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Universal Journal of Microbiology Research 3(4): 41-45, 2015 http://www.hrpub.org
DOI: 10.13189/ujmr.2015.030401
Wine Production from Mixed Fruits (Pineapple and
Watermelon) Using High Alcohol Tolerant Yeast Isolated
from Palm Wine
Okeke, B.C, Agu, K.C.*, Uba, P.O., Awah, N.S., Anaukwu C.G., Archibong, E.J.,
Uwanta, L.I., Ezeneche, J.N. Ezenwa, C.U. Orji, M.U.
Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University, Nigeria
Copyright © 2015 by authors, all rights reserved. Authors agree that this article remains permanently open access under the
terms of the Creative Commons Attribution License 4.0 International License
Abstract Mixed fruit wine (pineapple and watermelon)
was produced using Saccharomyces cerevisiae isolated from
palm wine. Primary and secondary fermentation of the fruits
lasted for 7 and 28 days respectively, during which aliquot
samples analysis of pH, titrable acidity, specific gravity,
alcohol content and reducing sugar were carried out using
standard procedures. Specific gravity of the wine was
observed to reduce drastically as the fermentation progresses.
The pH of the fruit must during the period of fermentation
ranged from 3.0 to 4.46. During the fermentation period,
consistent increase in alcohol content was observed with
time. At the end of the 28th day of fermentation, the alcohol
content was observed to be 3.2%. The titrable acidity of the
wine was observed to show steady trend with time
throughout the period of fermentation. This study showed
that acceptable wine can be produced from mixed fruits
pineapple and watermelon using yeasts especially
Saccharomyces cerevisiae isolated from palm wine.
Keywords Wine, Palm Wine, Pineapple, Watermelon,
Yeast, Fermentation
1. Introduction
Wine is any alcoholic beverage produced from juices of
variety of fruits by fermentative action of microorganisms
either spontaneously or seeding with a particular strain
mainly of yeast species to adopt a particular quality of wine.
Wine is one of the most recognizable high value added
products from fruits.
Most commercially produced wines are usually made
from fermented grapes; this fermentation process is not done
by introducing any chemicals or sugar but by adding
different species of yeast to the crushed grapes. Yeast has the
capability of converting grapes into an alcoholic compound
and removing the sugar content in it for the production of
different types of wines. Sometimes wines are produced
from different types of fruits like; Paw-Paw, mango,
Pineapple, Banana, Lemon, Watermelon etc., here the wine
so produced bears the name of the fruit or fruit mixture used
in its production1.
In the European Union, wine is legally defined as the
fermented juice of grapes3. Wine can be made from virtually
many plant matters that can be fermented3. Most fruits and
berries have the potential to produce wine. Wine making
involves the use of yeast to ferment the ‘must’ of a chosen
fruit or fruits for a number of days, depending on the
objective of the winemaker. The yeast which is the main
organism responsible for alcoholic fermentation usually
belongs to the genus Saccharomyces.
Palm wine is the fermented sap of the tropical plant of the
palmae family. It is produced and consumed in very large
quantities in the Southeastern Nigeria. It contains
nutritionally important components including amino acids,
proteins, Vitamins and sugar2. These make this wine a
veritable medium for the growth of a consortium of
microorganisms, where growth in turn, change the
physicochemical conditions of the wine giving rise to
competition and succession of organism. Ogbonna (1984)
and Onyedinma (1983) used palm wine isolates of
Saccharomyces cerevisiae to produce artificial palm wine
and beer, respectively.
Palm wine is tapped from the sap of Elaesi Species and the
sap of Raphia Species which contains a heavy suspension of
live yeasts and bacteria2. Most studies on palm win have
reported its potentials are source of yeast isolate for the
fermentation industries. Okafor, (2007) in his study isolated
seventeen yeast strains, four belonging to the species of
Candida, twelve to the genus of Saccharomyces and one to
Endomycopsis species.
The objective of this study is to produce wine from mixed
fruits: Pineapple and Watermelon to study the fermentation
42 Wine Production from Mixed Fruits (Pineapple and Watermelon) Using High Alcohol
Tolerant Yeast Isolated from Palm Wi ne
process of the wine and encourage its occurrence. pH,
temperature, Reducing sugar and filterable acidity tests were
all analyzed quantitatively in the course of this work.
2. Materials and Methods
2.1. Sample Collection
Ripe watermelon fruits (Citrullus lanatus var lanatus) and
pineapple fruits (Ananas comosus) were bought from Eke
Market, Awka, Anambra State. The fruits were washed
thoroughly with sterile water and rinsed with deionized
water containing 0.1% formaldehyde. Fresh palm wine was
obtained from a palm wine tapper in Amanseavillage, Awka,
Anambra State, Nigeria.
2.2. Isolation of Yeast from Palm Wine
Fresh palm wine was obtained from Amensea village
Awka, Anambra State and fermented for 24hours.The fresh
palm-wine was allow to sediment and the sediment was
inoculated onto Malt extract agar in duplicates. The plates
were incubated at room temperature for 24 hours.
