INTRODUCTION
An antimicrobial is an agent
that kills microorganisms or inhibits their growth. Antimicrobial medicines can
be grouped according to the microorganisms they act primarily against. For
example, antibacterial are used against bacteria and antifungals are used
against fungi. A wide array of antimicrobial classes that are used for animal
health and production are also used for human treatments. Those antimicrobials
genes that propagate from the use of antibiotics for animal populations may
spread and eventually hurt the efficacy of these same antibiotic when they are
used to treat people.
While all antimicrobial is a
potential human health hazard, the preserved efficacy of some human medicine
antibiotics is of higher priority. Antimicrobial resistance is only a threat to
public health when humans are infected with a resistant organism that is
difficult or impossible to treat. This is an issue seen more frequently with
human pathogens transmitted between humans (such as extremely drug resistant
tuberculosis, also called XDRTB, and MRSA). While outbreaks of resistant
foodborne pathogens have been reported, very few have been epidemiologically
traced back to the farm. Even fewer have been specifically associated with a
specific indication such as increased rate of gain or prevention of a
particular disease.
Bacteriocins comprise a large and
diverse group of ribosomally synthesized antimicrobial proteins or peptides.
Although bacteriocins can be found in numerous Gram-positive and Gram-negative
bacteria, those produced by lactic acid bacteria (LAB) have received special
attention in recent years due to their potential application in the food
industry as natural biopreservatives. Different classes of LAB bacteriocins
have been identified on the basis of biochemical and genetic characterization.
These bacteriocins have been reported to inhibit the growth of Listeria
monocyotogenes, Staphylococcus aureus, Enterococcus faecalis and Clostridium
tyrobutyricum.
Materials
and reagents
·
MRS broth
·
Sterile
filter paper disk
·
Forceps
·
Sterile
universal bottles
·
Cultures of
LAB and spoilage/pathogenic organisms
·
Bench-top
refrigerated centrifuge
·
Incubator
30oc and 37oc
·
UV/V is
spectrophotometer
·
Distilled
deionized water
·
Trypticase
soy agar
·
Brain heart
infusion agar
·
Yeast extract
Procedure
Part I: Determination
of bacteriocin activity via agar diffusion test
1. Three
petri dishes were labelled for each group.
2. A
petri dish was shared between two person.
3. Each
petri dish was divided into four parts and each side for one replicate.
4. A
strain of pathogen (Staphylococcus aureus) and a strain of lactic acid
bacteria (LAB) were used in this experiment.
5. 10
ml of trypticase soy-yeast extract agar (TSAYE) was added into each labelled
petri dish and the petri dish was moved in “8 shape” on the work surface to
ensure the agar was distributed evenly.
6. The
petri dish was left aside for solidification.
7. 2
ml of pathogen (Staphylococcus aureus) was innoculated into 10 ml of
brain heart infusion (BHI) agar.
8. The
innoculation process must be carried out quickly to avoid solidification of BHI
agar.
9. The
mixture is then loaded on top of the TSAYE agar layer and the petri dish was
moved in “8 shape” on the work surface to ensure the agar was distributed
evenly.
10. The
petri dish was left aside for solidification.
11. 50
ml of broth containing LAB cultures was centrifuged at 3500rpm for 10 minutes.
The supernatants will be used as extracellular extracts.
12. The
forceps were held by its handle and the tip of the forceps were heated with the
flame of a Bunsen burner.
13. A
sterile filter paper disk was aseptically picked up using sterile forceps.
14. The
paper disk was then dipped into Lactobacillus Fermentum containing
extracellular extracts. The excess extract was drained off completely.
15. The
paper disk was placed on top of the solidified BHI agar.
16. The
petri dishes were incubated for one day at 37°C.
17. Upon
incubation, the inhibition zones were measured (in cm) and the readings were
recorded.
Part II: Determination of bacteriocin activity via optical density
1. Universal
bottles were prepared.
2. Each
of the universal bottle was labelled with 0x, 2x, 10x, 50x ,100x and control.
3. Lactobacillus Plantarum
was mixed with MRS to be diluted to final volume of 5 ml in each universal
bottle labelled. The volume of the Lactobacillus Plantarum and MRS needed
for each universal bottle is shown as below:
|
0x
|
2x
|
10x
|
50x
|
100x
|
control
|
|
|
Volume of Lactobacillus Plantarum (ml)
|
5.0
|
2.5
|
0.5
|
0.1
|
0.05
|
0
|
|
Volume of MRS (ml)
|
0
|
2.5
|
4.5
|
4.9
|
4.95
|
5.0
|
|
Total (ml)
|
5.0
|
5.0
|
5.0
|
5.0
|
5.0
|
5.0
|
4. Each
of the mixture was then added with 5 ml of MRS and 1 ml of pathogen Escherichia
coli (E.coli).
