Journal of Microbiology and Biotechnology Research

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J. Microbiol. Biotech. Res., 2012, 2 (5):778-782
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ISSN : 2231 3168

CODEN (USA) : JMBRB4

Evaluation of the antibacterial potential of Vernonia Amygdalina on

foodborne pathogens isolated from kunu sold in Calabar, Nigeria
Enyi-Idoh1*, K. H., Etim1, S. E Ofor1, U. A. Mandor2, B. I. and Ogar1, M.
1
Department of Biological Sciences, Cross River University of Technology, Calabar. Cross River
State. Nigeria
2
Department of Medical Laboratory Sciences, University of Calabar. Calabr. Cross River State.
Nigeria
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ABSTRACT

The antimicrobial efficacy of the formaldehyde, petroleum ether and hexane extracts of Vernonia amygdalina on
bacteria isolated from a popular local beverage kunu sold in various locations in Calabar Metropolis was
evaluated. The antibacterial activity was compared with standard antibiotics. Bacteriological assessment of kunu
was conducted using standard sensitivity test methods. The results revealed the presence of various species of
bacteria including Bacillus cereus, Escherichia coli, Lactobacillus sp., Staphylococcus aureus, Streptococcus
feacalis, Bacteriodes fragillis, Clostridium perfringens, Bacillus subtillis, Corynebacterium sp. and Micrococcus
spp. The antibacterial activity of the formaldehyde extract of V. amygdalina inhibited some of these bacteria to a
greater extent than the other extracts and standard antibiotics. Plants are rich source of antibacterial agents, which
could be exploited in human disease management and many edible vegetables in Nigeria have been known to have
antibacterial potentials. The presence of bacteria and the level of antibacterial activity of V. amygdalina on them
signify the health benefit of this particular vegetable and thus the public health importance of the preparation and
consumption of kunu.

Keywords: Vernonia amygdalina, Staphylococcus, kunu, public health, antibiotics

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INTRODUCTION

Kunu is a common refreshing health drink hawked in many Nigerian cities and towns. It is made with cereals like
millet, sorghum and maize and thus can be used as a food drink. Cereals and cereal products which includes grains,
flours, bread and other bakery products contain organisms from soil, insects and other sources. Bacteria found on
freshly harvested grains include species of Pseudomonas, Micrococcus, Lactobacillus, Bacillus etc. and the total
number of bacteria per gram of grain could vary from a few to several millions. Kunu is however produced locally
by anyone with the knowledge involved and bottled in reusable empty plastic bottles originally used for bottled
water. The poor shelf life and crude packaging method employed in its production leaves a lot to be desired
microbiologically. Kunu which used to be consumed mainly in northern Nigeria is now widely accepted
everywhere in Nigeria (1, 2) and yet there is still inadequate information the microbial load of kunu and the health
implications that kunu poses. Effieuvwere and Akona, (1995) The microbiological assessment of kunun zaki sold
in Jos in northern Nigeria during its production process and its health implication has been done previously (3).

V. amygdalina is known as mujonso in Tanzania while in Nigeria the Edos call it oriwo, the Hausas call it chusar
doki and the Ibibios atidot. In Igbo language it is onubu while the Yoruba call it ewuro.

Vernonia amygdalina is a shrub that can reach twenty three feet in height when fully grown and grows throughout
tropical Africa and has been domesticated in many parts of West Africa and Nigeria, where it is locally known as

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bitter leaf and used as vegetable in soups (4). It has a grey or brown coloured bark. The bark has a rough texture and
is flaked. The branches of the shrub are brittle and break off easily. The green leaves are oblong to lanceolate in
shape, veined and bear pale soft hairs on the underside. V. amygdalina has small white flowers which bloom in
clusters. It is most often grown as a leaf vegetable. The therapeutic relevance of all parts of bitter leaf as a useful
folkmedicine and a popular vegetable adjunct in the delicious onubu soup has been reported by some researchers (5,
6, 7, 8).

Many plants used in traditional medicine in Nigeria are readily available in rural areas as every day edible
vegetables and at relatively cheaper cost than orthodox medicine. Due to a rise in the rate of food borne infections,
antibiotic resistance in bacteria and side effects of synthetic antibiotics, medicinal plants are gaining popularity over
standard antibiotics.

In this study, formaldehyde, hexane and petroleum ether extracts of V. amygdalina were screened for antibacterial
activity against bacterial isolates from the popular refreshing fermented drink called kunu.

MATERIALS AND METHODS

Sample collection
Kunu was bought from commercial vendors from four different locations in Calabar including Bogobiri, Etim Edem
Park, Watt Market and Ekpo Abasi Junction and taken to the laboratory for direct examination. V. amygdalina was
harvested from the botanical garden of Cross River University of Technology, Calabar, Nigeria.