Developing isolates were purified by repeated subculture
techniques and slides of pure culture were prepared for
microscopic observation and identification using lactophenol
cotton blue and the pure cultures were identified by their
morphological characteristics.
2.3. Inoculum Development
The pineapple and watermelon were washed thoroughly
with 0.1% sodium metabisulphite in water. The fruits were
cut, manually deseeded, blended and filtered to obtain the
juice. About 200ml of the juice (100ml of pineapple juice
and 100ml of Water melon juice) were introduced into a
clean sterile 500 ml conical flask and sterilized by
autoclaving. Upon cooling, 3 loopful of the yeast culture
isolated from the palm-wine was used to inoculate the juice
and incubated in a rotary shaker for 48 hours.
All procedures were done under aseptic condition.
2.4. Must Fermentation
The pineapple and watermelon were washed thoroughly
with 0.1% sodium metabisulphite in water. The fruits were
cut, manually deseeded, blended and filtered to obtain the
must.Aliquots of the extracted juice obtained and used for
pH, temperature, titrable acidity and reducing sugar analysis.
The must was transferred into a sterile 500 ml glass
fermenter, until it was ¾ filled. This was followed by the
addition of 0.4g Sodium metabisulphite, 100g of granulated
sugar (for fortification), 29.4g of 0.84% Ammonium
sulphate 4.2g of 0.12% potassium dihydrogen for yeast
supplementation. The juice was inoculated with yeast
obtained by inoculum development and the set-up allowed to
ferment for 28 days, with daily analysis of parameters such
as: pH, temperature, reducing sugar and titrable acidity.
2.5. Physiochemical Analyses
pH Determination
Ten ml of the “must” was put into a sterile beaker, and the
pH of the must determine using a pH a digital pH meter
(Model No: pH S-25)
2.6. Determination of Reducing Sugar
The quantitative estimation of reducing sugar of the wine
was determined using the method described by Miller (1971).
One ml of 3,5-Dinitrosalicyclic acid (DNS) was added to
1ml of supernatant of sample, in a test tube and the mixture
heated in boiling water for 10minutes. The test tube was
cooled rapidly in tap water and the volume adjusted to 12ml
with distilled water. A blank containing 1ml of distilled
water and 1ml of DNS was prepared. The optical density of
the sample was read against the blank in the
spectrophotometer or 540nm absorbance. The concentration
of reducing sugar in the supernatant was estimated from the
glucose standard curve.
2.7. Determination of Specific Gravity
Fifty ml specific gravity bottle was thoroughly cleaned
with distilled water, dried in an oven for 50oC and allowed to
cool. The weight of the cooled dried bottle (W1) was
recorded. The dried bottle was filled with deionized water
and surface of the bottle was cleaned with a cotton wool and
weighed as (W2).
The bottle was empty and cleaned twice with 10ml of the
“must” thereafter the bottle was filled to the brim with the
“must” and the bottle cleaned with cotton wool and weighed
as (W3). The specific gravity (S.G) was calculated
31
2 1�
–
. –
WW S
SG WW W
= =
Where
S = weight of volume of must (W3 – W1)
W = weight of volume of water (W2 – 1)
2.8. Estimation of Titrable Acidity
This was determined by the methods described by
Amerine and Ough14. 200ml of distilled water was
introduced into a sterile 500ml conical flask and boiled. 1ml
of 1% aqueous alcoholic phenolphthalein indicator solution
was added. This was titrated with 0.1M NaOH solution to
give a faint pink colour. 5ml of the “must” was pipette and
introduced into the boiling neutralized solution and titrated
again to the end point using the same 0.1M NaOH solution.
The titrable acidity was expressed as tartaric acid and was
calculated thus:
Universal Journal of Microbiology Research 3(4): 41-45, 2015 43
75 100
/100
100
V xM x x
Tartaric acid g ml xV
=
Where;
V= Volume of NaOH (final reading- initial reading).
M = Molarity of NaOH
V = Volume of “must”
3. Results
Tab le 1. Analysis of the fruit Juice
Parameters Mean values ± S.D
Pineapple Watermelon Mixed juice
pH 4.22 ± 0.01 5.07 ± 0.01 4.47 ± 0.01
Reducing sugars 0.80 ± 0.00 0.81 ± 0.00 0.71 ± 0.00
Specific gravity 43.91 ± 0.01 43.04 ± 0.06 40.93 ± 0.04
Tab l e 2 . Protein and Moisture content values of Pineapple and
Watermelon
Parameters Values (mean ± S.D)
Pineapple (per 100g) Watermelon (per 100g)
Protein content 0.55 ± 0.01 0.62 ± 0.01
Moisture content 86.05 ± 0.07 91.46 ± 0.01
Throughout the period of fermentation, pH of the must
was within the acidic range. This was irrespective of the
yeast strain used for fermentation. pH ranged from 4.46 to
3.2 (Fig.1). As shown in Fig.1, a steady increase in alcohol
content was observed in the fruit wine throughout the period
of fermentation with the fermenting yeast strain. The
increases were irrespective of the yeast strain and the fruit
used. At the end of the 28th day, of the fermentation, the
concentration of alcohol in the fruit wines were observed to
range from 0% to 3.0.
