LAB
5: DETERMINATION OF ANTIMICROBIAL EFFECT OF MICROBIAL EXTRACT
INTRODUCTION
An antimicrobial is a substance that kills
or inhibits the growth of microorganisms such as bacteria, fungi, or
protozoans. Antimicrobial drugs either kill microbes (microbiocidal) or prevent
the growth of microbes (microbiostatic). Disinfectants are antimicrobial
substances used on non-living objects or outside the body.
The history of antimicrobials begins with
the observations of Pasteur and Joubert, who discovered that one type of
bacteria could prevent the growth of another. They did not know at that time
that the reason one bacterium failed to grow was that the other bacterium was
producing an antibiotic. Technically, antibiotics are only those substances
that are produced by one microorganism that kill, or prevent the growth, of
another microorganism. Of course, in today's common usage, the term antibiotic
is used to refer to almost any drug that attempts to rid your body of a
bacterial infection. Antimicrobials include not just antibiotics, but
synthetically formed compounds as well.
The discovery of antimicrobials like
penicillin and tetracycline paved the way for better health for millions around
the world. Before penicillin became a viable medical treatment in the early
1940s, no true cure for gonorrhea, strep throat, or pneumonia existed. Patients
with infected wounds often had to have a wounded limb removed, or face death
from infection. Now, most of these infections can be cured easily with a short
course of antimicrobials.
However, with the development of
antimicrobials, microorganisms have adapted and become resistant to previous
antimicrobial agents. The old antimicrobial technology was based either on
poisons or heavy metals, which may not have killed the microbe completely,
allowing the microbe to survive, change, and become resistant to the poisons
and/or heavy metals.
Antimicrobial nanotechnology is a recent
addition to the fight against disease causing organisms, replacing heavy metals
and toxins and may some day be a viable alternative.
Infections
that are acquired during a hospital visit are called "hospital acquired
infections" or nosocomial infections. Similarly, when the infectious
disease is picked up in the non-hospital setting it is considered
"community acquired".
Certain groups of bacteria can produce
antimicrobial substances with the capacity to inhibit the growth of pathogenic
and spoilage microorganisms. Organic acids, hydrogen peroxide, diacetyl and
bacteriocins are included among these antimicrobial compounds. Interest in
naturally produce antimicrobial agents, such as bacteriocins, is on the rise,
since nowadys consumers demand "natural" and "minimally
processed" food.
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.
OBJECTIVE
To determine the antimicrobial effects of
extracellular extracts of selected LAB strains.
RESULT
PART 1 :
Determination of bacteriocin activity via agar diffusion test
 |
figure 1
presence of antimicrobial effects
|
 |
figure 2
presence of antimicrobial effects
STRAINS LAB
|
Strains of spoilage / pathogenic
bacteria
|
Inhibitio zone ( cm )
|
L.plantanum
|
S.aureus
|
0.0
|
K.pneumonia
|
0.9
|
P.aeruginose
|
0.9
|
L.casei
|
S.aureus
|
0.0
|
K.pneumonia
|
0.7
|
P.aeruginose
|
0.7
|
L.brevis
|
S.aureus
|
0.0
|
K.pneumonia
|
0.6
|
P.aeruginose
|
0.7
|
PART 2 :
Determination of bacteriocin activity via optical density
Dilutions
|
OD600 of
spoilage/pathogenic bacteria
|
S.aureus
|
K.pneumonia
|
P.aeruginosa
|
0X
|
-
|
-
|
-
|
2X
|
0.454
|
0.484
|
0.410
|
10X
|
0.610
|
0.696
|
0.620
|
50X
|
0.386
|
0.571
|
0.312
|
100X
|
0.238
|
0.464
|
0.265
|
Equation
|
y = 0.013x+0.278
|
y = 0.113x+0.111
|
y = -0.031x+0.532
|
OD600 of control
|
0.270
|
0.892
|
0.432
|
50% of OD600
|
0.135
|
0.446
|
0.216
|
AU/ml
|
-11
|
2.96
|
10.19
|
|
DISCUSSION
The lactic acid bacteria (LAB) comprise a clade of Gram-positive,
low-GC, acid-tolerant, generally non-sporulating, non-respiring rod or cocci
that are associated by their common metabolic and physiological
characteristics. These bacteria, usually found in decomposing plants and lactic
products, produce lactic acid as the major metabolic end-product of
carbohydrate fermentation. This trait has, throughout history, linked LAB with
food fermentations, as acidification inhibits the growth of spoilage agents. Proteinaceous
bacteriocins are produced by several LAB strains and provide an additional
hurdle for spoilage and pathogenic microorganisms.
Furthermore, lactic acid and other metabolic products contribute to the
organoleptic and textural profile of a food item. The industrial importance of
the LAB is further evinced by their generally recognized as safe (GRAS) status,
due to their ubiquitous appearance in food and their contribution to the
healthy microflora of human mucosal surfaces. The genera that comprise the LAB
are at its core Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and
Streptococcus as well as the more peripheral Aerococcus, Carnobacterium,
Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus, Vagococcus, and
Weisella; these belong to the order Lactobacillales.
PART 1
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 is 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 the zone 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.
Kirby-Bauer antibiotic testing (KB testing
or disk diffusion antibiotic sensitivity testing) is a test which uses
antibiotic-impregnated wafers to test whether particular bacteria are
susceptible to specific antibiotics. A known quantity of bacteria are grown on
agar plates in the presence of thin wafers containing relevant antibiotics. If
the bacteria are susceptible to a particular antibiotic, an area of clearing
surrounds the wafer where bacteria are not capable of growing (called a zone of
inhibition).
This along with the rate of antibiotic
diffusion are used to estimate the bacteria's sensitivity to that particular
antibiotic. In general, larger zones correlate with smaller minimum inhibitory
concentration (MIC) of antibiotic for that bacteria. This information can be used
to choose appropriate antibiotics to combat a particular infection.
PART 2
Optical density that is measured in the
spectrophotometer can be used as the measurement of the concentration of the
bacteria suspension. It is basically used for the measurement of transmittance
or reflectance of solution that are depending on their wavelength.
Optical density of this experiment
contain MRS medium and pathogenic bacteria was determined by using the
spectrophotometer of OD600. One from the
others type of LAB used is Lactobacillus plantirum. While the tree type of
pathogenic microorganism that had been used were S.Aureus, P.Aeruginosa and
K.Pneumonia.
The positive-controls that show higher
optical density than the other sample show antimicrobial effect. While the
negative-control was prepared in MRS with 2mL distilled water is for auto-zero
via spectrophotometer.
CONCLUSION
Bacteriocins from lactic acid bacteria are
of importance in bioconservation of various foods. Moreover, the use of more
than one LAB bacteriocin as a combination of biopreservative may have major
applications in improving food safety. In the present study, the inhibitory
effect of the cell-free filtrates of each of the 20 isolates was evaluated.
Antimicrobial activity was observed for 6 isolates, and only against Gram
positive bacteria. The biochemical nature of the antibacterial molecule
produced by S. thermophilus T2 was studied in both the cell-free supernatant
and the chloroform extract.
REFERENCE
http://en.wikipedia.org/wiki/Lactic_acid_bacteria
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