5. The
universal bottles were then incubated for 12-15 hours at 37°C.
6. Upon
incubation, the optical density of the pathogenic bacteria in each universal
bottle was measured at 600 nm using spectrophotometer.
Results:
Part 1: Determination of bacteriocin activity via agar diffusion test.
Strains of LAB: Lactobacillus plantarum
|
Strain of LAB
|
Strain of pathogenic bacteria
|
Inhibition zone (cm)
|
||
|
Lactobacillus Fermentum
|
Staphylococcus aureus
|
Diamater1 (cm)
|
Diameter 2 (cm)
|
Average diameter (cm)
|
|
0.95
|
0.80
|
0.88
|
||
|
0.60
|
0.70
|
0.65
|
||
|
0.73
|
0.76
|
0.75
|
||
|
0.65
|
0.64
|
0.65
|
||
|
0.52
|
0.45
|
0.49
|
||
|
0.50
|
0.30
|
0.40
|
||
Part II: Determination
of bacteria activity via optical density
|
Dilutions
|
OD600 of spoilage/ pathogenic bacteria
|
|
Strain 1 : E.coli
|
|
|
0x
|
0.254
|
|
2x
|
0.384
|
|
10x
|
0.351
|
|
50x
|
0.349
|
|
100x
|
0.428
|
|
Equation
|
y=0.00098x+0.33
|
|
OD600 of control
|
0.1370
|
|
50% of OD600
|
0.0685
|
|
AU/ ml
|
-266.84
|
DISCUSSION
Part 1: Determination Of Bacteriocin Activity Via Agar
Diffusion Test
1. Bacteriocin are
proteinaceous toxins produced by bacteria to inhibit the growth of
microorganisms such as fungi, protozoans and similar or closely related
bacterial strains by producing intraspecies antagonistic effects.
2. The larger the inhibition
zone (no bacteria growing area) on the agar medium, means that the bacteriocin
is effective on the pathogenic bacteria and vice versa.
3. The production of
bacteriocins, organic acids, free fatty acids, ammonia, diacetyl and hydrogen
peroxide by lactic acid bacteria (LAB) promotes bacterial interference. These
metabolites have been applied for many years to extend the shelf life of foods
in food industry by enabling acidification that will inhibit the growth of
spoilage agents. Besides that, proteinaceous bacteriocins produced by LAB
supress the growth of pathogenic microorganisms.
4. As for none inhibition
zone exist result are because not enough Lactobacillus fermentum are being applied
around the pathogenic bacteria. This is because without adequate numbers of
Lactobacillus fermentum, the point of critical mass which is needed cannot
occur and the bacteria will be unable to have the desired impact on the
symptoms being treated.
Part 2:
Determination Of Bacteriocin Activity Via Optical Density
1. The optical density (OD)
or called as scattering intensity is the measure of transmittance of an optical
medium for a given wavelength. The higher the OD the lower the transmittance,
the higher protection factor by a filter (e.g. goggles, viewing windows, etc.).
This factor allows us to calculate the cell density from any measured OD. A
spectrophotometer can be set at a wavelength of 420 – 660 nm. In this
experiment, the OD600 is measured. Typically, this wavelength must be
standardized and may need to be adjusted specifically to the material being
tested. Different vegetative cells and bacterial spores may not have the same
maximal absorbance wavelength.
2. One arbitrary (AU) is
known as the dilution factor of the extracellular extract that inhibited 50% of
the spoilage or pathogenic bacteria growth and expressed as AU/mL.
Abs600 = Z. Thus, 50% of Z = Z/2
y = mx + c ; Thus, x = (y-c)/m
When y = Z/2, Thus x = (Z/2 - c)/m
3. The positive control which
showed the growth of bacteria without extracellular extract of lactic acid
bacteria has been set up for each pathogenic bacteria. TheOD600 of the positive
control was then measured in order for us to investigate whether there is
inhibition of pathogenic bacteria activity by comparing the OD600 of the
samples. If the OD600 of the sample is less than OD600 of the positive control,
there will be inhibition of spoilage bacteria.
4. Based on our result, LAB
shows low inhibition activity on E.Coli. This might be due to we didn’t shake
the bottle of bacteria culture before pipetting especially for the positive
control. Therefore, the reading for positive control is lower than all the
samples of different dilution.
CONCLUSION
Some of the microorganisms
can synthesis certain substances that have antimicrobial effects. Bacteriocins
produced by lactic acid bacteria (LAB) as bio preservatives against both
Escherichia coli (E.coli) and Staphylococcus aureus. Lactic acid bacteria (LAB)
also synthesize bacteriocins that have antimicrobial effects. The use of strains
that produce multiple bacteriocins could be advantageous to limit the potential
emergence of bacteriocin-resistant populations.
REFERENCE
