Introduction
Bacteriocins are proteinaceous toxins produced by bacteria, it can inhibit the growth of closely or similar bacterial strains. It is also a naturally produced antimicrobial agent. Bacteriocins can be found in numerous bacteria however, the ones that are produced by lactic acid bacteria (LAB) are significant to the food industry. This is because, they have huge potential application as one of the natural biopreservatives. As interest in natural and minimally processed food is in demand, more research is done to cater for consumer needs.
Lactic acid bacteria (LAB) are characterized as Gram-positive cocci or rods, non-aerobic but aerotolerant, able to ferment carbohydrates for energy and lactic acid production. Thus, LAB bacteriocins can work via different mechanisms to exert an antimicrobial effect. 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 several microorganisms such as Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecalis and Clostridium tyrobutyricum.
To determine the antimicrobial effects of extracellular extracts of selected LAB strains.
Material and reagents
MRS broth
Sterile filter paper disk (50mm x 50mm)
Forceps
Sterile universal bottles
Cultures of LAB and spoilage / pathogenic organisms
Bench-top refrigerated centrifuge
Incubator 30˚C and 37˚
CUV/V is spectrophotometer
Distilled deionized water
Trypticase soy agar
Brain heart infusion agar
Yeast extract
Procedure
Results
Bacteriocins are proteinaceous toxins produced by bacteria, it can inhibit the growth of closely or similar bacterial strains. It is also a naturally produced antimicrobial agent. Bacteriocins can be found in numerous bacteria however, the ones that are produced by lactic acid bacteria (LAB) are significant to the food industry. This is because, they have huge potential application as one of the natural biopreservatives. As interest in natural and minimally processed food is in demand, more research is done to cater for consumer needs.
Lactic acid bacteria (LAB) are characterized as Gram-positive cocci or rods, non-aerobic but aerotolerant, able to ferment carbohydrates for energy and lactic acid production. Thus, LAB bacteriocins can work via different mechanisms to exert an antimicrobial effect. 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 several microorganisms such as Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecalis and Clostridium tyrobutyricum.
To determine the antimicrobial effects of extracellular extracts of selected LAB strains.
Material and reagents
MRS broth
Sterile filter paper disk (50mm x 50mm)
Forceps
Sterile universal bottles
Cultures of LAB and spoilage / pathogenic organisms
Bench-top refrigerated centrifuge
Incubator 30˚C and 37˚
CUV/V is spectrophotometer
Distilled deionized water
Trypticase soy agar
Brain heart infusion agar
Yeast extract
Procedure
(Refer to lab manual)
Results
Part I: Determination of
Bacteriocin Activity Via Agar Diffusion Test
Absence of inhibition zone
Strains
of LAB
|
Strains
of spoilage/pathogenic bacteria
|
Inhibition
zone(cm)
|
LAB
1
|
Staphylococcus aureus
|
No
inhibition zone
|
LAB
2
|
Staphylococcus aureus
|
No
inhibition zone
|
Reading of optical density from spectrophotometer
Dilution
|
OD600 of Spoilage/ Pathogenic Bacteria
|
|||
Reading
1
|
Reading
2
|
Reading
3
|
Average
|
|
0x
|
0.421
|
0.408
|
0.396
|
0.408
|
2x
|
0.912
|
0.903
|
0.885
|
0.900
|
10x
|
1.037
|
1.005
|
1.024
|
1.022
|
50x
|
0.955
|
0.962
|
0.961
|
0.959
|
100x
|
0.908
|
0.946
|
0.824
|
0.893
|
Equation
|
y=
- 0.0021x + 1.1
|
|||
OD600
of control
|
0.233
|
0.231
|
0.224
|
0.229
|
50%
of OD600
|
0.117
|
0.116
|
0.112
|
0.115
|
AU/ml
|
469.05
| |||
Discussion
Part I: Determination of Bacteriocin Activity Via Agar Diffusion Test
- Bacteriocins are
usually effective against gram-positive microorganisms.
