Introduction
Pathogenic and spoilage microorganisms had created many undesired effects
to human race. Antimicrobials are used to overcome these problems. Antimicrobial is a group of drugs includes antibiotics, antifungals,
antiprotozoals, and antivirals that are used to destroy
microorganisms or to prevent their development. They are
used in various aspects such as in agriculture, surgery, treatment and food
production. Some examples of antimicrobials which is critical important to
human are Penicillins,
Aminoclycosides
and Streptogramins.
Lactobacillales or lactic acid bacteria (LAB) are
microorganisms that produce lactic acid and proteinaceous bacteriocins which inhibit other
microorganisms to survive. They are gram-positive, acid-tolerant, non-sporeforming cocci, coccobacilli or rods and lack catalase. All LAB grow anaerobically, but unlike most anaerobes, they can grow in the presence of O2 as "aerotolerant
anaerobes". LAB bacteriocins contribute to the taste and texture of fermented products and inhibit
food spoilage bacteria by producing growth-inhibiting substances and large
amounts of lactic acid as acidification
inhibits the growth of spoilage agents. They are mainly used in
food processing such as making
yogurt, cheese, cultured butter, sour cream and sausage. Some examples of Lactobacillales are Lactobacillus, Lactococcus and Streptococcus.
Objective
To determine the antimicrobial effects of
extracellular extracts of selected LAB strains
Materials
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
UV/Vis spectrophotometer
Distilled deionized water
Trypticase soy agar (TSAYE)
Brain heart infusion agar (BHI)
Yeast extract
Procedure
(Refer to lab manual)
Results
Part 1: Determination of bacteriocin
activity via agar diffusion test
Strains of LAB
|
Strains of spoilage/pathogenic bacteria
|
Inhibition zone(cm)
|
LAB 1
|
Staphylococcus aureus
|
No
|
LAB 2
|
Staphylococcus aureus
|
No
|
Part 2: Determination of bacteriocin
activity via optical density
Dilutions
|
OD600 of spoilage/pathogenic bacteria
|
|||
Strain
: Staphylococcus aureus
|
||||
Reading
1
|
Reading
2
|
Reading
3
|
Average
|
|
0x
|
0.453
|
0.445
|
0.476
|
0.458
|
2x
|
0.671
|
0.665
|
0.673
|
0.670
|
10x
|
0.970
|
1.014
|
1.004
|
0.996
|
50x
|
1.090
|
0.995
|
0.950
|
1.012
|
100x
|
0.887
|
0.948
|
0.945
|
0.927
|
Equation
|
y = 0.0031x + 0.7115
|
|||
OD600 of control
|
0.247
|
0.225
|
0.229
|
0.234
|
50%
of OD600
|
0.124
|
0.113
|
0.115
|
0.117
|
AU/ml
|
-191.774
|
Discussion
Part 1: Determination of bacteriocin
activity via agar diffusion test
From this experiment, the volume of the LAB
extracellular extract and MRS needed for each universal bottle is shown as
below:
Dilution
Mixture
|
0x
|
2x
|
10x
|
50x
|
100x
|
Control
|
LAB Extracellular Extract (ml)
|
5.0
|
2.5
|
0.5
|
0.1
|
0.05
|
0
|
MRS Broth (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
|
Antimirobials are the substance that produced by a
certain microorganism to prevent other microorganisms to compete resources with
them. In this experiment, bacteriocin, lacic acid, diacetyl and hydrogen
peroxide act as the antimicrobials which is produced by Lactic Acid Bacteria
(LAB) are tested by antimicrobial susceptibility testing to test their ability
to inhibit the growth of SA (Staphylococcus aureus) culture. The goals of
antimicrobial susceptibility testing are to detect possible drug resistance in
common pathogens and to assure susceptibility to drugs of choice for particular
infections. The effectiveness of LAB antimicrobials towards the growth of SA
can be determined by measuring the inhibition zones around the LAB-staining
paper disks. Inhibition zones are the places where the growth of a certain
microorganism is inhibited by antimicrobial. The larger the inhibition zones,
the higher the degree of sensitivity of SA to LAB antimicrobial.
However, in our experiment, the inhibition zones
around the LAB paper disks cannot be determined as there is no growing of SA
culture. This is probably caused by the concentration of SA spoilage culture is
too dilute. In our experiment, we mixed 2 ml of SA culture into 100 ml of BHI
agar but refer to our lab manual, we should mix 20 ml of SA culture into 100 ml
of BHI agar. The low concentration of SA culture causes their growth in agar is
totally suppressed by LAB antimicrobials. This results we cannot measure the
degree of sensitivity of SA to LAB antimicrobial. Thus, we can at least
speculate there is a suppression of LAB to SA culture as SA cannot grow in the
agar with LAB. Besides, the bacteriocins produced by the lactic acid
bacteria(LAB) may not strong enough.
Part 2: Determination of bacteriocin
activity via optical density
Optical density can be measured by using the spectrophotometer.
Optical density is used as a measure of the concentration of bacteria in a
suspension. The spectrophotometer is an instrument which measures the amount of
light of a specified wavelength which passes through a medium; it is used to
determine the amount of light scatter. When visible light passes through a cell
suspension, the light is scattered. Greater degree of scatter proves that more
bacteria are present.
A spectrophotometer can be set at a wavelength of
420-660nm. Normally the OD600 is measured. OD600 is an abbreviation indicating
the optical density of a sample measured at a wavelength of 600 nm. OD600 is also
preferable to UV spectroscopy when measuring the growth over time of a cell
population. It is because at this wavelength, the cells will not be killed as
they would under too much UV light.
spectrophotometer
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.
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 experiment, the strain we used is Staphylococcus aureus. It is a
gram-positive coccal bacterium that is frequently found in the respiratory
tract and on the skin.
The result we obtained shows that the optical
density decreases slightly when the dilution is 100 x. However, the graph still
shows the positive gradient. This indicates the positive inhibition on the
growth pattern of the bacteria. It means the higher the concentration of
extracellular extract, the higher concentration of bacteriocin, the lower the
growth rate of bacteria. The inaccurate result might due to human error, we did not use the pipette correctly that causes the volume of MRS and Staphylococcus aureus taken incorrect. Besides, when we transfer the extracellular extracts
into the mixture, we should take the supernatant instead of pellet, if not it
might affect the accuracy of result. The expectation of the result is when the serial
dilution increasing, the optical density increasing too. The graph plotted
should be in linear positive gradient.
Conclusion
LAB is used to produce bacteriocin which can inhibit
the growth of bacteria (antimicrobial effect). Nowadays, the usage of LAB is getting attention as it
exerts a strong antagonistic activity against many microorganisms, including
food spoilage organisms and pathogens. The graph of determination of bacteria
activity via optical density showing a positive gradient indicates positive
inhibition of Staphylococcus aureus results
in high optical density.
References
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