YASMIN SYAFIKAH
BT RAZALI ( 111435 )
INTRODUCTION
Certain
groups of bacteria can produce antimicrobial substances with the capacity to
inhibit the growth of pathogenic and spoilage microorganisms. Organic acid,
hydrogen peroxide, diacetyl and bacteriocins are included among these
antimicrobial compounds. Interest in naturally produced antimicrobial agents
such as bacteriocins, is on the rise, since nowadays consumers demand natural
and minimally processed food.
Bacteriocins
comprise a large and diverse group of ribosomally synthesized antimicrobial
protein 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 monocytogenes, Staphylococcus aures,
Enterococcus faecalis and Clostridium tyrobutyricum.
Part
I: Determination of bacteriocin activity via agar diffusion test
The agar
diffusion test, or the Kirby-Bauer disk-diffusion method, is a means of
measuring the effect of an antimicrobial agent against bacteria grown in
culture.
The
bacteria in question are swabbed uniformly across a culture plate. A
filter-paper disk, impregnated with the compound to be tested, is then placed
on the surface of the agar. The compound diffuses from the filter paper into
the agar. The concentration of the compound will be highest next to the disk,
and will decrease as distance from the disk increases. If the compound is effective
against bacteria at a certain concentration, no colonies will grow where the
concentration in the agar is greater than or equal to the effective
concentration. This is thezone of inhibition. Thus, the size of the zone of
inhibition is a measure of the compound's effectiveness: the larger the clear
area around the filter disk, the more effective the compound.
Part
II: Determination of bacteriocin activity via optical density
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, you would determine the optical density at a particular wavelength
that correlates with the different phases of bacterial growth. Generally we
will want to use cells that are in their mid-log phase of growth. Typically the
OD600
is measured.
OBJECTIVE
To determine
the antimicrobial effect of extracellular extracts of selected LAB strain.
RESULT
PART I
Calculations
Inhibition zone :
Serial dilutions of extracellular extract
Y axis :
Abs600 or OD600
X axis : Serial dilutions of
extracellular extract
m and c :
Constants
One
arbitrary unit (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 .
Therefore , x = ( Y – c)/m
When Y = Z/2
, thus, x= (Z /2-c)
Strains
of LAB
|
Strain
of spoilage
|
Inhibition
zone (cm)
|
Lp
|
Kp
|
0.7
|
Sa
|
-
|
|
Pa
|
0.6
|
|
Lb
|
Kp
|
0.7
|
Sa
|
-
|
|
Pa
|
0.8
|
|
Lc
|
Kp
|
0.6
|
Sa
|
-
|
|
Pa
|
0.7
|
Strain
of LAB
|
Strains
of spoilage/pathogenic bacteria
|
Inhibition
zone (cm)
|
LAB
species
|
Sa
|
-
|
Kp
|
1.0
|
|
Pa
|
1.05
|
PART
II
Sa
Dilutions
|
Abstarct
|
0x
|
0
|
2x
|
0.735
|
10x
|
1.032
|
50x
|
0.621
|
100x
|
0.502
|
Kp
Dilution
|
Abstract
|
0x
|
0
|
2x
|
0.637
|
10x
|
0.691
|
50x
|
0.641
|
100x
|
0.765
|
Pa
Dilutions
|
Abstract
|
0x
|
0
|
2x
|
0.71
|
10x
|
0.076
|
50x
|
0.804
|
100x
|
0.528
|
DISCUSSION
PART I
Lactic acid bacteria produce
a variety of antagonistic factors that include metabolic end products,
antibiotic-like substances and bactericidal proteins, termed bacteriocins. The
range of inhibitory activity by bacteriocins of lactic acid bacteria can be
either narrow inhibiting only those strains that are closely related to the
producer organism, or wide, inhibiting a diverse group of Gram-positive
microorganisms.
The extracts of the lactic
acid bacteria gave zones of inhibition onto the indicator pathogen strains
tested. In the agar well diffusion assay a linear relationship existed between
response (diameter or area of the zone of inhibition). The strains inhibited
were Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa . The diameter of inhibition zone obtained
from Klebsiella pneumoniae was 1.0 cm while the diameter of inhibition zone
obtained from Pseudomonas aeruginosa was
1.05 cm but for Staphylococcus aureus the result can be obtained due to mistake
from procedure but the diameter of inhibition zone should be bigger than Klebsiella
pneumoniae and Pseudomonas aeruginosa . Staphylococcus
aureus was a gram positive bacteria while Klebsiella pneumoniae and Pseudomonas
were gram negative bacteria. The result showed us that the diameter of
inhibited zone in Staphylococcus aureus was larger than others. This was
because a gram positive indicator bacterium was much more sensitive to
bacteriocin of lactic acid bacteria strains than gram negative indicator
bacteria. The resistance of gram negative bacteria was attributed to the
particular nature of their cellular envelope, the mechanisms of action
described for bacteriocin bringing in a phenomenon of adsorption.
According to Bhunia et al. (1991) the
pediocin(bacteriocin produced by
Pediococcusacidilactici) interacts with lipoteichoic acids absent in
gram negative bacteria. These molecules play the role of site of necessary not
specific reception to produce the bactericidal effect. Some bacteriocins
produced by gram positive bacteria had a broad spectrum of activity. These
variations of sensibility were due to the characteristic of indicators strains
and thus in level of hurt caused by the inhibitive factor.
