOVA were analyzed using SPSS version 20.0 at p<0.05% significant level.