-LAB bacteriocins may be
inefficient to inhibit Gram-negative organisms because the outer membrane
hinders the site for bacteriocin action, which is the cell membrane
- Different mechanisms of action have been proposed for
bacteriocins: alteration of enzymatic activity, inhibition of spore germination
and inactivation of anionic carriers through the formation of selective and
non-selective pores
- Staphylococcus aureus is
a gram -positive coccal bacterium .Although it has thick cell wall made of polysaccharides and proteins but it is easily
digested by acid particularly acetic acid produced by lactic acid bacteria
(LAB).
-If there is a circle around the
paper disk or colony of lactic acid bacteria (LAB),
that means bacteriocin is able to inhibit the growth of Staphylococcus aureus . This is the zone of inhibition.
- The larger the inhibition zone means that the bacteriocins is effective on the pathogenic bacteria and vice versa.
-For our lab result, there is no
inhibition zone.
This is because not enough lactic acid bacteria (LAB)
are being applied around the
pathogenic bacteria. Without adequate numbers of lactic acid bacteria (LAB), 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.
-The bacteriocins produced by the lactic acid bacteria(LAB)
is not strong as antibiotics.
Part II : Determination
Of Bacteriocin Activity Via Optical Density
-The
serial dilution of extracellular extract is done as shown in the table below.
- Serial dilutions are used to accurately create highly diluted
solutions as well as solutions for experiments resulting
in concentration curves with a logarithmic scale.
0x (ml)
|
2x (ml)
|
10x (ml)
|
50x (ml)
|
100x (ml)
|
Control (ml)
|
|
EE
|
5
|
2.5
|
0.5
|
0.1
|
0.05
|
0
|
MRS
|
0
|
2.5
|
4.5
|
4.9
|
4.95
|
5
|
Total:
|
5
|
5
|
5
|
5
|
5
|
5
|
- Optical density, measured in a spectrophotometer, can be
used as a measure of the concentration of bacteria in a suspension. As visible
light passes through a cell suspension the light is scattered. Greater scatter
indicates that more bacteria or other material is present. The amount of light
scatter can be measured in a spectrophotometer. Typically, when working with a
particular type of cell, we would determine the optical density at a particular
wavelength that correlates with the different phases of bacterial growth.
Generally we will use cells that are in their mid-log phase of growth.
Typically the OD600 is measured.
- One arbitrary (AU) is defined as the dilution factor of the
extracellular extract that inhibited 50% of the spoilage/pathogenic bacteria
growth and expressed as AU/ml.
Control : 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
From the graph, m = - (1.1-0.893) ÷
100 = -0.0021
From the graph, c = 1.1
Thus, y = -0.0021x + 1.1
When y = 0.115 , x = ( 0.115- 1.1 )
÷ - 0.0021
x = 469.05
Thus, AU/ml = 469.05
-From the result, the graph shows that as the serial dilution increases, the optical density decreases. This shows that there is negative inhibition of the pathogenic bacteria. This might be caused by the human error. When we transfer the extracellular extracts into the mixture, we should take the supernatant instead of pellet, we should not shake the broth containing LAB cultures after centrifuge because this will mix the supernatant and pellet together.
-The control shows lower reading
than all sample of dilution. This might be due to we didn’t shake the bottle of
bacteria culture before pipetting especially for the control.
-The
other reason is using distilled
water during serial dilution. The distilled water is colourless. When the LAB is diluted with
distilled water, the optical colour density will become very much lower
compared to the normal colour density of LAB. Thus, the results obtained is wrong.
- Therefore, the
peptone is suggested to replace with the distilled water in the serial dilution
as the colour of peptone is quite similar with the culture.
-Actually there should be a
positive inhibition on the growth pattern of the bacteria. Generally, as the serial of dilution increases, the optical density will
increase. It shows lactic acid bacteria (LAB) has
strong microbial effect on the pathogenic bacteria which is Staphylococcus aureus.The high concentration of extracellular extract and bacteriocins result
in low growth rate of bacteria.
The results shows the potential of Lactic Acid Bacteria (LAB) as biopreservatives against both Gram postive (Escherichia coli) and Gram negative (Staphylococcus aureus) bacteria. Bacteriocin was extracted by centrifuge method is to determine for its antagonistic activity. Lactic Acid Bacteria (lactobacilli) will produce various substances
such as bacteriocin to inhibit the growth of pathogenic microorganisms. Lactic acid bacteria (LAB) also synthesize bacteriocins that have antimicrobial effects.
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