ABSTRACT

The natural fermentation of apple under anaerobic condition was

investigated. This was done by soaking 45g of chopped apple in 100ml of sterile
distilled water, all in a fermenter for a given period of 5days. The fermentation
process, which was limited to 5days, was monitored daily starting from day 0 to
day 4. Some parameters like; titrable acid, pH, fungal count and bacterial count
were monitored daily. It was then realized that as the fermentation proceeded, there
was increase in the value of the titrable acid and decrease in pH as well as the
growth of yeast increased at the expense of bacterial growth.

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A project on the fermentation of apple carried out by IBILEYE, Ajibola Ayodeji (MCB/06/9106)
INTRODUCTION

Fermentation can be defined as the chemical transformation of organic

compounds with the help of enzymes to form new products, which are useful to
mankind.
Different fermentation processes are used depending on the desired product,
and many of these processes have been described in the literature. Some forms of
fermentation are naturally occurring, meaning the ferment, where the yeast, mold,
or bacteria, is already in the reactant. In other situations, the ferment must be
carefully added under specific conditions. Still, the fermentation process is the
same; sugar molecules in the substance are converted by the yeast, mold, or
bacteria to something else, such as alcohol or lactic acid or malic acid
In addition to being used in drinks and food, the process of fermentation is
used to make products far more important. Fermentation products have both
industrial and medicinal applications. Antibiotics, including penicillin and
riboflavin vitamins, are created by fermentation. Citric acid created by
fermentation is used in metal cleaners and in food preservation. This acid is
naturally occurring in citrus fruits, but it is uneconomical to obtain directly from
fruits. By fermenting molasses, the same acid is produced cheaply and efficiently
(Bamfort, 2000).
This laboratory experiment subjects apple to natural fermentation by leaving
apple to ferment without adding any starter culture or inoculum. This is done to
ascertain the kind of organisms associated with the alcoholic fermentation of apple.
In alcoholic fermentation, yeast converts glucose to ethanol and carbondioxide.
yeast
C6H12O6 2CH3CH2OH (l) + 2CO2 (g)
The apple is the pomaceous fruit of the apple tree, species Malus domestica
in the rose family (Rosaceae). It is one of the most widely cultivated tree fruits, and

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A project on the fermentation of apple carried out by IBILEYE, Ajibola Ayodeji (MCB/06/9106)
the most widely known of the many members of
genus Malus that are used by humans. The tree
originated in Western Asia, where its wild ancestor,
the Alma, is still found today. There are more than
7,500 known cultivars of apples, resulting in a range of desired characteristics.
Cultivars vary in their yield and the ultimate size of the tree, even when grown on
the same rootstock.
MATERIALS AND METHODOLOGY
Collection of substrate (apple) and fermenter
Apple fruits were purchased from road side hawkers in Oluyole industrial
estate, Ibadan, Oyo state, Nigeria and improvised fermenter was used.
Materials
Apple, fermenter, syringes, sterile distilled water, sodium hydroxide
(NaOH), pH meter, pipette, conical flask, indicator (phenolphthalein), Petri dishes,
Agar (potato dextrose agar and nutrient agar), autoclave, weigh balance and
incubator.
Preparation of plates
Five fermenter were set and 100ml of distilled water was dispensed into
each of the fermenter. The apple was chopped into pieces, 45g of the pieces was
weighed using a weigh balance and soaked in each of the fermenter containing
distilled water.
Determination of pH and titrable acid.
The pH and titrable acid (TA) were taken for 0th (0hrs), 1st (24hrs), 2nd
(48hrs), 3rd (72hrs), 4th (96hrs) day. A 1ml of water from the soaked apple was put
in the Petri dishes and the same volume into other Petri dishes. Some sets of the
samples inside the Petri dishes were overlaid with nutrient agar while the other sets
were overlaid with potato dextrose agar. The plates were then incubated at 370C for
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A project on the fermentation of apple carried out by IBILEYE, Ajibola Ayodeji (MCB/06/9106)
24 hours for nutrient agar and 250C for 72 hours for potato dextrose agar. After
which the bacterial and fungal counts were taken.
A 20ml of the sample was titrated against 0.1M of sodium hydroxide, one
drop of the indicator; phenolphthalein was added to the sample before the titration
process began. The pH meter was calibrated and standardized before the electrode
was inserted into 25ml of the solution (the water in which the apple is soaked) in a
clean beaker and the pH was read. This same procedure was repeated daily.
Several pH values were read for a solution but the value recorded is the mean of
three values read.
RESULTS AND DISCUSSION
The result of the titrable acid (TA) (g/ml) shows that there is an increase in
the acidity.
As the fermentation proceeds, the pH decreases tending towards strong acidity
level. A graph of pH, TA, fungal count and bacterial count was plotted against time
(days).
This result reveals that the growth of bacteria and fungi is inversely
proportional and also that the bacterial population tends to diminish as the acid
becomes much stronger, which gives room for yeast to increase in number. At this
time, yeast outnumbered bacteria owing to the concentration of acid produced. It is
read that lactic acid bacteria are the colonizers at the start of the fermentation and
the eventual increase in the concentration of acid produced by these bacteria
reduces their number while the growth of yeast is favoured (Adams et al., 2000).
The titrable acid (g/ml) is inversely proportional to the pH of the acid
produced. It is observed that as the fermentation process tarries, there is reduction
in pH and appreciation in the value of titrable acid (g/ml). The observation of the
nutrient agar plate from the culture of the 0th day revealed higher population of
gram positive cocci; most of these cocci are clustered, a characteristic feature of
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A project on the fermentation of apple carried out by IBILEYE, Ajibola Ayodeji (MCB/06/9106)
Streptococci while some are straight chain, a characteristic feature of
Staphylococci. The bacterial culture plate of the 1st day showed the presence of
gram negative bacilli, which may be opportunists- Pseudomonas aeruginosa,
Escherichia coli as a result of environmental contamination.

