2. It is mainly produced from the lala palm (Hyphaene
coriacea) by cutting the stem and collecting the sap. In
part of central and western Democratic Republic of the
Congo, palm wine is called "malafu" (Wikipedia, 2010).
Composition of palm wine
The main ingredient of the fresh palm sap is sucrose,
which is about 12-15% by weight. There is little
reducing sugar such as glucose, fructose, maltose and
raffinose which are present. In addition to sugar, the
sap also contains 0.23% protein, 0.02% fat, mineral
matter and ascorbic acid 5.7 mg/100ml (Nwokeke,
2001). Generally, palm wine is good for the body.
According to Bassir (1982), palm wine’s major
constituents are; carbohydrate, organic acid, protein,
vitamin C and Ash.
Fermentation of palm wine
The palm wine fermentation is a lactic alcoholic acetic
one, involving lactic acid bacteria, yeast and acetic acid
bacteria as well as Zymomonas and Leuconostoc which
are normal flora of the sap (Okafor, 1972; Faparusi and
Bassir, 1972; Faparusi, 1973). The types and numbers
of organisms encountered vary widely, even from tree
to tree (Theivendirarajah et al., 1977).The fermentation
of raphia palm wine is considered an in-expensive and
effective means of food production in Nigeria, fresh
palm sap are usually converted to palm wine during
storage. Bacteria and yeasts usually contaminate the
juice as it is tapped and there are changes in
biochemical composition of the palm wine. Previous
studies on the microbiology of E guineensis and R.
hookeri have incriminated several bacteria and yeast
flora to be involved in the fermentation process
(Faparusi and Bassir, 1972a; Okafor, 1972ab; Okafor,
1975b; Eze and Ogan, 1987; Amanchukuru et al., 1989;
Ejiofor, 1994; Orimaiye, 1997; Nester et al., 2004).
Lactic acid bacteria (LAB) are responsible for many
fermented foods such as sauerkraut, pickles and
yogurt. They have also been isolated from palm wines
at various states of fermentation. Many species of LAB
do not conduct malolactic fermentation and their growth
in wine can cause some serious wine spoilage
(Bousbouras et al., 1971). Types of lactic acid bacteria
found in palm wine belongs to three genera, namely:
• Leuconostoc - Heterofermentative cocci, oval or
spherical, occur in pairs or chains.
• Pediococcus - Homofermentative cocci, often found
in tetrads.
• Lactobacillus - Homofermentative or
heterofermentative rods, found singly or in chains.
These organisms are gram positive, catalase negative,
non-sporing cocci, coccobacilli or rods. They are
microaerophilic that means they grow well under
conditions of low oxygen content. Since they can grow
under low oxygen conditions, they can grow throughout
the wine (as opposed to on the surface of the wine)
even though the container is kept full. The bacteria can
metabolize sugars, acids and other constituents in wine
and produce several compounds. Some of these are
undesirable and constitute spoilage.
Upon fermentation by the natural microbial flora, the
sugar level decreases rapidly as it is converted to
alcohol and other products (Obire, 2005) whereas, the
sap becomes milky-white due to the increased microbial
suspension resulting from the prolific growth of the
fermenting organisms (Okafor, 1975a,b). During
fermentation, sugar is hydrolyzed to glucose and
fructose. Reducing sugar is found only in trace amounts
at the beginning, increases as sucrose is hydrolyzed.
During the first 24hrs more than half of the total sugars
are fermented, with sucrose always used most rapidly.
At this time, many consumers prefer to stop
fermentation with the pH about 5. Acetic, lactic acids
are produced along with ethanol during fermentation.
After about 36 to 48 hrs, the concentration of ethanol
reaches a maximum of 5.0 to 5.28% v/v; the
concentration of ethanol soon start to increase in the
palm wine as is stored for a longer period. The pH of
the original palm sap is about 7.2 and after 8 hrs, it falls
to 5.5 to 5.8, at which stage of fermentation by the
yeast Saccharomyces cerevisiae is most vigorous
(Okafor, 1972). By the time the alcoholic content is
about 5.0%, the pH has fallen to around 4.0 to 4.3.