Bacteriological examination of kunu samples

Bacterial assessment was done for each of the sample using standard bacteriological procedures including serial
dilutions, Grams reaction, sugar fermentation and IMVIC test to identify bacterial isolates.

Extract preparation
Fresh and air-dried leaves of V. amygdalina were used. Leaves were air-dried for several days away from direct
sunlight until brittle enough to break. Formaldehyde, petroleum ether and hexane were used for extracting freshly
harvested macerated and previously air-dried grounded leaves of V. amygdalina. The crude extracts of the leaves
were prepared using standard procedures with slight variations (9) involving soaking powdered leaves (20g) in
100ml of solvents for 24h at room temperature. The mixture was filtered and the filtrate evaporation using a rotary
evaporator (STUARC SCIENTIFIC, ENGLAND). The residue was retained as the crude extract.

Test for antibacterial activity of V. amygdalina.

The disc diffusion technique using the Kirby-Bauer method as described in Prescott et al., (2005) (10) was used in
testing the kunu isolates for their antimicrobial sensitivities. Sensitivity discs were punched from Whatman No. 1
filter paper. The discs were sterilized and afterwards impregnated with the crude extracts of V. amygdalina (11). The
kunu bacterial isolates were re-suspended in Mueller Hinton broth and incubated for 4h. About 0.3mls of the
suspension was dispensed into sterile Petri dishes and molten Mueller Hinton agar No.2 pour over the plate and
stirred briskly to make an even distribution of bacteria in culture. The extract impregnated disks were then placed on
the surface of each plate and incubated at 37 C for 24 h. Standard antibiotics sensitivity discs were used as controls
for comparison with the extracts. The plates were observed for zones of inhibition after incubation followed by
measurements and calculation of the mean zones. Minimum inhibitory and bactericidal concentration values were
also determined by broth dilution assay.

RESULTS AND DISCUSSION

The probable and identified bacterial isolates and their percentage occurrence in the samples are given in Tables 1
and 2. Bacteria identified include Bacillus spp, Escherichia coli, Lactobacillus spp., Staphylococcus aureus,
Streptococcus spp., Bacteriodes spp., Clostridium perfringens, Bacillus subtillis, Corynebacterium spp. and
Micrococcus spp. Staphylococcus aureus had the highest occurrence (30%) among the isolates from the kunu
samples followed by Bacillus spp. (20%). Micrococcus spp. and Lactobacillus spp. were not significantly present in
the samples and are therefore not reckoned with in this evaluation even though the presence of Lactobacillus spp.
maybe responsible for the slightly acidic nature of kunu. In a recent study of the microbial succession of foofoo,
another staple food in Nigeria (12) 8 microbial strains were isolated including the following, Bacillus subtilis,
Lactobacillus plantarum, Corynebacterium manihoti, Leuconostoc

mesenteroides, Enterobacter aerogenes, Aspergillus niger, Geotrichum sp. and Saccharomyces cerevisiae.

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Table 1: Bacteriological examination of kunu samples and identified isolates

SAMPLE GRAM DESCRIPTION CATALASE OXIDASE INDOLE CITRATE MR PROBABLE

ORGANISM
NA* I G- ve rods occurring in + + - + + Bacteriodes spp
chains
NA II Short rods, Long slender Lactobacillus Spp
rods without spores Corynebacterium
Spp
NA III Clustered appearance, Staphylococcus
Curved big head with tiny aureus
drum stick appearance with Clostridium
spores perfringes
NA IV Gram positive, Black Streptococcus spp
colonies
NA V G+ve bacillus Clostridium
perfringes
Bacillus subtilis
NA VI In clusters, Red coloured - - + + - Gram-ve short
cells rods.
E. coli
NA VII Long slender rods + - Lactobacillus spp,
Corynebacterium
spp
*=Nutrient agar

Table 2: Bacterial isolates and frequency of occurrence

Bacteria isolates % occurrence

Escherichia coli 10
Bacteriodes spp. 10
Corynebacterium spp. 15
Streptococcus spp. 10
Bacillus spp. 20
Clostridium perfringens 10
Staphylococcus aureus 30

The zones of inhibition obtained for the crude extract of V. amygdalina on the test bacterial isolates are shown in
Table 3. The highest activity was observed in the formaldehyde extract against E. coli. Petroleum ether extracts did
not have any activity against the isolates while hexane extract activity was comparatively lower against isolates than
formaldehyde extracts. This outcome shows that leaf extracts of V. amygdalina possess inhibitory potentials against
kunu bacterial isolates.