Table 1. Relationship between titrable acid, pH and microbial count during the
fermentation of apple.

Days pH TA Bacterial Fungal Volume Organisms Organisms

in (g/ml) count on count on of isolated on isolated on
hours Nutrient Potato sample Nutrient Potato
agar Dextrose used in agar Dextrose
(cfu/ml) agar TA (ml) agar
(sfu/ml)
0 4.3 0.36 160 90 20 GPC Mould
24 4.1 0.38 200 182 20 GNB Yeast
48 3.8 0.40 190 362 20 GPB and Yeast
GPC
72 3.6 0.60 175 420 20 GPB Yeast
96 3.4 0.80 120 382 20 GPB Yeast
Keys:
GPC – gram positive cocci
GNB – gram negative bacillus
GPB – gram positive bacillus

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A project on the fermentation of apple carried out by IBILEYE, Ajibola Ayodeji (MCB/06/9106)
The bacterial culture plate of the 2nd day revealed the presence of both gram
positive cocci, which may be Staphylococcus aureus, Streptococcus lactis and
gram positive bacillus, which may be Corynebacterium spp. The bacterial culture
plate of the 3rd day also revealed the growth of gram positive bacillus.
On the 4th day, the bacterial culture plate revealed the growth of gram
positive bacillus and there was an appreciable growth of yeast, which is suspected
to be related to Saccharomyces; (Saccharomyces cerevisiae, Saccharomyces
oviformis, and Saccharomyces steineri). Apple may undergo two different types of
fermentation when subjected to natural fermentation; it can be acid fermentation,
which produces more acid (malic, lactic or citric acid) as end product or alcoholic
fermentation, which produces alcohol (ethanol) as the end product. Fermentation
of apple occurs in the presence of yeast, mold or bacteria depending on the nature
of the product produced (Garbutt, 1997).
This experiment revealed that the number of yeast increases over time as the
acidity increases while the number of bacteria decreases as acidity increases over
time (although the number of yeast tends to diminish with time if fermentation
process tarries). This connotes the ability of yeast to survive at a lower pH as
against bacteria. More so, this experiment was carried out without using starter
culture or busterring the fermentation process.

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A project on the fermentation of apple carried out by IBILEYE, Ajibola Ayodeji (MCB/06/9106)
 GRAPHICAL REPRESENTATION OF RESULTS

The graph of pH against days in hours

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4.5
pH values of the fermentation process

0, 4.3
4 24, 4.1
36, 3.8
3.5 48, 3.6
96, 3.4
3
2.5
2 Series1
1.5
1
0.5
0
0 20 40 60 80 100 120
number of days in hours

Fig 1. The graph of pH of fermentation against number of days in hours

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A project on the fermentation of apple carried out by IBILEYE, Ajibola Ayodeji (MCB/06/9106)
 The graph of titrable acid (TA) against days in
hours
0.9
0.8 96, 0.8
0.7
titratble acid (g/ml)

0.6 48, 0.6

0.5
0.4 24, 0.3836, 0.4
0, 0.36 Series1
0.3
0.2
0.1
0
0 20 40 60 80 100 120
number of days in hours

Fig 2. The graph of titrable acid against number of days in hours

The graph of bacterial count (cfu/ml) against

days in hours
250

200 24, 200

Bacterial count (cfu/ml)

36, 190
48, 175
0, 160
150

96, 120
100 Series1

50

0
0 20 40 60 80 100 120
number of days in hours

Fig 3. The graph of bacterial count against number of days in hours

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A project on the fermentation of apple carried out by IBILEYE, Ajibola Ayodeji (MCB/06/9106)
 The graph of fungal count against days in
hours
450
48, 420
400
96, 382
350 36, 362
Fungal count (sfu/ml)

300
250
200
24, 182 Series1
150
100 0, 90
50
0
0 20 40 60 80 100 120
number of days in hours

Fig 4. The graph of fungal count against number of days in hours

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A project on the fermentation of apple carried out by IBILEYE, Ajibola Ayodeji (MCB/06/9106)
CONCLUSION
Fermentation process involving the production of foods and drinks is highly
significant to mankind, because myriads of new products are derived from the
technology of fermentation. The end product of the fermentation of apple in this
experiment (wine) can be drunk if pasteurized following normal procedures. Much
as the result of this experiment complies with those usually gotten (production of
cider from apple juice), it is discovered that apple can undergo natural fermentation
without addition of starter culture, follow the pathway chosen by the ambient
environmental condition to produce useful end product(s).

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A project on the fermentation of apple carried out by IBILEYE, Ajibola Ayodeji (MCB/06/9106)
 References
1. Adams, M. R. and Moss M. O. (2000); Food Microbiology, 2nd Edition, pg
80-98, Royal Society of Chemistry, London.
2. Bamforth, C. W. Chemistry Educator [online] 2000, 5, 102-112;
3. Garbutt, J (1997) Essentials of Food Microbiology, pg 210, Hodder Arnold,
London.

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A project on the fermentation of apple carried out by IBILEYE, Ajibola Ayodeji (MCB/06/9106)