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. C6H12O6
yeast

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 in hours pH TA (g/ml) Bacterial Fungal Volume Organisms Organisms isolated on isolated on Nutrient in agar Potato Dextrose agar

count on count on of Nutrient agar (cfu/ml) Potato Dextrose agar (sfu/ml) sample used

TA (ml)

0 24 48

4.3 4.1 3.8

0.36 0.38 0.40

160 200 190

90 182 362

20 20 20

GPC GNB GPB GPC

Mould Yeast and Yeast

72 96 Keys:

3.6 3.4

0.60 0.80

175 120

420 382

20 20

GPB GPB

Yeast Yeast

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
5 pH values of the fermentation process 4.5 0, 4.3 4 3.5 3 2.5 2 1.5 1 0.5 0 0 20 40 60 80 100 120 Series1 24, 4.1 36, 3.8 48, 3.6 96, 3.4

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 0.7 titratble acid (g/ml) 0.6 0.5 0.4 0, 0.36 0.3 0.2 0.1 0 0 20 40 60 80 100 120 36, 24, 0.38 0.4 Series1 48, 0.6 96, 0.8

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 0, 160 96, 120 100 Series1

Bacterial count (cfu/ml)

24, 200 36, 190 48, 175

150

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
450 400 Fungal count (sfu/ml) 350 300 250 200 150 100 50 0 0 20 40 60 80 100 120 0, 90 24, 182 Series1 48, 420 36, 362 96, 382

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)