The study aims to isolate the yeast strains that could be used effectively as baker’s yeast and compare them with the commercial baker’s yeast available in the market of Nepal. A total of 10 samples including locally available sources like fruits, Murcha, and a local tree “Dar” were collected from different localities of Bhaktapur, Kavre, and Syangja districts of Nepal, respectively. Following enrichment and fermentation of the samples, 26 yeast strains were isolated using selective medium Wallerstein Laboratory Nutrient Agar. From the differential tests which included morphological and microscopic observation and physiological and biochemical characterization such as nitrate reduction and lactose utilization tests, 8 strains were selected as possible
Yeast exists in natural environment like plant tissues, fruits, grains, leaves, dung, soil, and other fermented products [
Although the consumption of baker’s yeasts in Nepal is rising every year, the country imports its entire requirement of baker’s yeast mostly from China and Europe. Nepal imports the baker’s yeast of 10 million US dollar worth in 2013 [
Murcha is a mixed starter culture which contains mixture of fermenting yeasts, saccharifying molds and acidifying bacteria [
Ten samples which include 6 fruit samples, that is, sugarcane
Enrichment was done to increase the number of native microflora present in the sample by adding 27% (w/v) sucrose in the crushed fruit samples and was allowed to ferment for 3 days. The prepared samples were plated on Wallerstein Laboratory Nutrient Agar medium supplemented with chloramphenicol (0.01%) at pH 5.5 and allowed to grow for 72 hours. Through morphological examination, two distinct yeast colonies, that is, white and green, were selected. The selected colonies were subsequently subcultured on Yeast Malt (YM) Agar medium supplemented with chloramphenicol (0.01%) to obtain pure isolates.
Maltose is the principle carbon source during bread fermentation. Thus, baker’s yeast strains are good utilizers of maltose [
A single colony of yeast was mixed in a droplet of sterile distilled water on glass slide and smeared until the smear dried off. The smear was then stained using diluted lactophenol cotton blue dye, air-dried, and observed under light microscope at 100x magnification.
A well isolated colony was inoculated in nitrate broth (peptone 10 g, KNO3 10 g in 1000 ml distilled water). It was incubated at 30°C for 48 hours. After incubation, 5 drops of both reactive 1 (
Yeast cells were grown at 30°C for 3 days into Yeast Fermentation Broth (YFB) (peptone 7.5 g/L, yeast extract 4.5 g/L; 1 ml of 1.6% (w/v) bromothymol blue as an indicator) with autoclaved 6% (w/v) lactose. The Durham tubes were also placed into the media to trap the carbon dioxide released. The changes from green to yellow indicated that yeast using the carbon source, that is, lactose.
In this test, the isolates were subsequently grown in different conditions that mimicked the various stresses. Firstly, the isolates were grown onto YPG medium and incubated at 30°C for 3 days. From that, a single colony was transferred and grown on YPG medium and incubated at 37°C for another 3 days. Again, a colony was selected and subcultured to YPG 8% (v/v) ethanol and incubated at 30°C for 3 days. A single isolated colony was further subcultured on YPG supplemented with 20% (w/v) glucose and incubated under the same conditions. Finally, yeast cells were transferred on YP medium supplemented with 2% (w/v) sucrose and 8% (v/v) ethanol and incubated under the same conditions.
The yeast isolates were grown on lead acetate medium (40 g/L glucose, 5 g/L yeast extract, 3 g/L peptone, 0.2 g/L ammonium sulphate, 1 g/L lead acetate, and 20 g/L agar) and incubated at 30°C for 7 days.
The yeast isolates were inoculated in 10 ml of YPG broth and incubated at 30°C for 3 days. They were agitated to observe the flocculation forming.
Yeast isolates were cultured on YPG agar and incubated at 25°C, 30°C, 37°C, and 45°C for 72 h. Growth was observed and analyzed [
The carbohydrate utilization test was performed using broth (peptone: 10 g; NaCl: 5 g; phenol red: 0.018 g; distilled water: 1000 ml; carbohydrate: 10 g) along with inverted Durham tubes in the broth. The carbohydrates used were dextrose, fructose, lactose, galactose, maltose, and sucrose. The media were inoculated with yeast strains and incubated for 24 hrs. The color change from red to yellow indicated the fermentation using carbon sources.
Yeast isolates were cultured on YPD broth containing 30, 40, and 50% dextrose and incubated at 30°C for 48 hours. The cell density of different yeast isolates in response to high dextrose concentration was taken.
Yeast isolates were grown in YPG broth containing 3 different concentrations of ethanol, that is, 10%, 13%, and 15% (v/v), respectively, and incubated at 30°C for 72 hours.