Faint sulphur like odour may also be present.
According to Faparusi, Bassir and Okafor(1991 and
1987), the bacteria that are most predominant in palm
wine after fermentation are Micrococcus, Leuconostoc,
Lactobacillus and Acetobacter; while the predominant
yeasts usually identified are Saccharomyces and
Candida spp. The types of bacteria present appear to
depend on the stage of fermentation and the
composition of the sap (Bassir, 1962; Okafor, 1977).
Although alcohol production is common among yeasts,
it is rare among bacteria (Ingraham and Ingraham,
2004).
Uses of palm wine
Palm wine has several nutritional, medical, religious
and social uses which have been reported to have
increasingly enhanced the demand for this natural
product (Faparusi, 1966; Odeyemi, 1977; Ikenebomeh
and Omayuli, 1988; Uzogara et al., 1990; Iheonu,
2000),. The use of palm wine in religion ceremonies is
common to many cultures and regions. Libations (that is
gift of palm wine to a god) often included palm wine.
Palm wine plays an important role in many ceremonies
in parts of Nigeria such as among the Igbo (or Ibo)
peoples, and elsewhere in central and western Africa.
Guests at weddings, birth celebrations, and funeral
wakes are served generous quantities.
Palm wine is often infused with medicinal herbs to
remedy a wide variety of physical complaints (medical
uses), Epidemiological studies have consistently
Akinrotoye KP 5
3. demonstrated that moderate consumption of palm wine
is statistically associated with a decrease in death due
to cardiovascular diseases such as heart failure,
although excessive consumption of palm wine with high
alcohol content has adverse health effects (Lindberg
and Amsterdam, 2008). As a token of respect to
deceased ancestors, many drinking sessions begin with
a small amount of palm wine spilled on the ground.
Palm wine is enjoyed by both men and women,
although women usually drink it in less public venues.
Palm wine also serves a culinary use, In the Indian
State of Kerala, palm wine is used in leavening (as a
substitute to yeast) a local form of hopper called the
vellai Appam, a staple among the Nasrani Christians.
Palm wine is mixed with rice dough and left over night
to aid in fermentation and expansion of the dough
causing the dough to rise overnight, making the bread
soft when prepared. In Kerala, palm wine is vending
under the licence issued by the excise department and
it is an industry having more than 50,000 employees
having a welfare board under the labour department. It
is also used in the preparation of a soft variety of Idli,
which is famous in the parts of Karnataka and Goa in
India. Palm wine tapping has also provided jobs for
many rural dwellers from generations (David, 2003; and
Berkowitz, 1996).
Antibacterial properties of palm wine
In Thailand, fermented plant beverages (FPBs) such as
palm wine produced by lactic acid fermentation are
consumed by many Thai people who believe that these
products have a high nutrient value and some
antimicrobial activities. Thailand is located in the
tropical zone and diarrheal diseases caused by food
borne infection and food toxicity is endemic (FAO/WHO,
2002) and locally produced FPBs are thought by some
Thai people to be able to protect themselves from these
food borne illnesses. This present work is to found out
if palm wine can be used to treat diarrhoeal diseases by
evaluating its antibacterial potential on common
diarrhoeagenic bacteria in this part of this country.
Objectives of this research work
• To investigate the antibacterial potential of palm
wine on common bacteria that cause diarrhoea as
an alternative to antibiotic treatment.
• To study the succession of microorganisms
involved in the fermentation of palm wine.
• To monitor total titratable acidity and pH values of
palm wine during fermentation.
MATERIALS AND METHODS
Source of palm wine samples used
Freshly top-tapped palm wine from Raphia palm tree
(Raphia hookeri) was brought from a local palm wine
tapper in Akure metropolis. The freshly tapped samples
were collected using sterilized labelled 1Litre capacity
sample bottles with screw caps. This method of
collection according to Bassir (1962) and Obire (2005),
reduces fermentation rate considerably before the palm
wine is taken into the laboratory for investigation.
