ISSN 1553-3468
2005 Science Publications
Corresponding Author: Matook Saif Mokbel, Department of Biochemical Science and Technology, Faculty of Agriculture,
Kagoshima University, 1-21-24, Korimoto, Kagoshima 890-0065, Japan, Tel: + 81-99-285-8639
125
Antimicrobial assay:
Antimicrobial assay was
measured using the methods of Mokbel and
Hashinaga[9]. Five species of bacteria were used:
Bacillus cereus IFO 13597, Salmonella enteritidis IFO
3313, Escherichia coli IFO 13168, Bacillus subtilis IFO
3009, Staphylococcus aureus IFO 3761.
The bacteria stock culture media were maintained
on nutrient hard agar (peptone 1g, meat extract 0.5 g,
sodium chloride 0.25 g and agar 1 g 100 mL 1 H2O) at
4C. The bacterial culture medium was prepared as
following, nutrient broth (peptone 0.5 g, meat extracts
0.25 g, sodium and chloride 0.25 g 50 mL 1 H2O) and
soft agar medium (peptone 0.5 g, meat extracts 0.25 g,
sodium chloride 0.125 g and agar 0.2 g 50 mL 1 H2O)
adjusted to pH 6.6 and autoclaved at 121C for 20min.
To determine the antibacterial activity the
microorganisms were cultured in nutrient broth at 36C
overnight. Three-five L of the inocula (spores) was
added to 3.5 mL soft nutrient agar and well shaken. The
soft nutrient agar was then added on a Petri dish
containing 15 mL hard agar. Filter paper discs (6 mm in
diameter),
were
impregnated
with
different
concentrations (0.1 to 1 mg discs 1) and allowed to dry
completely for 10-15 min and then evenly placed on the
surface of previously inoculated agar. The Petri dishes
were incubated at 36C for 24 hr. Chloramphenicol was
used as a positive control.
Fig. 1:
Separation scheme of the antioxidant and antimicrobial substance from green banana peel
Thin-layer
chromatography
analysis
(TLC)
analysis: TLC analysis for crude extracts or silica gel
column chromatography fractions was performed on
aluminium sheets (20x20 cm) of silica gel 60 F254 plate
which were developed with appropriate solvents for
each sample such as (CHCl3:MeOH:hexane (5:1:0.5),
CHCl3:MeOH:H2O (5:1:0.5), CHCl3:acetone:MeOH:
H2O (5:3:4:1) or 1-butanol:acetic acid:MeOH:H2O
(2:1:0.5:1)). The resulting bands were located using
UV-light and 10% sulfuric acid spray followed by
heating in the oven for 5-10 min at 120C.
Gas
chromatography/
mss
spectrometry:
Molecular weight was determined using gas
chromatography (Thermo Finnign Polaris Q) equipped
with an EI ion source coupled to an MS (Polaris Q).
Helium was used as gas carrier, and the injector
temperature was kept at 250C.
127
Peel extract
BHA
Control
Absorbance, 500 nm
1.5
1.0
0.5
0.0
0
Time, days
BHA
Control
Absorbance, 470 nm
1.5
1.0
0.5
0.0
0
50
100
150
Time, min
Bacteria
Gram positive bacteria
Staphylococcus aureus
Bacillus subtilis
Bacillus cereus
Gram negative bacteria
Salmonella enteritidis
Escherichia coli
12 0.4
10 0.3
11 0.1
0
0
0
0
0
0
3 0.1
3 0.2
2 0.4
0
0
0
0
0
100.2
9 0.2
0
0
0
0
10.0
2 0.2
0
0
Values are means S.D of 9 paper disc for each three replication were used.
Sterile paper disc (6 mm diameter) was impregnated with fruit tissue extracts (1 mg disc 1).
Antibacterial activity of the compounds isolated from green banana peel using agar disc diffusion and MIC methods
Isolated compounds
Standard antibiotics
-------------------------------------------------------------------------------------------------------------------------------------------
Microorganisms
Gram positive bacteria
Staphylococcus aureus
Bacillus subtilis
Bacillus cereus
Gram negative bacteria
Salmonella enteritidis
Escherichia coli
-sitosterol
12-hydroxystrearic acid
--------------------- ---------------------------DDa
MICb
DDa
MICb
Palmitic acid
---------------------DDa
MICb
d-Malic acid
---------------------DDa
MICb
Succinic acid
-----------------------DDa
MICb
Chloramphenicol
------------------------DDa
MICb
7.1 0.3
6.2 0.2
6.7 0.4
330
270
250
10 0.5
11 0.3
12 0.2
NT
NT
NT
>1000
>1000
>1000
>1000
>1000
>1000
11 0.6
10 0.9
11 0.2
180
160
140
5.1 0.3
4.8 0.2
5.7 0.4
620
580
650
25 0.5
24 0.3
26 0.7
5.5
5.5
5.5
5.8 0.3
6.8 0.4
350
300
9 0.1
8 0.4
NT
NT
>1000
>1000
>1000
>1000
9 0.1
8 0.4
280
420
4.7 0.3
4.1 0.4
730
750
20 0.5
19 0.5
5.5
5.5
a
DD, agar disc diffusion method. Sterile paper (6 mm diameter) were impregnated with fruit tissues 0.1mg/disc.Diameter of inhibition zone (mm)
(unincluding disk diameter of 6 mm). NT, not tested. bMIC, minimum inhibitory concentration; values given as ppm for the isolated compounds
and chloramphenicol antibiotics as a standard. Values are means S.D of 9 paper disc for each three replication were used.
too. The doublets at 5.24 and 4.65 ppm are due to the
anomeric protons of glucose, -glucose H-1 and
beta-glucose H-1, respectively; the triplet at 3.25 ppm is
due to the -glucose H-2. The doublet at 4.12 ppm is
the only resonance signal clearly assigned to fructose.
The resonance signals in the region from 3.98 ppm are
mainly due to overlapping resonances of ring protons of
sucrose, fructose and glucose. This results
corresponding with 1H-NMR data of Raffo et al.[15].
However, sucrose, fructose and glucose recorded low
antioxidant activities as measured by DPPH free radical.
Glycoside and monosaccharide components isolated
(compound 6 and 7) had a higher antioxidant activity
than sucrose, glucose, fructose or control at
concentration 100 mg mL 1.
CONCLUSION
Ethyl acetate and water soluble fractions of green
banana peel displayed high antimicrobial and
antioxidant activity. Most of the compounds isolated
from
green
peel
-sitosterol,
malic
acid,
12-hydroxystrearic acid and succinic acid, which
showed significant antibacterial activities and low
antioxidant activities. While, those compounds isolated
from water soluble extracts glycoside and
monosaccharide components displayed significant
antioxidant and low antimicrobial activity.
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