S.A. Adeola et al / Research Journal of Biology (2012), Vol. 02, Issue 05, pp.

138-144

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Research Paper

Antimicrobial Activity of Ocimum basilicum and its

Inhibition on the Characterized and Partially Purified
Extracellular Protease of Salmonella typhimurium
S.A. Adeola*1, O.S. Folorunso1 and K.O. Amisu2
1

Department of Biochemistry and 2Department of Microbiology, Faculty of Science, Lagos State University, LagosBadagry Expressway Ojo, Nigeria. P.O. Box 0001 LASU Post Office Ojo Lagos State
Tel: +234-802-308-1364
E-Mail: adesegunadeola@yahoo.com; oluwafemi.folorunso@lasu.edu.ng

Abstract
The volatile oil of Ocimum basilicum, at the preliminary stage, showed a wide range of antibacterial activity
(10.50.5 to 19.50.5mm) against thirteen different enteric bacteria with highest sensitivity against Pseudomonas
aeruginosa but Providential alcalifaciens and Providential rettgeri were resistant to the oil. The minimum
inhibitory/bactericidal concentrations (MIC/MBC) results showed the oil to inhibit Klebsiella pneumoniae,
Pseudomonas aeruginosa, and Escherichia coli at 50%v/v. The MIC for most of these pathogens ranged
from 3.12-25%v/v, while Proteus vulgaris and Salmonella typhimurium was 1.56%v/v. Citrobacter fruendii has the lowest
value, 0.39%v/v. The MBC of the volatile oil was within one twofold dilution of MIC for each organism.
The crude enzyme had optimal activities at pH and temperature of 7.9 and 44o C respectively. The oil showed a
noncompetitive inhibition against the activity of the extracellular protease of Salmonella typhimurium and
apparently decreased reaction rate from 5.6 x 103 mol/min (absence of inhibitor) to 3.6 x 103 mol/min (presence of
inhibitor), while the Km remained 2.2 mg/ml. Purification fold of 6.06 and enzyme activity of 382.0 mol/min
as compared to the crude extract were achieved. The volatile oil of Ocimum basilicum, therefore, exhibited a wide
range of antibacterial activity and showed a noncompetitive inhibition against the extracellular protease of Salmonella
typhimurium.
Keywords: Ocimum basilicum, Volatile Oils, Salmonella typhimurium, Extracellular Protease

1. Introduction
Ocimum basilicum (Lamiaceae) is widely distributed in
tropical and warm temperate region. It is a multi-purpose
medicinal herb commonly used in folk medicines to treat
different diseases like upper respiratory tract infections,
diarrhea, headache, ophthalmic, skin disease, pneumonia,
cough, fever and conjunctivitis (Keita et al, 2001).
Previous studies showed that the essential oil of Ocimum
species grown in Rwanda, displayed antimicrobial activity
(Janssen et al, 1989). It has been also reported that the
volatile oil of this plant contained mostly phenol,
particularly thymol (Gills, 1992 and Sofowara, 1993) and

it is probably responsible for its reported antimicrobial

action.
Salmonellosis is a disease caused by Salmonella species
(WHO, 1997). The clinical course of human salmonellosis
is usually characterized by acute onset of fever, abdominal
pain, diarrhea, nausea and sometimes vomiting.
Salmonella typhimurium is a Gram-positive rod-shaped
motile bacterium. It is widely spread in animals especially
in poultry and swine. Salmonella typhimurium among
others, causes gastroenteritis which is often mild self-

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limiting but can be severe in the young, the elderly and

patients with weakened resistance. The infection requires a
large array of different virulence factors (Hardt et al,
1998).
Proteases play a crucial role in a numerous
pathological processes. Arthritis, tumour invasion and
metastasis, infections and a number of degenerative
diseases have been linked with the involvement
of one or more proteolytic enzyme (Brown, 1994).
Microbial proteases have been proposed as virulence
factors in a variety of diseases caused by microorganisms
(Jones, 1998). Proteases are classified according to
their catalytic mechanisms (Dunn, 2001). Identification
and characterization of microbial proteases are prerequisites for understanding their role in the
pathogenesis of infectious diseases as well as to improve
their application in biotechnology (Lantz & Ciborowski,
1994).
Several works have been done on the antimicrobial
properties of volatile oils against several pathogenic
organisms. However, no literature is available
on the kinetics of the inhibition of extracellular
protease by the volatile oil of Ocimum basilicum.
Therefore, this work was undertaken to partially
purify, characterise and study the kinetics of the
extracellular protease of Salmonella typhimurium with the
use of the volatile oil of Ocimum basilicum as potent
inhibitor.