Source of test organisms
E.coli 0157:H7, Salmonella typhi, Staphylococcus
aureus, Shigella dysentariae, and E.coli were collected
from post-graduate research laboratory and then
confirmed on different media to ascertain their identity
e.g MacConkey, Sorbitol McConkey, Eosin methylene
blue (EMB) agar etc.
Types of material used
The glasswares used in this research work include
petri-dishes, test-tubes, pipette, and beakers. These
were washed with detergent and rinsed well with clean
water. They were then sterilized in the oven at 160
0
C
for 2hours. The other materials used include nutrient
agar (NA), potatoes dextrose agar (PDA), De Man
Rogosa Sharpe agar (MRS agar), and Nutrient broth.
These were prepared according to manufacturer’s
specification and sterilized in the autoclave at 121
0
C for
15minutes before use. Other materials used include
Hydrogen peroxide, universal bottles, syringes,
centrifuge and beam-balance, Antibiotics
(chloramphenicol, tetracycline, ampicillin, ampiclox, and
ciprofloxacin).
Standardization of test-organisms
Standardization of the test organisms was carried out
by culturing the organisms on Nutrient broth for
24hours, after which they were centrifuged at 3000rpm
for 5minutes. The supernatant was then decanted and
10ml of sterile distilled water added and then re-
centrifuge for 5minutes to get the harvested cells. Serial
dilution of the harvested cells was then carried out and
plated on Nutrient agar and then incubated at 37
0
C for
24hours, after which the colonies were counted and
reported.
Assessment of growth inhibitory activity of palm
wine on the test organisms
This was done using agar diffusion method of Adebolu
and Adaramola (2010). One millilitre of standardized
cells of each of the test organisms was taken using a
sterile syringe and placed into sterile petri-dishes
(different organism per plate). Each plate was then
overlaid with 20ml nutrient agar, carefully swirled to
allow even distribution of the organisms within the agar
and were allowed to gel before 5 wells (8 mm in
diameter) were bored in the agar with the aid of a sterile
cork borer. Palm wine samples that were diluted in
10folds and undiluted were put into the wells; 0.1 ml per
well and different type for different well. Sterile distilled
water was used as control and the plates were
incubated at 37
0
C for 24h. The diameter of the zones of
6 Elite Res J. Bio M.
4. inhibition around the wells containing the palm wine
was determined and recorded. This assay was
repeated every 24h using fermented palm wine to
determine the effect of fermentation on the growth
inhibitory effect of the palm wine.
Antibiotic sensitivity assay
The antibiotics used are; Chloramphenicol,
Ciprofloxacin, Tetracycline, Ampiclox and Ampicillin.
Serial dilution of the test organisms was carried out and
1ml of each one was dispensed aseptically into different
petri-dishes. Sterilized Nutrient agar was poured into
the plates and allows to gel, wells were made on the
plates and 0.1ml of the antibiotics which has been
dissolved in 820ml of water (30µg/0.1ml) was then
dispensed into the wells and incubated at 37
0
C for
24hours. Diameter of zones of inhibition was recorded.
Isolation and characterization of bacteria present in
the fermented palm wine
Bacteria were isolated by direct streaking of raphia
palm wine samples on Nutrient agar, the plates were
incubated at 37
0
C for 24hours, and then sub-cultured
on De Man Rogosa Sharpe (MRS agar) incubated
anaerobically for 24 hours. Pure cultures of the isolates
were kept on nutrient agar slants and placed inside
refrigerator until used.
Gram staining
In the Gram reaction, the cells are first heat fixed and
then stained with a basic dye, crystal violet, which is
taken up in similar amounts by all bacteria. The slides
are then treated with lugol’s iodine (mordant) to fix the
stain, washed briefly with 95% alcohol (de-stained), and
finally counterstained with a paler dye of different color
(safranin). Gram-positive organisms retain the initial
violet stain, while gram-negative organisms are
decolorized by the organic solvent and hence show the
pink counter stain. The difference between gram-
positive and gram-negative bacteria lies in the ability of
the cell wall of the organism to retain the crystal violet.
