This document summarizes a study that isolated yeast from apple, orange, and banana fruits to determine their ability to produce alcohol at different glucose concentrations. Yeast was successfully isolated from the fruits and grown in culture. When fermented in media with varying glucose concentrations from 5-70g/L, the yeast produced differing amounts of biomass and alcohol. The yeast from apple produced the most biomass (0.38g/100mL) and alcohol (0.412g/100mL) at 70g/L glucose. Yeast from banana produced the highest alcohol (0.362g/100mL) at 70g/L glucose as well. Higher glucose concentrations generally resulted in greater yeast growth and alcohol production.

1. Production of alcohol by yeast isolated from apple, orange and banana
IJFNS
Production of alcohol by yeast isolated from apple,
orange and banana
Zahida Nasreen*, Shaista Jabeen, Muafia Shafique, Shumaila Usman, Tehseen Yaseen,
Ammara Yasmeen and Saima Nazir
1*,2,3,4,5,6,7
Pakistan Counsel of Scientific and Industrial Research Lahore, Pakistan
The purpose of this study was to isolate wild yeast strains present in different fruits (apple,
orange and banana) and to determine the yeast growth and the amount of alcohol production
at various glucose concentrations. Three fruits namely apple, banana and orange were
selected as natural sources for yeast isolation. Medium used for isolation of yeast from fruits
was consisting of 50 glucose, 3 malt extract, 3 yeast extract, 5 peptone and 15g/L agar. For
fermentation MGYP medium used with different glucose concentrations of 5, 10, 30, 50 and
70g/L. Inocula were prepared by loop transfers from stock slants to 50 mL of the 20 percent
medium in 500-mL Erlenmeyer flasks. The results showed that with higher concentration of
glucose (30g/L) higher amount of alcohol (0.22g/100mL) was produced. Similarly, the yeast
isolated from apple showed maximum yeast biomass (0.38g/100mL) at 70g/L glucose
concentration and minimum on 50g/L (0.03g/100mL) glucose concentration. In conclusion, the
wild yeast produce higher ethanol amount in case of banana fruits.
Keywords: Alcohol, fruit fermentation, yeast growth, glucose concentrations, malt extract, wild yeast
INTRODUCTION
Yeast is a unicellular eukaryotic fungus, very common in
the environment and is mostly saprophytic. It has been
classified as Ascomycetes or basidomycetes under fungi
taxonomy Kreger-van, et al., 1984) and there are about
1500 species of yeast (Kurtzman, et al., 2006, and
Barnett, et al., 1990). The commercial importance of
strains of yeast species Saccharomyces cerevisiae has
made it a model organism of study in both research and
industrial applications (Legras, et al., 2007). Fermenting
wild yeast species are being isolated from the natural
sources for over decades and is being used in various
fermentation processes. Yeast has been isolated from
variety of natural sources like leaves, flowers, fruits etc
(Spencer, et al., 1997; Davenport, et al., 1980; Tourna,
2005; Li et al., 2008). Being a sugar-loving
microorganism, it is usually isolated from sugar rich
materials. Fruits contain high sugar concentration and
hence yeast species are naturally present on these and
can be easily isolated from fruits. Distinct wild yeast
species are supposed to be present and associated with
different fruits in natural environments (Spencer, et al.,
1997). Because of yeast fermentative characteristic,
there is always a need for yeast strains with better
features of fermentation especially high ethanol tolerance
for production of ethanol as bio fuel on commercial scale
(Colin, et al., 2006).
*Corresponding author: Zahida Nasreen, Pakistan
Counsel of Scientific and Industrial Research Lahore,
Pakistan. Tel.: 0092 -042-9230688, Fax: 99230705,
zahidanassreen_pcsr@yahoo.com
International Journal of Food and Nutrition Sciences
Vol. 1(2), pp. 016-019, December, 2014. © www.premierpublishers.org ISSN: 2167-0434x
Research Article

2. Production of alcohol by yeast isolated from apple, orange and banana
Nasreen et al. 016
Moreover, besides S. cerevisiae, there is always a search
on for new wild/non toxic-fermentative yeast species for
their further industrial exploitation in fermentation
industry, baking industry, therapeutic production etc
(Alvarenga, 2011: Legras, et al., 2007).
The present study was intended to determine the
potential of the waste of ripe banana, orange and apple
fruits for alcohol production. The outcome of this study
may expand the utility of banana, orange and apple
wastes. That would not only ensure a cleaner
environment but also create more job opportunities,
reduce seasonal losses of the fruits and serve as a
substitute for produced alcohol by increasing their
production.
This research study reports on ethanol production from
the juices of apple, orange and banana.
MATERIALS AND METHODS
All chemicals used in the experiment were of highest
purity and were obtained from Sigma Chemical Company
(St. Louis, MO), Merck limited.
Yeast source, Isolation and medium
Fruit samples (banana, apple and orange) were obtained
from local sources of Lahore Pakistan and mostly
naturally decaying/fermented samples were preferred.