2. Materials and Methods

2.1. Plant Materials
Ocimum basilicum plants (Lamiaceae) was obtained at
Adoff, Off LASU-Isheri Road, Lagos State Nigeria. The
plant samples were identified and authenticated at the
department of Botany, Faculty of Science, Lagos State
University, Ojo Lagos State, Nigeria. The plant was airdried for 5 days before extraction.
2.2. Microorganisms
All the microorganisms used in this work were
obtained from the Nigeria Institute of Medical Research
(NIMR), Yaba, Lagos Nigeria and maintained on nutrient
agar petri-dishes at 4 oC. These microbes were
Staphylococcus aureus, Escherichia coli, Pseudomonas
aeruginosa,
Salmonella
typhimurium,
Salmonella
paratyphimurium, Klebsiella pneumoniae, Shigella
dysenteriae, Enteroheamorrhagic Escherichia coli
(EHEC), Poteau vulgaris, Enterobacter aerogenes,
Citrobacter fruendii, Providencia alcalifaciens and
Providencia rettgeri.

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2.3. Extraction of Volatile Oil

The volatile oil from Ocimum basilicum plants was
extracted by hydrodistillation method as described by
Lawrence & Reynolds (1993). The five-day-air-dried
leaves of Ocimum basilicum were squeezed into pieces and
packed into the 5 L 34/35 quick fit round bottom flask
containing 1.5 L of water with fixed Clevenger. The oil
was extracted at a steady temperature of 80 oC for 3 hours
and was collected over 2 mL of n-hexane. The oil was kept
tightly in a sample bottle and stored at 4 oC.
2.4. Microbial Sensitivity Test of the Volatile Oil
The sensitivity test of the volatile oil of Ocimum basilicum
was carried out using a disc diffusion technique on Muller
Hinton agar as described by Meena & Sethi (1994) with
little modification. Sterile 5 mm diameter paper disc
soaked with the volatile oil was placed gently on the
media, which had been freshly inoculated with each of the
organisms. The plates were incubated for 24 hours at 37
o
C. The results were recorded by measuring the zone of
growth inhibited by the oil.
2.5. Minimum Inhibitory Concentration (MIC) and
Minimum Bactericidal Concentration (MBC)
The MIC and MBC of the volatile oil of Ocimum
basilicum were carried out using microbroth dilution
method as described by Janssen et al (1989). A colony of
each organism was added to 200 L of susceptible test
broth containing two-fold serial dilution of the volatile oil
using Tween 80 ( 0.5%v/v) as diluent in a microtitre plate
(21.5 cm x 17 cm). The plate was incubated at 37 oC for
18-24 hours. Each of the microwell on the plate was
inoculated on a freshly prepared Muller Hinton agar where
MIC and MBC were determined.
The MIC was the lowest concentration of the oil sample
that prevented visible growth of the microorganisms in the
required medium. This is also called Minimum Lethal
Concentration (MLC). The MBC was considered as the
lowest concentration of the oil that yielded no colonial
growth of the subcultured microorganisms in the required
medium under favourable conditions.
2.6. Extraction of Crude Enzyme
The extracellular protease of S. typhimurium was
extracted by the method described by Makino et al (1981).
A colony of the microbe was inoculated into the Muller
Hinton broth and incubated for 24 hours at 37 oC. The
broth was centrifuged (Kendros PicoBiofuge, Heraeus)
9000 rpm for 10 minutes at room temperature. The
supernatant, which was cell-free, was stored in a sample
bottle at 4 oC.