Motility test
Motility is an inheritable phenotype and is a useful
criterion for identification and classification of bacteria.
This test was done to demonstrate whether isolate is
motile or not, culture used for this test was broth culture
grown within (18-24hours) old of motile
microorganisms, then 10ml of prepared nutrient agar
was poured into a universal bottles and allow to solidify.
A sterile syringe was then dipped inside the broth
culture of the organisms grown and then used to stab
the solidified agar in the middle; the universal bottle was
then incubated at 37
0
C for 24h and examined for motile
action. If the organism diffuses around the agar then it
is motile but if there was no diffusion, then the
organisms is said to be non-motile.
Catalase test
This was performed by dipping a portion of colonies
using a sterile toothpick on 3% Hydrogen Peroxide
solution and the emergence of air bubbles indicated
positive result. Catalase is an enzyme that converts
hydrogen peroxide into water and oxygen. The
presence of catalase can be easily detected by the slide
method. A drop of 3 percent hydrogen peroxide is put
on a slide and the bacteria are emulsified in the drop.
The presence of bubbles is evidence of the production
of oxygen.
Coagulase test
A drop of plasma (contained in an EDTA bottle) was
placed on a clean slide thta has been sterilized and a
culture suspension that has been grown for 24hours
was emulsified in the plasma and mixed for 30secs. A
positive result shows clumping or agglutination.
Isolation and characterization of fungi from
fermented palm wine
Fungi and yeasts were isolated by direct streaking of
palm wine samples on potatoes dextrose agar
supplemented with antibiotics. Plates were incubated at
room temperature (25 – 27
0
C) for 72hours. The isolates
were kept on slants of the same medium and
refrigerated until use. The identification of yeasts was
according to the general methods described by Lodder
(1990).
Determination of pH and TTA (total titratable acidity)
pH determination
pH was measured using digital pH meter after
homogenizing 5ml of the palm wine samples in 40ml of
distilled water. The pH meter was standardized with
buffer solution; the buffer solution was prepared with pH
buffer powder of pH 4.00 at 25
o
C which is dissolved in
250ml distilled water. The electrode of the pH meter
was immersed in a glass beaker containing the sample;
Readings were obtained from the photo-detector of the
pH meter.
Titrable Acidity Determination
For TTA measurement, a 5ml of the palm wine samples
was homogenized in distilled water (20ml). The
solution was titrated with 0.05M sodium hydroxide
solution to a faint but definite pink colour; 5mls of the
sample was titrated to a pink color with the 0.05M
NaOH, using 3 drops of 1% phenolphthalein as
indicator.
The total acidity is expressed as lactic acid as shown
below;
Akinrotoye KP 7
5. TTA = M NaOH × ml NaOH × 0.09 × 100 / ml of palm
wine sample
Where TA = Titratable acidity, M NaOH = molarity of
NaOH used, ml NaOH = amount (in ml) of NaOH used;
0.09 = equivalent weight of lactic acid.
RESULTS
Growth Inhibition of the Selected Test Bacteria by
the Palm Wine Used.
The palm wine used in this assay inhibited the growth of
all the selected diarrhoeagenic bacteria used. The
inhibition ranged from 2.2mm-28.2mm. Palm wine
subjected to fermentation at room temperature (30±2
0
C)
for 7 days (168 hours) exerted the greatest inhibitory
effect on all the test bacteria while the freshly tapped
recorded the least inhibitory effect (Table 1, 2, 3 and 4).
Plates 1, 2, 3 show the growth inhibition of some of the
test organisms by palm wine.
Number of cells (cfu/ml) of the test bacteria used for
the growth inhibitory assays.
For Salmonella typhi- 1.11×10
7
cfu/ml, Shigella
dysentariae- 2.49×10
7
cfu/ml, Staphylococcus aureus-
1.40×10
7
cfu/ml, E.coli 0157:H7- 2.68×10
7
cfu/ml, E.coli-
1.82×10
7
cfu/ml.
pH of the palm wine samples used
The pH of the palm wine samples used ranged from
6.20-2.86. The pH of the freshly tapped palm wine was
6.20 but by the 7
th
day it has decreased to 3.3 (fig.1)
Total Titratable Acidity (TTA) of the palm wine
samples used
The TTA value of the palm wine samples used ranged
from 1.98-6.05. The TTA value of the freshly tapped
palm wine was 1.96 but by the 7
th
day it has increased
to 6.05 (fig. 2).