YPD broth modified with respective sugars was used. Cell density of yeast isolates in sucrose and glucose media was compared. The cell density was measured using DEN-1B Grant bio Densitometer. 18 phi test tubes were used for generating data and data were obtained in McFarland standards.
Cultivation of the yeast strains was done in conical flasks in 250 ml media containing yeast extract, peptone, and sucrose. The flasks were kept in shaker incubator at 30°C for 72 hours and yeast pellets were collected after centrifugation at 10,000.
For each strain, 50-gram wheat flour was weighed and 1% salt was mixed with the flour. 6% sugar was dissolved in lukewarm water and 0.6 g yeast pellets were inoculated in the sugar solution to allow its activation. The activated yeast solution was poured in the flour and mixed well. The DY5 which is a commercial yeast strain was used as a positive control, whereas the dough without any yeast was used as a negative control. Proofing was done by incubating the dough at 30°C for 2 hours. It was then baked in hot air oven at 180°C for 20 minutes.
Besides, to assess the rise of dough level, about 10 g of dough mixture was kept in measuring cylinder. It was incubated and the level was noted every half hour.
The colonies exhibiting characteristics such as creamy to white color, fluffy, and smooth margin were selected as tentative
The isolation tests as described previously were used for identification of
Microscopic observation of yeast colonies from different sources as observed at 100x magnification.
DY5
FAPW
SUG1
MUR3B
ENG
DPSW
Therefore, the screening of
All strains except JAK3 and FAPW showed flocculation property (Table
Flocculation test.
S. number | Samples | Flocculation | Hydrogen sulfide production |
---|---|---|---|
|
MUR3B | Yes | ++ |
|
ENG | Yes | + |
|
DPSW | Yes | −− |
|
FAPW | No | ++ |
|
DY5 (SC) | Yes | +++ |
|
SUG1 | Yes | + |
|
JAK3 | No | +++ |
|
SUGW | Yes | + |
+++: intensive response; ++: moderate response; +: low response; −−: no response.
The viability of isolated strains was checked under spontaneous stress conditions such as temperature 37°C, ethanol 8%, sucrose 2%, and glucose 20% for 15 days. It is necessary to check the capacity of baker’s yeast to survive these stress conditions as they mimic the ethanol, osmotic, and temperature stress that yeast faces during baking. In the presence of these stresses, yeast becomes impaired and its survival denotes that yeast can carry out fermentation even in these harsh conditions [
Stress exclusion tests on yeast cell for temperature and cell osmotic pressure in high concentration of ethanol and sugar.
Samples | Growth into different media | Use of yeast as potential leavening agent | ||||
---|---|---|---|---|---|---|
YPG | Temperature 37°C | Ethanol 8% (v/v) | YPG 20% (v/v) glucose | YPS 2% (v/v) sucrose + ethanol 8% (v/v) | ||
MUR3B | +++ | +++ | +++ | +++ | ++ | Yes |
ENG | +++ | ++ | +++ | +++ | ++ | Yes |
DPSW | +++ | +++ | +++ | ++ | ++ | Yes |
FAPW | +++ | +++ | +++ | +++ | ++ | Yes |
DY5 (SC) | +++ | +++ | +++ | +++ | ++ | Yes |
SUG1 | +++ | +++ | +++ | +++ | ++ | Yes |
JAK3 | +++ | ++ | +++ | −− | −− | No |
SUGW | +++ | +++ | ++ | ++ | ++ | Yes |
+++: intensive response; ++: moderate response; −−: no response; YPG: yeast peptone glucose medium; YPS: yeast peptone sucrose medium.
In response to temperature change, all the strains including commercial yeast strain were able to grow at temperatures 25°C, 30°C, and 37°C. Strains JAK3 and SUGW showed feeble growth at 37°C. However, the strain SUG1 was able to survive at higher temperature (45°C) too at which even the commercial strain failed to grow (Table
Growth and inhibition of yeast isolates at different growth temperatures.
S. number | Samples | Temperature | |||
---|---|---|---|---|---|
25°C | 30°C | 37°C | 45°C | ||
|
MUR3B | +++ | +++ | +++ | −− |
|
ENG | +++ | +++ | +++ | −− |
|
DPSW | +++ | +++ | ++ | −− |
|
FAPW | +++ | +++ | +++ | −− |
|
DY5 (SC) | +++ | +++ | +++ | −− |
|
SUG1 | +++ | +++ | +++ | ++ |
|
JAK3 | +++ | ++ | ++ | −− |
|
SUGW | +++ | +++ | ++ | −− |
+++: intensive response; ++: moderate response; −−: no response.