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2.7. Determination of the Total Protein and Protease

Activity of the Crude Enzyme Extract
The total protein of the crude enzyme extract was
determined using a method described by Lowry et al
(1951) while the protease activity was assayed for by the
method of Folin & Ciocalteu (1927) using casein as
the substrate. This was done by adding 5.0 mL of alkaline
solution containing a mixture of 50 mL of solution A (20 g
sodium trioxocarbonate IV and 4 g sodium hydroxide
in 1.0 L) and 1.0 mL of solution B (5 g cupper II
tetraoxosulphate VI pentahydrate and 10 g sodium
potassium tartrate in 1.0 L) to 0.1 mL of crude enzyme
extract and mixed. The reaction solution was allowed to
stand for 10 minutes at room temperature and 0.5 mL of
Folin Ciocalteaus phenolic reagent (50 %v/v) was added.
The solution was mixed thoroughly and the absorbance
was read at 750 nm after 30 minutes using Spectronic-21
(Bausch and Lomb). BSA was used as standard protein
(0.20 mg/ml).
Protease activity was carried out by adding 5.0 mL
of casein solution (0.6%w/v in 0.05 M Tris buffer
at pH 8.0) to 0.1 mL of the crude enzyme extract and the
mixture was incubated for 10 minutes at 37 oC. The
reaction mixture was stopped by adding 5.0 mL of a
solution containing 0.11 M trichloroacetic acid, 0.22 M
NaCl and 0.33 M acetic acid mixed in ratio 1:2:3. The
turbid solution was filtered and 5.0 mL of alkaline
solution was added to 1.0 mL of the filtrate and mixed
thoroughly. The reaction mixture was allowed to stand
for 10 minutes and 0.5 mL of Folin Ciocalteaus phenolic
reagent was added to it. The absorbance was read at 750
nm after 30 minutes using spectrophotometer-21 (Bausch
and Lomb). L-tyrosine solution (0.20 mg/ml) was
used as standard for the protease activity. All reagents
used were of analytical grade. One Unit of protease
activity was defined as the amount of enzyme required to
liberate 1.0 mol of tyrosine in 10 minutes at 37 oC.
The specific activity was expressed in mol/min/mg
protein.
2.8. Determination of the Optimum pH
The method adopted was described by Makino et al (1981)
with little modification. Protease activity was assayed
using 0.6% casein solution in 0.05 M Tris buffer solution
(pH 6.0-8.5) at 37 oC.
2.9. Determination of Optimum Temperature
As described by Makino et al (1981), protease activity was
assayed under varying temperature (30-60 oC) using 0.6%
casein solution in 0.05 M Tris buffer at pH 8.0.
2.10. Inhibitory Assay

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The method used was described by Makino et al (1981)

with a slight difference. This was carried out by adding 0.1
mL of the crude protease extract and 0.1 mL of 3.5 %v/v
of the volatile oil (as inhibitor) in 0.5 % v/v Tween 80
solutions concomitantly to different concentration of
casein solution (0.2-1.0 %w/v) in 0.05 M Tris buffer, pH
8.0 and the reaction mixture was incubated at 37 oC for 10
minutes. The reaction was stopped and the protease assay
was carried out on each of the filtrate. The procedure was
repeated without an inhibitor.
2.11. Dialysis
The crude enzyme extract was dialyzed (SIGMA Dialysis
Tubing Cellulose Membrane, D9402), at room
temperature, with 55 %w/v saturated solution of
ammonium sulphate for 48 hours in 0.05 M Tris buffer
solution (pH 8.0). The solution was then centrifuged
(Kendros PicoBiofuge, Heraeus) at 9000 rpm for 10
minutes to separate the residue and this residue was further
dialyzed (SIGMA Dialysis Tubing Cellulose Membrane,
D9402) with 50 %w/v saturated solution of ammonium
sulphate for another 48 hours in 0.05 M Tris buffer (pH
8.0). At each stage, both total protein and protease activity
were assayed.
2.12. Gel Filtration
Three gram of Sephadex G-100 was soaked in distilled
water for 72 hours. The soaked gel was poured into a
capillary tube (20 x 2 cm) with a flow rate of 1.5 mL/min
and this was used to separate 50 %w/v ammonium
sulphate dialysate crude protease extract.

3. Results
The dried leaves of Ocimum basilicum, 587.5 g, produced
1.0 mL of concentrated volatile oil. The antimicrobial
susceptibility test of the volatile oil against thirteen
different enteric pathogenic organisms was shown in
Figure 1.
Among the thirteen different microorganisms used, only
Providencia alcalifaciens and Providencia rettgeri
remained insensitive to the volatile oil. The MIC and MBC
of the volatile oil against the thirteen different enteric
bacterial were shown in Table 1. Citrobacter fruendii
remained most sensitive among the thirteen organisms
while E. coli, Klebsiella pneumoniae and Pseudomonas
aeruginosa responded to higher concentration of the
volatile oil. Providencia alcalifaciens and Providencia
rettgeri showed no response to the volatile oil.
Figures 2 and 3 showed the effects of pH and temperature
on the activity of the extracellular protease of Salmonella
typhimurium. The enzyme exhibited optimum activity at

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pH 7.9 and at 44 oC. In addition, 0.1 mL of 3.5 %v/v

dilution of the volatile oil using 0.5 %v/v Tween 80 was
tested as an inhibitor against the extracellular protease
activity of Salmonella typhimurium at 37 oC and pH 8.0.