TABLE 1: Growth inhibitory effect of undiluted palm wine on selected diarrhoeagenic bacteria.
Diameters of Zones of Inhibition (mm)
Days of fermentation
Antibiotics
Salmonella
typhi
Shigella
dysentariae
Staphylococcus
aureus
Esherichia coli
0157:H7
Escherichia coli
0 7.0 8.9 2.2 7.0 5.0
1 8.5 10.0 3.5 10.0 6.4
2 12.7 16.0 14.0 13.0 10.0
3 13.8 17.6 14.7 16.0 12.2
4 14.0 19.2 15.3 16.2 13.6
5 15.0 21.0 18.0 17.0 17.0
6 16.5 25.0 22.0 18.2 23.5
7 19.2 28.2 23.6 20.0 26.4
Ampicillin 0.0 10.0 0.0 0.0 0.0
Ampiclox 0.0 12.0 0.0 0.0 0.0
Tetracycline 12.0 5.0 0.0 0.0 0.0
Chloramphenicol 0.0 12.0 4.0 3.0 7.0
Ciprofloxacin 17.0 18.0 8.0 12.0 15.0
TABLE 2: Growth inhibitory effect of diluted palm wine (1:10) on selected diarrhoeagenic bacteria.
Diameters of Zones of Inhibition (mm)
Days of fermentation
Antibiotics
Salmonella
typhi
Shigella
dysentariae
Staphylococcus
aureus
Esherichia coli
0157:H7
Escherichia coli
0 2.8 1.8 1.1 2.0 2.4
1 3.0 3.5 1.8 2.4 3.5
2 3.4 5.0 2.7 3.1 4.0
3 4.0 6.5 3.0 3.6 4.8
4 4.5 7.6 3.5 4.1 5.5
5 4.7 8.0 4.0 6.8 6.2
6 5.8 9.3 4.4 7.0 7.0
7 7.2 11.2 6.3 8.1 9.5
Ampicillin 0.0 10.0 0.0 0.0 0.0
Ampiclox 0.0 12.0 0.0 0.0 0.0
Tetracycline 12.0 5.0 0.0 0.0 0.0
Chloramphenicol 0.0 12.0 4.0 3.0 7.0
Ciprofloxacin 17.0 18.0 8.0 12.0 15.0
8 Elite Res J. Bio M.
6. TABLE 3: Growth inhibitory effect of diluted palm wine (1:1,000) on selected diarrhoeagenic bacteria.
Diameters of Zones of Inhibition (mm)
Days of fermentation
Antibiotics
Salmonella
typhi
Shigella
dysentariae
Staphylococcus
aureus
Esherichia coli
0157:H7
Escherichia coli
0 0.0 0.0 0.0 0.0 0.0
1 0.0 0.0 0.0 0.0 0.0
2 0.0 0.0 0.0 0.0 0.0
3 0.0 0.5 0.0 0.0 0.0
4 0.0 0.6 0.0 0.0 0.0
5 0.0 0.8 0.0 0.0 0.0
6 0.0 0.9 0.0 0.0 0.0
7 0.5 1.0 0.6 0.8 0.5
Ampicillin 0.0 10.0 0.0 0.0 0.0
Ampiclox 0.0 12.0 0.0 0.0 0.0
Tetracycline 12.0 5.0 0.0 0.0 0.0
Chloramphenicol 0.0 12.0 4.0 3.0 7.0
Ciprofloxacin 17.0 18.0 8.0 12.0 15.0
TABLE 4: Growth inhibitory effect of diluted palm wine (1:10,000) on selected diarrhoeagenic bacteria.