Fruit sample 100g was taken in a sterile mortar and
crushed to a fine paste by mixing with sterile water. Then
mixture was kept for overnight at normal room
temperature so that natural wild yeast present on fruit
samples to grow. Delete line. The medium was placed in
Erlenmeyer flasks containing 400 glucose, 10 g/L yeast
extract medium, and incubated with shaking 30o
C at
250rpm until good growth had taken place.
Then the cultures were streaked on 200 glucose, 10
yeast extract, 20g/L agar plates, and individual colonies
were picked from these and transferred to stock slants.
The stock slants were prepared with a medium consisting
of 200g/L glucose (reagent grade), 10 yeast extract, 1
urea, and 20 g/L agar. During this study, stock cultures
were transferred every 2 weeks. After 48 to 72h growth,
stock slants were prepared and used as inocula to
prepare fresh slants.
Sterilization of Culture Media
The fermentation media was optimized by varying the
concentration of glucose (5, 10, 30, and 50g/L) as
fermentation media were made up in approximately 10,
20, and 30 per cent glucose concentration and 5, 10, and
15g/L yeast extract, respectively.
After preparing the media, the media was processed in
order to homogenize all the contents. After homogenizing
the media was poured in test tubes, the mouth of the test
tubes were covered with cotton plugs and secured with
aluminum foil. The medium was then sterilized by
autoclaving at 121o
C at 15lb/ inch2
pressure for 15
minutes.
Inoculation
A loop full of liquid portion from each sample was
streaked (Quaternary streaking) in plate (with replica)
containing MGYP medium (yeast extract 3, malt extract
3, peptone 5, glucose 10 and agar 30g/L), pH 6.4
(phosphate buffer system) and incubated at 26°C for 2
days.
Optimization of Fermentation Medium
The fermentation media was optimized by varying the
concentration of glucose (5, 10, 30, and 50g/L) as
fermentation media were made up in approximately 100,
200, and 300 g/L glucose concentration and 5, 10, and
15 g/L yeast extract, respectively. Inocula were prepared
by loop transfers from stock slants to 50 ml of the 20 per
cent medium in 500-ml Erlenmeyer flasks. These were
incubated at 30
o
C for 72h on a rotary shaker (250 rpm).
Inoculum (3.5mL) was used with 50 ml of fermentation
medium in 500-ml Erlenmeyer flasks. These were
incubated at 35
o
C. Instead of closing the Erlenmeyer
flasks with cotton plugs, a double disc was used to
provide a more uniform material for gas passage.
Analyses were made at 24h intervals required for the
fermentation. Fermentation and incubation was continued
until most of the glucose had been used.
Analytical methods:
Evaporation during the fermentation was variable
depending to a large extent on aeration rate and length of
fermentation. Before analyses were made, the
fermentation broth was diluted to the original volume. The
media of yeast cells was centrifuged. The supernatant
was used for further analyses. Glucose was determined
by the copper reduction method of Shaffer and Somogyi,
(1933). Ethanol was determined by the Conway micro
diffusion method as described by Neish (1952).
RESULTS
Yeast isolated from the various fruits (apple, orange and
banana) were established in a pure culture on MGYP
medium. After 48h, white to cream colored colonies
appeared on agar plates. The colonies were smooth,
shiny and flat with entire margin. Observation of the
colonies under microscope showed that the obtained
budding/ oblong shaped colonies were of yeast and
belonged to the same morphological group. The cells
were globes to ovoid, mostly sub globes, occurring singly

3. Production of alcohol by yeast isolated from apple, orange and banana
Int. J. Food Nutr. Sci. 017
Table 1. Yeast fermentation (Apple, Orange and Banana biomass) with respect to glucose concentrations after 4 days.
Apple Orange Banana
Glucose
conc.
(g/100mL)
Initial weight
Yeast
Biomass
(g/100mL)
Final weight
yeast
Biomass
(g/100mL)
Initial weight
Yeast
biomass
(g/100mL)
Final weight
yeast
biomass
(g/100mL)
Initial weight
yeast
biomass
(g/100mL)
Final weight
yeast
biomass
(g/100mL)
Control 0.11 0.04 0.02 0.00 0.004 0.002
0.5 1.16 0.06 3.29 0.29 2.40 1.70
1 1.17 0.19 0.95 0.11 3.69 0.33
3 1.20 0.10 2.68 0.21 0.80 0.22
5 0.72 0.03 1.66 0.02 1.64 1.08
7 2.05 0.38 1.17 0.77 2.35 1.36
Table 2. Alcohol productions in Apple, Orange and Banana Samples
Apple Orange Banana
Glucose
conc.
(g/100mL)
Alcohol
produce
(g/100mL)
Alcohol
produce
(g/100mL)
Alcohol
produce
(g/100mL)
Control 0.001 0.002 0.003
0.5 0.069 0.072 0.042
1 0.074 0.088 0.036
3 0.082 0.091 0.031
5 0.086 0.035 0.133
7 0.412 0.362 0.252
or in pairs. Short, branched chains of cells were also
identified. But pseudo hyphae and true hyphae were
missing. Yeast cell biomass and alcohol were prepared
from fruits wastes. Yeast isolated from fruits were than
fermented by optimizing the medium (MGYP) by varying
the glucose concentration (5, 10, 30, 50, 70g/l) in an
incubating shaker at 30o
C in 250mL flasks, after 72 h.