In sugar utilization test, change of color to yellow signifies that the organisms utilize sugar to produce acidic products and gas which is trapped in Durham tubes. The orange color indicates the partial fermentation of sugars. All the isolates were able to ferment sugars to different extent (Table
Sugar utilization test.
Yeast strains | Dextrose | Fructose | Galactose | Maltose | Sucrose | |||||
---|---|---|---|---|---|---|---|---|---|---|
Color | Gas | Color | Gas | Color | Gas | Color | Gas | Color | Gas | |
DY5 | Yellow | +++ | Yellow | ++ | Yellow | ++ | Yellow | +++ | Yellow | +++ |
DPSW | Yellow | +++ | Orange | ++ | Yellow | ++ | Yellow | ++ | Yellow | +++ |
MUR3B | Yellow | +++ | Yellow | +++ | Yellow | +++ | Yellow | +++ | Yellow | +++ |
ENG | Yellow | ++ | Yellow | ++ | Yellow | + | Orange | ++ | Yellow | +++ |
SUG1 | Yellow | +++ | Yellow | +++ | Yellow | ++ | Yellow | +++ | Yellow | +++ |
FAPW | Orange | + | Orange | − | Orange | ++ | Orange | ++ | Yellow | − |
JAK3 | Orange | − | Orange | + | Yellow | +++ | Yellow | + | Orange | + |
SUGW | Yellow | ++ | Orange | + | Orange | − | Yellow | +++ | Yellow | ++ |
+: ≤one-third of Durham tube; ++: greater than one-third and ≤two-thirds of Durham tube; +++: greater than two-thirds; −: no gas production.
In baking process, it is very important for a yeast strain to survive the minimal osmotic pressure [
Comparison of osmotolerance (a) and alcohol tolerance (b) of different yeast isolates with commercial yeast.
The effective size, calculated using eta squared, was 0.917 for 30%, 0.909 for 40%, and 0.883 for 50% glucose concentration. Post hoc comparisons using Tukey’s HSD test indicated that the mean cell density for DPSW in both 30% and 40% glucose concentration was significantly lower than that of DY5. The mean cell density of other strains did not differ significantly compared to that of commercial dry yeast (DY5) in the three given glucose concentrations. There was significant decrease to the biomass of all strains in 50% glucose concentration with respect to the biomass of respective strains at 40%. In 30% and 40%, glucose concentration MUR3B and ENG shows the same level of tolerance to that of DY5. This means they can tolerate a higher osmotic pressure than the other strains including commercial yeasts.
The one-way between-groups analysis was performed to observe the alcohol tolerance of different yeast strains, as measured by cell density in presence of different alcohol concentrations (Figure
The growth of different strains was checked in glucose and sucrose by measuring the cell density (Figure
Comparison of different yeast isolates with commercial yeast in presence of sugar. Cell density comparison of different yeast isolates with commercial yeast in presence of glucose and sucrose.
The invertase activity for the strain MUR3B was found to be the highest (Figure
(a) Invertase activity of different yeast strains; (b) standard curve of mg of glucose versus absorbance.
The rise of dough level or the speed of fermentation process was assessed by noting the rise of dough mixture kept in measuring cylinder (Figure
Comparison of leavening effect of different yeast strains.
The final fermented dough with SUG1 and ENG was comparable to that by the commercial strain DY5. The cross-sectional examination of the dough from all strains also showed similar crumb textural property (Figure
Properties of baked dough.
S. number | Sample | Color of crust | Color of crumb | Aroma |
---|---|---|---|---|
|
MUR3B | Light Brown | Creamy | +++ |
|
ENG | Light Brown | Creamy | ++ |
|
DY5 | Light Brown | Creamy | +++ |
|
SUG1 | Light Brown | Creamy | +++ |
+++: baking aroma of commercial active dry yeast; ++: intensity of aroma being less than that of commercial active dry yeast.
Baked dough from different isolated strains: SUG1, MUR3B, ENG, and DY5.
This study showed that three isolated strains of
The authors declare that there are no conflicts of interest regarding the publication of this paper. Also the funding body mentioned in Acknowledgments does not lead to any conflicts of interest regarding the publication of this paper.
The authors thankfully acknowledge Kyushu University Research Grant for funding the research project on “Fermented Foods of Nepal” and Department of Biotechnology, Kathmandu University, for providing research space. They are grateful to Ms. Asmita Shrestha and Mr. Sandeep Adhikari of Department of Biotechnology, Kathmandu University, for their support during the research and documentation. They are thankful to Mr. Himal Shrestha and Mr. Aman Maharjan of Department of Natural Science, Kathmandu University, for their support in data analysis.