Diameters of Zones of Inhibition (mm)
Days of fermentation
Antibiotics
Salmonella
typhi
Shigella
dysentariae
Staphylococcus
aureus
Esherichia coli
0157:H7
Escherichia coli
0 0.0 0.0 0.0 0.0 0.0
1 0.0 0.0 0.0 0.0 0.0
2 0.0 0.0 0.0 0.0 0.0
3 0.0 0.0 0.0 0.0 0.0
4 0.0 0.0 0.0 0.0 0.0
5 0.0 0.0 0.0 0.0 0.0
6 0.0 0.0 0.0 0.0 0.0
7 0.0 0.0 0.0 0.0 0.0
Ampicillin 0.0 10.0 0.0 0.0 0.0
Ampiclox 0.0 12.0 0.0 0.0 0.0
Tetracycline 12.0 5.0 0.0 0.0 0.0
Chloramphenicol 0.0 12.0 4.0 3.0 7.0
Ciprofloxacin 17.0 18.0 8.0 12.0 15.0
Plate 1: - Growth inhibition of Staphylococcus aureus by
fermented Palm wine
A = Undiluted; B = 1:10 dilution; C = 1:1,000 dilution; D
= 1:10,000 dilution; E = Control (sterile distilled water).
Plate 2: - Growth inhibition of Esherichia coli 0157:H7
by fermented Palm wine
A = Undiluted; B = 1:10 dilution; C = 1:1,000 dilution; D
= 1:10,000 dilution; E = Control (sterile distilled water).
Akinrotoye KP 9
7. Plate 3: - Growth inhibition of Shigella dysentatriae by
fermented Palm wine
A = Undiluted; B = 1:10 dilution; C = 1:1,000 dilution; D
= 1:10,000 dilution; E = Control (sterile distilled water).
Fig 1: - pH value of fermenting palm wine
Fig 2: - TTA value of fermenting palm wine
Identity of the bacterial isolates from the palm wine
used
Seven different types of bacteria were isolated from palm
wine, they are; Micrococcus sp., Leuconostoc mesenteriodes.,
Lactobacillus plantarum., Lactobacillus lactis, Proteus
vulgaricus, Pseudomonas aeruginosa. The result of the
characterization can be seen in Table 5.
Identity of fungi isolated from the palm wine used
Six types of fungi were isolated from the palm wine used, they
are; Saccharomyces cerevisiae, Aspergillus niger, Penicillium
spp, Aspergillus fumigatus, Neurospora crassa, Aspergillus
flavus. The result of the charcaterization of the isolates can be
seen in Table 6. A replicate of this fungal isolates is
represented in plate 4.
TABLE 5: Characteristics of Bacteria Isolated from the Palm Wine Used
ISOLATES
Tests A B C D
Gram’s reaction +ve +ve +ve +ve
Cellular Morphology coccus coccus Rod Rod
Growth on MRS agar ND ND +ve +ve
Growth on MacConkey ND ND ND ND
Catalase +ve -ve -ve -ve
Coagulase -ve -ve -ve -ve
motility -ve -ve -ve -ve
Glucose fermentation
(Acid/gas production)
-ve -ve +ve -ve
Probable Organisms
Micrococcus
spp.
Leuconostoc
dextranicum
Lactobacillus
plantarum
Lactobacillus
lactis
10 Elite Res J. Bio M.
8. TABLE 5: Characteristics of Bacteria Isolated from the Palm Wine Used (Continued)
ISOLATES
Tests E F G
Gram’s reaction -ve -ve +ve
Cellular Morphology Rod Rod Bacilli
Growth on MRS agar ND ND +ve
Growth on MacConkey -ve -ve ND
Catalase +ve +ve -ve
Coagulase -ve -ve -ve
motility +ve -ve -ve
Glucose fermentation (Acid /
gas production)
-ve -ve +ve
Probable Organisms
Proteus
vulgaricus
Pseudomonas
aeruginosa
Leuconostoc
mesenteroides
TABLE 6: IDENTITY OF FUNGI ISOLATED FROM THE PALM WINE USED
Description of the isolates Probable Organisms
Black mycelia growth and fully extended from the growth medium Aspergillus niger
The texture is velutimous, sporulation very heavy. The appearance is grayish-green
while the reverse is pale yellow. Septate mycelium bearing single conidiophores which
are branched near the apex, ending
Penicillium notatum.