Figure 1. Wine Analyses
In the case of specific gravity of the fruit wine gradual
decrease in valves were observed throughout the period of
fermentation. These decreases were observed to be
irrespective of the yeast strain and the fruits used in the wine
production. Between 21 days to 28 days of the fermentation,
specific gravity valves were observed to range from 0.922 to
0.901kgm-3.
Fig.1 also showed the trend in titrable acidity, this was
observed to show a steady increase with time throughout the
period of fermentation. At the 28th day of fermentation, acid
concentration in the fruit wine was observed to increase from
the initial concentration ranges of 0.55% to 1.01%. In the
case of reducing sugar of the wine during the period of
fermentation, the values of the reducing sugar of the wine
were observed to be decreasing. It was high on the first day
which was the start of the wine fermentation and due to the
presence of sugar in the fermenting wine, the reduction in the
reducing sugar is due to the activities of the fermenting yeast
on the wine and the production of alcohol. The reducing
sugar ranged from 0.712g on the first day to 0.202g on the
28th day.
4. Discussion
In this research work, the choice of the fruits: Pineapple
and Watermelon were deliberate. From table 1, it was
observed that the moisture content of the fruits ranged from
86g to 91.45g and accounts for their high perishable nature
and their short shelf life under normal storage condition6.The
fruits contained reasonable amount of carbohydrate, which
gives an account of their high caloric value.
This work has shown that the pH ranges of the pineapple,
watermelon, and mixed fruit juice used for the production of
the wine for this research were 4.22 ± 0.01, 5.07 ± 0.01 and
4.47 ± 0.01 respectively, while there was no significant
difference in the values of the reducing sugars amongst the
three samples. In the case of Specific Gravity, there was no
obvious difference between pineapple and watermelon, but
diifered slightly with the value obtained for mixed fruits.
Also revealed are consistent increases in acidity (titrable)
of the wine throughout the period of fermentation. Studies
have shown that during fermentation of fruits, low pH is
inhibitory to the growth of spoilage organisms but creates
conducive environment for the growth of desirable
organisms. Also, low pH and high acidity are known to give
fermenting yeasts competitive advantage in natural
environments4. The titrable acidity of the final wine is
expected to be between 0.5 to 1.0%.
In this study, the results of titrable acidity of the wine fell
within this limit.In order to supplement the sugar content of
the “musts” granulated sugar which is from cane sugar and a
source of “sucrose” was part of the additives. Reports have
shown that the major problem associated with the use of
tropical fruits in wine production is their low sugar content8.
Remarkable amount of alcohol was produced from the
fruit wines during fermentation with the yeast strain used.
This trend was consistent during the course of fermentation.
In general, the percentage alcohol produced from the fruits
used for the fermentation by the yeast strain was above 2%
which is comparable with moderate grape wines 9,5,11. The
44 Wine Production from Mixed Fruits (Pineapple and Watermelon) Using High Alcohol
Tolerant Yeast Isolated from Palm Wi ne
performance and potential of the yeast strain as substituent
for the commercial baker’s yeast was measured by the
amount of alcohol produced. High alcohols are known to be
important precursors for the formation of esters, which are
associated with pleasant aromas10.
In the present study, the amount of alcohol produced by
the isolate the yeast from the palmwine did not show any
difference from palm wine10.
Reports have shown that alcoholic fermentation leads to a
series of by-products in addition to ethanol. Some of the
by-products include carbonyl compounds, alcohols, esters,
acids and acetals, all of them influencing the quality of the
finished product. The composition and concentration levels
of the by-products can vary widely (mg/L to hundreds of
mg/L)12,13.
In this study, pH of the fruit wine throughout the period of
fermentation ranged from 3.0 to 3.5. A similar observation
has been reported by Reddy and Reddy; in their study on
mango fruit, Optimum pH value for quality wine production
was 4.04.
The specific gravity of the fruit wine produced in this
study reduces as the fermentation days of the wine increases.
After the 28 days fermentation the specific gravity of the
wine reduced drastically to 0.901kgm-3 this was due to the
type of yeast used in the wine production. Saccharomyces
cerevisiae isolated from palm wine has been reported to
reduce specific quality of fruit wines during fermentation9.
The short shelf-life of beverages are however a major
problem faced by their producers and consumers in
Africa32,33.
The specific gravity values of the wine were observed to
diminish by significant value of p ≤ 1.01 ± 0.011. The type
and aroma produced during wine production is reported to
depend on yeast, environmental factors and
physico-chemical characteristics of the “musts”.
Acknowledgements
I acknowledge the entire Laboratory staff of the
Department Of Applied Microbiology and Brewing for their
efforts in the Isolation, characterization and identification of
the Yeast (Saccharomyces cerevisiae) used for this study.
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