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The kinetic result was shown in Figure 4. The volatile oil

showed a noncompetitive inhibition against the activity of
the extracellular protease. The rate of enzyme catalysis
reduced because Vmax was reduced in the presence of the
inhibitor.
The extracellular protease of Salmonella typhimurium was
partially purified using dialysis and using gel size
exclusion chromatography (Figure 5 and Table 2). A
purification fold of 6.06 and enzyme activity of 382.0
mol/min as compared to the crude extract were achieved.

4. Discussion

Figure 1. Antimicrobial Susceptibility Test of the Volatile Oil of

Ocimum Basilicum Against Thirteen Different Strains of
Enteric Pathogenic Bacterial

The antimicrobial susceptibility tests of volatile oil of

Ocimum basilicum was reported for both Gram-positive
and Gram-negative enteric pathogenic bacteria. The
inhibition zones for all the enteric pathogens used in this
study were in the range of 10.50.5 to 19.50.5 mm.
Similar, reports have been observed in the work of Janssen
et al (1989). Pseudomonas aeruginosa was the most
sensitive microbes among the rest with the highest
inhibition zone of 19.50.5 mm while Proteus vulgaris,
Salmonella paratyphimurium and Staphylococcus aureus
showed similar inhibition zone value of 10.50.5 mm, the
lowest inhibition zone. Providencia alcalifaciens and
Providencia rettgeri were considered resistance to the
volatile oil since no inhibition zone was observed after 24
hours of incubation. For a century, a large number of

Table 1. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the Volatile Oil Extracted From of
Ocimum Basilicum against Thirteen Different Strains of Enteric Pathogenic Bacteria

Organisms

MIC (%v/v)

MBC (%v/v)

Citrobacter fruendii
Enteroheamorrhagic Escherichia coli (EHEC)
Enterobacter aerogenes
Escherichia coli
Klebsiella pneumoniae
Providencia alcalifaciens

0.39
6.25
12.50
50.00
50.00
-

0.78
12.50
25.00
100.00
100.00
-

Providencia rettgeri
Proteus vulgaris
Pseudomonas aeruginosa
Salmonella paratyphimurium
Salmonella typhimurium
Shigella dysenteriae
*Staphylococcus aureus

1.56
50.00
3.13
1.56
25.00
12.50

3.13
100.00
6.25
3.13
50.00
25.00

* Gram Positive Bacteria

Most Sensitive Microorganisms

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S.A. Adeola et al / Research Journal of Biology (2012), Vol. 02, Issue 05, pp. 138-144

essential oils and their phytoconstituents have been

investigated for their antimicrobial properties against many
strains of protozoan, bacteria and fungi (Mendosa et al,
1997 and Jones et al, 1998).

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lowest value of 0.39 %v/v, hence most sensitive to the

lowest concentration of the volatile oil. The volatile oil
inhibited Salmonella paratyphimurium, Staphylococcus
aureus, Enterobacter aerogenes, EHEC and Shigella
flexneri with MIC values ranging from 3.12-25.0 %v/v
(Table 1). The MBC of the volatile oil against the growth
of thirteen strains of enteric bacteria were estimated as a
value MIC corresponding to that organism and this was
found to fall within one twofold dilution of the MIC (Table
1).