It is creamish in colour and oval in shape (spore). It has a branched cell, sometimes
white in clour.
Saccharomyces
cerevisiae
It is whitish in colour and wooly in nature. Neurospora crassa
Brown mycelia growth, An upright conidiophores that terminates in a davate swelling,
bearing phialides at the apex or radiating from the entire surface; conidia are 1-celled
and globose.
Aspergillus fumigates
An upright conidiophores that terminates in a davate swelling, bearing phialides at the
apex or radiating from the entire surface; conidia are 1-celled and globose.
Aspergillus flavus
Plate 4: Isolation of fungi using PDA agar
supplemented with antibiotics
Succession of the Isolated Microorganisms in the
Palm Wine
When the palm wine was freshly brought (Day 0),
Saccharomyces cerevisae and Proteus vulgaricus were
isolated from it. By day 1, the organisms isolated has
increased to four; this include Aspergillus niger, Penicillium
sp., Lactobacillus lactis and Saccharomyces cerevisae. By
day 2, the organisms isolated are Aspergillus niger,
Micrococcus sp., Pseudomonas aeruginosa. By day 3, the
organisms isolated are Aspergillus flavus, Lactobacillus lactis
and Leuconostoc dextranicum. By day 4, the organisms
isolated are Lactobacillus lactis, Penicillium notatum.,
Lactobacillus plantarum. On day 5, organisms such as
Leuconostoc dextranicum, Neurospora crassa were isolated.
On day 6, organisms such as Penicillium sp., Lactobacillus
plantarum, Leuconostoc mesenteroides were isolated from
the fermenting palm wine. On day 7, Aspergillus fumigatus,
Lactobacillus plantarum, Lactobacillus lactis were isolated
from the palm wine.
The succession of both bacteria and fungi isolates is
represented in Table 7.
Akinrotoye KP 11
9. TABLE 7: Succession of the Isolated Microorganisms in the Palm Wine during fermentation for 168hours
DURATION OF
FERMENTATIONS
TYPES OF MICROORGANISMS
DAY O Aspergillus niger, Saccharomyces cerevisae, Proteus vulgaricus
DAY 1 Aspergillus niger, Penicillium sp., Lactobacillus lactis,
DAY 2 Aspergillus niger, Micrococcus sp., Pseudomonas aeruginosa
DAY 3 Aspergillus flavus, Lactobacillus lactis and Leuconostoc dextranicum
DAY 4 Lactobacillus lactis, Penicillium sp., Lactobacillus plantarum
DAY 5 Leuconostoc dextranicum, Neurospora crassa,
DAY 6 Penicillium sp., Lactobacillus plantarum, Leuconostoc mesenteroides
DAY 7 Aspergillus fumigatus, Lactobacillus plantarum, Lactobacillus lactis.
DISCUSSION AND CONCLUSION
In this study, the effect of palm wine from Raphia palm
trees (Raphia hookeri) on diarrhoeic bacteria was
investigated. The palm wine used inhibited the growth
of all the test organisms. The highest inhibition was
observed with the palm wine subjected to natural
fermentation for 7 days on all the five organisms used.
The sharp drop in pH of palm wine in the first few days
from 6.20 to 3.42 (fig.1) might likely be responsible for
the inhibition. Palm-wine has been reported by many
workers (Ekundayo, 1969; Faparusi, et al., 1973; Rose,
1977; Swings and De Ley, 1977; Okafor 1977) to have
many microorganisms such as lactic acid bacteria
which metabolise the sugar present in the wine to
produce alcohol and organic acid with a consequent
decrease in pH of the palm-wine samples. Rokosu and
Nwisienyi (1980) suggest that, the organic acid
produced at this pH is lactic acid, because there is
usually a predominance of lactic acid bacteria at this
stage of fermentation (Fooks and Gibson, 2002).