The yeast isolated from apple showed maximum yeast
biomass (0.38g/100mL) at 70g/L glucose concentration
and minimum on 50g/l (0.03g/100mL) glucose
concentration. The variation of yeast biomass at different
glucose concentration was in Table 1. Similar results
have led to the tentative application of orange fruit
biomass production ranged from 70g/L glucose
(0.77g/100mL) to 50g/L glucose supplementation (Table
1).
Table 1 shows the different values of biomass production
from banana fruit. The highest yeast biomass was
recorded in 50g/l glucose concentration and lowest one
was on 30g/L glucose concentration (0.22g/100mL).
Alcohol production from apple, orange and banana
fruits.
The result of alcohol (ethanol) production from fermented
apple, orange and banana fruits showed that the
increased glucose conc. enhance the ethanol yield.
Same concentration of glucose (50-70g/L) was used to
produce ethanol from yeast isolated from fruits (apple,
orange and banana). In this study maximum ethanol
production by the yeast was recorded from apple
(0.412g/100mL) at 70g/l glucose concentration where as
minimum yield (0.069g/100mL) was obtained at 50g/l of
glucose concentration (Table-2).
The volume of ethanol in orange was formed
0.031g/100mL in 50g/L glucose while the largest volume
of ethanol was 0.252g/L 100mL in 70g/L glucose. The
variation in ethanol yield obtained from fermentation of
orange isolate was shown in table 2.
Lastly, the alcohol estimation from banana isolate
fermentation gave maximum ethanol yield
(0.362g/100mL) at 70g/L of glucose concentration and
0.072g/100mL) at 50g/L of glucose concentration. The
variation in ethanol yield obtained from fermentation of
banana isolate was given in table 2.
DISCUSSION
Apple, orange, banana and other fruits locally available
and thus serve as readily available raw materials for the
separation of ethanol yeasts. Eghafona (1999) isolated
various strains of indigenous yeasts capable of producing
ethanol from local fermented pineapple juice. Bansal and
Singh (2003) and Hossain et al (2014) did comparative
study on ethanol production from molasses using
Saccharomyces cerevisiae and Zymomonas mobilis.

4. Production of alcohol by yeast isolated from apple, orange and banana
Nasreen et al. 018
Glucose with different amounts (50, 10, 30, 50, and 70g/l)
was used as a sole sugar in the MGYP medium; the
consequences showed that the maximum yeast biomass
and maximum ethanol yield was obtained at high glucose
concentration. Similar result was shown by Xin, et al
2003. Viability of Saccharomyces spp also studied by
Moaris (1996) and Aguilar (2010). In 50% glucose,
reported a viability of 10-98.8% in different strains of
yeast.
The proximate analysis of various fruits (Apple, orange
and banana) was not determined in this study, but Essein
et al (2005) recorded crude protein and crude fat
contents of 78 and 116g/L, respectively in banana peels.
Protein is an essential nutrient for yeast growth while fat
is vital for the structure and biological functions of the cell
and can be utilized as alternative source of energy by the
cells.
The isolates exhibited high flocculation ability, and
fermented higher amount of glucose to yield appreciable
amount of ethanol. In present experiment, the alcohol
estimation from banana isolate fermentation gave
maximum ethanol yield (0.362g/100mL) at 70g/L of
glucose concentration and minimum yield
(0.072g/100mL) at 50g/l of glucose concentration. Brooks
(2008), isolated yeast strains from ripe banana peels for
ethanol production and found, that isolates fermented
40% glucose at 30
o
C to yield 3.6 and 5.8% ethanol
respectively.
Temperature is one of the major constrains that
determines the ethanol production. In this study the
experiment was carried out at 30
o
C with 70g/L glucose
concentration. The maximum amount of ethanol
produced by yeast cells using 70g/L glucose was
1.36g/100mL. Sree (2000) were also carried out
fermentation with initial glucose concentration of 150, 200
and 250g /litre at 30o
C the maximum amount of ethanol
produced by yeast cells using 150,200 and 250g/L
glucose was 72.5,83 and 93g ethanol per litre at 30o
C
after 48h. Similarly, Yadav et al (1997) also obtained high
ethanol yield (40g/l/h) at 30o
C. So present study was also
carried out at 30o
C in 250 mL conical flasks for 72h and
obtained the highest ethanol yield (0.412g/100mL) at 7
percent glucose concentration which was the maximum
concentration used in this experiment.
ACKNOWLEDGEMENT
The authors thank to the PCSIR labs. for financial
support.
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Accepted 17 September, 2014.
Citation: Nasreen Z, Jabeen S, Shafique M, Usman S,
Yaseen T, Yasmeen A, Nazir S (2014). Production of
alcohol by yeast isolated from apple, orange and banana.
International Journal of Food and Nutrition Sciences 1(2):
016-019.
Copyright: © 2014. Nasreen et al. This is an open-access
article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium,
provided the original author and source are cited.