In Table 4, the minimum inhibitory and bactericidal concentrations are given. The plant shows good inhibitory
activity against the isolates tested.

The antibiogram profile of the isolates shows that the leaves of V. amygdalina have greater antibacterial activity
than most conventional antibiotics tested as presented in Table 5. This agrees with a previous observation (13) that
some plant extracts are more potent than some antibiotics.
Table 3: Antibacterial activity of V. amygdalina on kunu isolates

Isolates/Solvent extracts Fresh sample Air-dried sample

FE* HE PE FE HE PE
Zones of Inhibition (mm)
E. coli 31 20 - 40 20 -
Corynebacterium spp. 40 - - 40 - -
S. aureus - - - 30 20 -
Bacillus subtilis 40 - - 33 25 -
Clostridium perfringes 40 - - 30 - -
*
FE Formaldehyde extract, HE Hexane extract, PE Petroleum ether extract

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Table 4: Minimum inhibitory and minimum bactericidal concentrations

Isolates/plant concentration(g/ml) Minimum inhibitory concentration Minimum bactericidal concentration

E. coli 25 12.5
Corynebacterium spp. 50 12.5
S. aureus 25 12.5
Bacillus subtilis 25 -
Clostridium perfringes 100 -

It has also been recorded (14) that acetone and alcohol extracts of V. amygdalina showed higher antifungal activity
against S. cerevisiae than ciprofloxacin and gentamycin. Acetone-ethanol extract of the leaf and stem of V.
amygdalina has also previously been shown to have a higher zone of inhibition for Klebsiella sp isolated from urine
at 20 mg/ml than ciprofloxacin at the same concentrations (15).

Antimicrobial activities in plants have been reported to be as a result of bioactive components present in the plants,
such as alkaloids, saponins, tannins, anthraquinones, sterioids, flavonoids (16, 17, 18).
Leaf extracts of V. amygdalina in this study has been shown to inhibit bacteria except for petroleum ether extracts,
indicating its broad spectrum activity as an antibiotic.

Table 5: Antibiogram profile of test organisms showing zones of inhibition (mm)

Isolates/Antibiotics CPX10 NB10 CN10 AML20 S RD20 E CH30 APX20 LEV CEP NA SXT PN AU PEF OFX10
(mg) 30 30 20 10 30 30 30 30 10
E. coli 34 ND* 14 ND 24 ND ND R R R 12 10 12 18 12 28 30
Corynebacterium 10 10 R R R R 12 R 10 R ND ND ND ND ND ND ND
spp.
S. aureus 18 22 24 16 24 16 22 16 36 R ND ND ND ND ND ND ND
B. subtilis 24 16 18 R R R R R R 30 ND ND ND ND ND ND ND
C. perfringes 12 16 10 12 R R R R R R ND ND ND ND ND ND ND
* - ND = not done
Key: CPX Ciprofloxacin, NB Norfloxacin, CN Gentamycin, AML Amoxil, S Streptomycin, RD Rifampicin, E Erythromycin, CH
Chloramphenicol, APX Ampicloxacillin, LEV Levofloxacin, OFX Tarivid, PEF Peflacin, AU Augmentin, CEP Ceporex, Na
Nalidixic acid, SXT Septrin, PN Ampicillin

This result corresponds to earlier works (2, 3) which isolated similar bacteria from kunu sold in Jos area of Nigeria.
These bacteria can therefore be assumed to be common food pathogens. S. aureus and E. coli are common food
poisoning bacteria (19) and their presence in kunu signifies a serious health implication in terms of the level of
hygiene maintained during its preparation and the potential for a large scale bacterial epidemics. However, lactic
acid bacteria of the genus Lactobacillus sanfransiscensis, L. farciminis, L. fermentum, L. brevis, L. plantarum, L.
amylovorus, L. reuteri, L. pontis, L. panis, L. Alimentarius and Weissella cibaria has been shown to be involved in
fermentation of cereals (20).

Earlier assessment of the antibacterial activity of V. amygdalina leaves extract on selected food pathogens (21)
revealed relatively high zones of inhibition at low concentrations, proving that the plants can be medically useful.

CONCLUSION

Until now, kunu production and marketing has remained a cottage affair. The benefit and need for a cool and
refreshing drink of kunu during the heat of the day cannot be overemphasised. However, considering the rich
nutritional content of the popular food drink, kunu serves as a fodder for bacterial growth and proliferation, a fact
that needs serious and urgent quality control attention and the need for government regulatory agencies to wade into
the cottage industry with possible potentials for better production and packaging process for kunu, thus ensuring a
healthier and more nutritious food drink made from Nigerian grown cereals.

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