Therefore, the inhibition might be as a result of the
production of lactic acid by lactic acid bacteria, there
have been reports that lactic acid bacteria (LAB) exert
strong antagonistic activity against food spoilage and
pathogenic bacteria, particularly those involved with
food borne diseases (Ivanova et al., 2000; Sjogren et
al., 2003; Chung and Yousef, 2005). Another one is
bacteriocins which have been reported to have cidal
effects on closely related organisms (Atozie et al., 1980;
Kang and Fung, 1999; Chung and Yousef, 2005;
Savadogo et al., 2006)
Also revealed are consistent increases in acidity
(Titratable acidity) of the palm wines throughout the
period of fermentation as shown in which increases
from 1.98 to 6.05. Studies have shown that during
fermentation of palm wine, high titritable acidity is
inhibitory to the growth of spoilage organisms but
creates conducive environment for the growth of
desirable organisms. Also, high acidity is known to give
fermenting yeasts competitive advantage in natural
environments (Reddy and Reddy, 2005). The titratable
acidity of palm wine is expected to be between 0.5 to
1.0% (Snell and Ettre, 1974). In this study, the results of
titratable acidity in the palm wines fell within this limit
(Plutowska and Wardencki, 2008; Duarte et al., 2010).
In this study, seven bacterial species were obtained;
which includes Micrococcus sp., Leuconostoc
mesenteriodes., Lactobacillus plantarum., Lactobacillus
lactis, Proteus vulgaricus, Pseudomonas aeruginosa
and six fungi isolates identified to belong to the genera
Saccharomyces cerevisae, Aspergillus niger,
Penicillium notatum., Aspergillus fumigatus., Aspergillus
flavus and Neurospora crassa.The occurrence of these
microbial isolates in the palm wine samples, however,
supports the reports made by Faparusi and Bassir
(1971), Okafor (1975a, b) and Ikenebomeh and
Omayuli (1988) because they also isolated these
organisms. Other isolates gotten from the palm wine
includes Neurospora crassa, Pseudomonas
aeruginosa. Proteus vulgaricus and Aspergillus niger.
The isolation of Pseudomonas aeruginosa and Proteus
vulgaricus may be through unhygienic handling of palm
wine and while the isolation of Neurospora crassa and
Aspergillus niger might likely be from the laboratory
environment. Aspergillus niger is ubipuitous in soil and
is commonly reported from indoor environment (Okafor,
1972).
The gradual but progressive decrease in the level of the
individual isolates as fermentation progresses could be
associated with the progressive increase in the level of
the acidity of the wine as fermentation progresses as
well as the obvious changes in the physicochemical
quality that characterize the quality of palm wine such
as high-sugar concentration and alcoholic content (Eze
and Ogan, 1987; Ikenebomeh and Omayli, 1988).
In conclusion, this work has been able to show that
palm wine has antibacterial activity against the
diarrhoeagenic bacteria in this research work.
Moreover, fermentation duration plays significant role in
the antibacterial activity. The longer the duration of
fermentation, the more effective the palm wine in
inhibiting the growth of these test organisms. Since
palm wine has potent antibacterial activity on the test
bacteria, it is suggested that it can be used as an
alternative measure for the control of the diarrhoea
produced by these organisms in the absence of
antibiotics.
12 Elite Res J. Bio M.
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APPENDICES
APPENDIX I: pH of palm wine samples used during fermentation
Sample Hours of fermentation pH value
0 Freshly tapped 6.20
1 24hrs 5.31
2 48hrs 4.02
3 72hrs 3.92
4 96hrs 3.42
5 120hrs 3.36
6 144hrs 3.25
7 168hrs 2.86
APPENDIX II: TTA OF PALM WINE SAMPLES USED DURING FERMENTATION
Sample Hours of fermentation TTA value
0 Freshly tapped 1.98
1 24hrs 2.26
2 48hrs 2.59
3 72hrs 3.04
4 96hrs 3.28
5 120hrs 4.51
6 144hrs 5.76
7 168hrs 6.05
APPENDIX III: COMPARISON OF TTA AND pH VALUES OF FERMENTING PALM WINE
14 Elite Res J. Bio M.