PART
II
The activity of bacteriocins was difficult
to quantify and was dependent on the determination method. The most widely used
techniques were based on the evaluation of growth inhibition caused on a
sensitive bacterial strain, either in solution or in agar plates. A
standardized bioassay does not exist because bacteriocinswere significantly
different among them, making difficult the use of a common standard, and
results depend on experimental conditions. The advantages of using this method
were the elimination of diffusion related problems, its quickness, commodity
and low cost. Major experiment errors came from the cell sedimentation and
interference of sample colour. In addition, time of reaction was generally
critical and the relationship between the bacteriocin concentration and the
inhibitory response usually follow a sigmoidal curve making use of complex
regression models of limited practical application.The logarithm of the dose
while a non-linear equation was used to model the sigmoidal dose/response curve
in photometric assays (PA). The dose/response curves were used to define
titters of the standard solutions in arbitrary units and to develop
quantitative assays for all the bacteriocins.
Generally, the OD value is
higher from the 100X dilution. It indicated that the LAB has stronger
antimicrobial effect on the pathogenic bacteria compare to a more diluted
solution. The OD600
values in 100X dilutions had the highest value in both pathogenic bacteria
where Staphylococcos aurens is 0.270 Klebsiella
pneumonia is 0.829 and Pseudomonas aeruginosa is 0.432.So, the more diluted the
extracellular extract, the greater the value of OD600. The graphs of Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa
are obtained by plotting the optical
density of spoilage/pathogenic bacteria Staphylococcus aurensor, Klebsiella pneumoniae and Pseudomonas
aeruginosa against different serial
dilutions of extracellular extract. The graph demonstrated a less linear
relationship between serial dilutions of extracellular extract and OD600, with the regression analysis giving
an R2 value of 0.138 for Staphylococcus aureus.While the graph of Klebsiella pneumonia
demonstrated a more linear relationship between serial dilutions of
extracellular extract and OD600, with the regression analysis giving an R2 value of 0.612 and for Pseudomonas
aeruginosa is 0.245 .
CONCLUSION
In summary, the
present results clearly suggest the potential usefulness of the bacteriocins
produced by Lactic acid bacteria (LAB) as bio preservatives against both
Escherichia coli and Staphylococcus aureus. Both acidification and the
production of hydrogen peroxide by LAB were ruled out as the source of the
inhibition. LAB competed with spoilage microorganisms, such as certain
Gram-negative bacteria for nutrients or space with. Moreover, the shelf-life of
food products could extend the production of organic acids, hydrogen peroxide,
low molecular weight metabolites (such as diacetyl and bacteriocins) due to
their inhibiting effect on the growth of spoilage and pathogenic bacteria. For
these reasons, antagonistic effects against pathogenic bacteria exhibited by
harmless LAB applied interest as bio preservatives – with the subsequent
reduction in the use of antibiotics – in future aquaculture activities.
Bacteriocins have
been defined as proteinaceous substances exhibiting bactericidal activity
against closely related species. Currently they are receiving increased
attention because of their inhibitory activity against food spoilage and
food-borne pathogenic bacteria such as Listeria monocytogenes. Commercial nisin
preparations have been evaluate in food systems. It was now widely used as bio
preservatives in the food industry due to their antibacterial properties. This
allowed a more strict microbial control of a variety of commercial food
products.
Determination of
bacteriocin activity by the agar diffusion assay in which inhibition zones are
produced in plates in a procedure similar to that of antibiograms, is
undoubtedly the most commonly used despite the inconveniences and limitations
of its application. The performance of the method, which is laborious and
time-consuming, depends largely on human ability and judgment and precision
can’t be achieved when inhibition zones are unclear or not perfectly circular.
Diffusion-related difficulties of the active substance represent another
important limitation of agar diffusion assays. The need to eliminate
diffusion-related problems associated with the agar techniques, introduced
liquid medium methods, which make use of indicator organisms and quantify the
bacteriocin concentration from the percentage of growth inhibition in the
indicator organism. Since then, applications of turbidometric assays by
spectrophotometer can be found in a number of reports in which, as with the
agar diffusion assay, various indicator microorganisms were used, in procedures
that show large variability regarding bacteriocin extraction, general
experimental conditions and definition of the bacteriocin unit. Sensitivity
limits and linearity of responses to various bacteriocin levels vary
significantly among different test-microorganisms in both bioassays, the lower
sensitivity limits depending on both the test-microorganism and the applied
method. Very low nisin concentrations, e.g. 1 IU/ml, were more safely
determined in the turbidometric assay (spectrophotometer) through determination
of the percentage of inhibition of growth of the indicator microorganism. This
method proved to be more suitable for determination of nisin in processed food
samples.
Although the agar
diffusion assay is the most widely used method in routine measurements of
bacteriocin activity, turbidometry (spectrophotometer) offers a simpler, faster
and more reliable alternative since diffusion related problems are eliminated,
the degree of human intervention and judgment is low, and very low bacteriocin
concentrations can be quantified.
REFERANCES
4.
Pearson International Biology, 8th Edition, Campbell, Reece
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