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STUDIES OF ANTIBACTERIAL PROPERTIES AND BIOACTIVE COMPOUNDS

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afifah azmi

on 30 October 2013

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Transcript of STUDIES OF ANTIBACTERIAL PROPERTIES AND BIOACTIVE COMPOUNDS

INTRODUCTION
LITERATURE REVIEW
Ability to prolong life, prevent disease, preserve good health and also promote beauty.

Prevention for cancer, heart disease, lowers blood pressure, cholesterol & also prevent ulcers and kill bacteria.
LITERATURE REVIEW
Seaweeds also rich in bioactive compounds that potentially become functional ingredients for both human and animal health applications (Gupta et al., 2010)

(Cordeiro et al,2006) seaweed represents a potential source of antimicrobial substances due to their diversity of secondary metabolites with antiviral, antibacterial and antifungal activities.


METHODOLOGY
STUDIES OF ANTIBACTERIAL PROPERTIES AND BIOACTIVE COMPOUNDS (Protein) FROM MARINE SEAWEED (Padina sp)
NOOR AFIFAH BT AZMI
SB10045

Bachelors of Applied Sciences (Hons) Industrial Biotechnology
METHODOLOGY
RESULTS & DISCUSSION
Padina sp
Classified based on their pigments and
colouration that exists in the plant.

Scientific Name:
Padina Sp
.
Common Name: Scroll Algae, Potato Algae
Depth Collected: 3 - 40 feet

This species of seaweed
can be eaten
in some place and also been used in
traditional medicine
.

Padina sp also is the only one brown seaweed that incorporated calcium.

Contains more than
72 trace minerals.


Padina sp
Substance or compound that has a biological activity if it has direct effects on living organism
s.
Bioactive Compounds
An agent that inhibits bacterial growth or kills bacteria.

The term is often used synonymously with the term antibiotic
Antibacterial Properties
Pulau Besar, Mersing in Johor Bahru.





Seaweed collection
Crude protein
isolation
QUANTITATIVE & QUALITATIVE ANALYSIS
Lowry Method
Accurate methods for quantifying proteins.
Measure the absorbance of the
unknown sample at a wavelength of
600nm
Seaweeds were carefully
chosen
--> removed the epiphytes
--> endoparasitic infections and undesired particles
Air dried under the shade
for 2 weeks
Gently brushed under running
seawater, rinsed with distilled
water

Sample ground with homogenizer
Padina sp added with distilled water and kept at 4ºC for 12 h
centrifugation at 8000rpm for 40min,
at 4 ºC.
Supernatant was collected and kept at 4ºC
Escherichia coli
Salmonella typhi Pseudomonas aeruginosa
Staphylococcus aureus Bacillus Subtilis
ANTIBACTERIAL ACTIVITY
Centrifuge at 8000rpm for
40 mins at room temperature.

Supernatant was collected and combined with previous supernatant

Crude extract +
25% of cold (TCA)

Kept in an ice bath for 30 min.

Supernatant was discarded

8000 rpm for 40 mins at room temperature

Rinsed
with 10% of cold TCA &
centrifuged 8000 rpm, at 4ºC for 5 min

-> Supernatant was discarded

-> Added 5% of TCA and go for last centrifugation

1 ml of 1N NaOH


1 hour with occasionally shaking room temperature

Crude protein
obtained
Tubes incubated at room
temperature for 30 mins

Absorbance was determined
by spectrophotometre

Six test tubes were prepared with BSA of 20,30,40,50 and 60 µl into five respectively test tubes.

2 ml of solution which consists of 48 ml 2% Na2CO3 in 0.1 N NaOH, 1ml of 1% NaK Tartrate in H2O and 1ml of 1% NaK Tartrate in H2O was added
Tubes were incubated for 10 mins

0.2 ml of dilute Folin-phenol
was added to each tube

Supervisor : Dr.Natanamurugaraj
Antibacterial Activity
MIC
Broth tube dilution method

24h of incubation at 37 ºC,
tubes were evaluated by
turbidity of the broth.
Method for determining the total protein
in a sample.

4 test tubes
1)BSA
2)Gelatin
3)Glycine and
4)Crude sample.
Each tube was added with 1ml of biuret
reagent. (observed for any the
colour changes.)

Biuret Method
Bioactive Compound by using GC-MS Analysis & HPLC
1µl of algae extract was injected into the GC-MS with Helium carrier gas
-> flow rate of 1.50 ml/min; injector and
->column oven temperature 280ºC and 80ºC;
injection mode ‘split’ and split ratio 20:1.

HPLC
-> Acetonitrile & deionised water.
->Filter first by the vacuum pump and membrane use was PTFE membrane filter. By using injection method of 20µl with flow rate of 1ml per minute and around 45 min for
each sample to be completed.
The wavelength used was 278nm-340nm.


Results & Discussion
BIOACTIVE COMPOUNDS FROM GS-MS AND HPLC

Crude protein of Padina sp

Lowry Test

Qualitative analysis of protein already been carried
out by Biuret’s test.

Determined the lowest concentration of crude extract
against specific bacteria.

MINIMAL INHIBITORY
CONCENTRATION (MIC)


Data of Minimal inhibitory concentration (MIC) for
reaction of gram-negative E.coli.

D) Zone of Inhibition of Staphylococcus aureus

C) Zone of Inhibition Pseudomonas aeruginosa
Antibacterial Activity

A) Zone of Inhibition Salmonella Typhi







B) Zone of Inhibition Escherichia coli
Conclusion
The result shown that :
-> Padina sp clearly has the antibacterial
properties.

->Aided of HPLC and GC-MS shown more strong evidence towards the antibacterial properties
and bioactive compounds that present
in seaweed.

E) Zone of Inhibition of Bacillus subtilis
QUALITATIVE & QUANTITATIVE
ANALYSIS
CONCLUSION
0.163 = 20.2µg approximately per 1ml
(Rollins et al., 2000) mentioned that tube dilution is the standard method for determining the levels of resistance to an antibiotic
MIC= 80µg/ml with the level of OD is 0.410
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