Figure 2. Effect of pH on the Activity of Extracellular Protease of

Salmonella typhimurium

Figure 4. Lineweaver Burke Plot of Volatile Oil of Ocimum basilicum

as Inhibitor Against the Activity of the Extracellular
Protease of Salmonella Typhimurium

Figure 3. Effect of Temperature on the Activity of Extracellular

Protease of Salmonella typhimurium

The MIC and MBC of the volatile oil against the growth of
these enteric bacteria indicated that Klebsiella
pneumoniae, Pseudomonas aeruginosa and Escherichia
coli have MIC value of 50.0 %v/v as compared to a
relatively low value of 1.56 %v/v for Proteus vulgaris and
Salmonella typhimurium. Citrobacter fruendii has the

Figure 5. Elution profile of Sephadex G-100 Purification of the

Extracellular Protease of Salmonella typhimurium

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Extracellular protease of Salmonella typhimurium was

characterized and partially purified. The optimum enzyme
activity of 5.9 x 103 mol/min was observed at pH 7.9
(Figure 2). This showed that the enzymatic activity of
extracellular protease of Salmonella typhimurium was
most favourable at slightly alkaline pH. The activity of this
enzyme over a range of pH 6.9-8.0 has made it possible to
survive physiological pH in the human body system and is,
therefore, regarded as virulence factors of the pathogenic
microbes (Buchwald et al, 2002). Most of the enteric
bacteria have been reported to have their optimal activities
at pH 6.0-8.0 (Makino et al, 1981). In addition, this
enzyme showed an optimum activity of 5.4 x 103 mol/min
at 44 oC, Figure 3. This might probably explained why
some of the enteric pathogens are relatively thermostable.
Similar results have been reported by Vinci et al (2005)
and Kobayashi et al (1995), while working with the
extracellular protease of Alkalophilic bacillus species,
KSM-K16 and Streptomyces species, strain C5-A13.

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precipitation (Table 2). The specific activity of the

Sephadex G-100 purified enzyme was 382.0 mol/min/mg
proteins. The elution chromatogram (Figure 5) of G-100
showed a peak each for total enzyme assay and total
protein
The extracellular protease of Salmonella typhimurium has
been partially purified, characterized and shown to be
inhibited by the volatile oil of Ocimum basilicum and this
oil has also been confirmed to have a wide range
antibacterial effect on pathogenic organisms.

5. Conclusion
The volatile oil of Ocimum basilicum elicited antivirulence
activity against the activity of extracellular proteolytic
enzyme of Salmonella typhimurium, a common infectious
enteric pathogenic organism. The extracellular protease of
this pathogen was partially purified and its kinetic showed

Table 2. Purification Profile of the Extracellular Protease of Salmonella typhimurium

Purification Steps
Crude Extract
65 % (NH4)2SO4
precipitation
50 % (NH4)2SO4
precipitation
35 % (NH4)2SO4
precipitation
Sephadex G-100

Total
Proteins
(mg)
100
65
45
19
10

Total Activity
(mol/min)

Specific Activity
(mol/min/mg Protein)

Yield
(%)

Purification
(Fold)

6300
5915
4046
4010
3820

63.0
91.0
89.9
211.1
382.0

100
93.9
64.2
63.7
60.6

1
1.44
1.46
3.35
6.06

Double reciprocal plot of Lineweaver Burke analysis was

used to predict the effect and type of inhibition elicited by
this volatile oil, Figure 4. The volatile oil exhibited a
noncompetitive inhibition against the activity of the
extracellular
proteolytic enzyme
of
Salmonella
typhimurium with Vmax reducing from 5.6 x 103 mol/min
in the absence of inhibitor to 3.6 x 103 mol/min in the
presence of inhibitor. The Km was 2.2 mg/ml. These
signified that an increase in the substrate concentration
might not relief the enzyme of the inhibitor because the
inhibitor was occupying a separate site, apart from the
active site, on the enzyme and this was the reason why
affinity of the enzyme for substrate was not affected.
Reports had earlier been noticed in the use of phosphor,
sulphonyl and metallic compounds such as Cu2+, Pb2+ and
Hg2+ as potent inhibitors against pathogenic extracellular
proteases (Morihara, 1974 and Makino et al, 1981).
Sephadex G-100 size-exclusion chromatographic technique showed approximately 6.1 fold purification from the
initial culture broth with a percentage recovery of 60.6%
(Table 2). The specific activity of 50% (NH4)SO4
precipitation was slightly lower than 65% (NH4)SO4

a noncompetitive inhibition in the presence of the volatile

oil extracted from Ocimum basilicum. In vivo analysis is
highly recommended for further analysis on the
antivirulence effect of the medicinal plant oil and its
inhibitory mechanism against the activity of extracellular
protease.

Acknowledgement
We sincerely appreciate the effort of Mr O.D.
Omonigbeyin of the National Institute of Medical
Research, Yaba Lagos State, Nigeria for providing isolates
used in this work.

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