Name : Teoh Leong Sin
Matric no: 111433
LAB 5: Determination of Antimicrobial
Effects of Microbial Extracts
Introduction:
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 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 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 monocytogens, Staphyloccus aureus, Enterococcus faecalis and
Clostridium tyrobutyricum.
Objective:
To determine
the antimicrobial effects of the extracelluar extracts of selected LAB strains.
Results:
Part 1:
Determination of bacteriocin activity via agar diffusion test
The sample result that shows that there is inhibition zone or presence of antimicrobial effect.
The sample result that shows that there is no inhibition zone or absence of antimicrobial effect.
Strains of Lab
|
Strains of spoilage/pathogenicbacteria
|
Inhibition zone(cm)
|
Lactobacillus casei
|
S.aureus
|
(0.6+0.6)/2 = 0.6
|
P.aeruginosa
|
(1.3+0.9)/2 = 1.15
|
|
K.pneumonia
|
(1.0+1.3)/2 = 1.1
|
|
Lactobacillus brevis
|
S.aureus
|
0
|
P.aeruginosa
|
0
|
|
K.pneumonia
|
(0.7+0.8)/2 = 0.75
|
|
Lactobacillus planterum
|
S.aureus
|
0
|
P.aeruginosa
|
0
|
|
K.pneumonia
|
(0.9+0.9)/2 = 0.9
|
Part 2 : Determination of bacteriocin activity via optical density
Serial dilution of extracellular extract
Dilutions
|
OD600 of spoilage/pathogenic
bacteria
|
||
Strain 1 : S.aureus
|
Strain 2 : P.aeruginosa
|
Strain 3 : K.pneunomia
|
|
2x
|
0.699
|
0.854
|
0.828
|
10x
|
0.698
|
0.990
|
0.943
|
50x
|
0.590
|
0.780
|
0.625
|
100x
|
0.168
|
0.553
|
0.512
|
Equation
|
y= -0.0054x + 0.7572
|
y= -0.0038x + 0.9468
|
Y= -0.004x + 0.8849
|
OD600 of control
|
0.508
|
0.129
|
1.156
|
50% of OD600
|
0.254
|
0.0645
|
0.578
|
AU/ml
|
93.19
|
232.18
|
77.85
|
Discussion:
1)An anti-microbial is a substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, and protozoans
2)The Lactic acid bacteria(LAB) comprise a clade of Gram-positive,acid-tolerant,generally non-sporulating, non-respiring rod or cocci that are associated by their common metabolic and physological characteristics.Lactic acid bacteria are rod-shaped bacilli or cocci characterizd by an increased tolerance to a lower pH range.This aspect partially enables LAB to outcomplete other bacteria in a natural fermentation as they can withstand the increased acidity from organic acid production. LAB are amongst the most important groups of mircroorgnisms used in the food industry.
3)Part 1 determination of bacteriocin activity is mainly due to lactic acid and other acid production.
The agar diffusion test is means of measuring effect of an antimicrobial agent against bacteria grown in culture. 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 is a measure of the compound's
effectiveness: the larger the clear area around the filter disk, the
more effective the compound.
4)Three types of LAB have been used in the experiments are Lactobacillus casei, Lactobacillus brevis, Lactobacillus plantirum. Three types of pathogenic microorganisms have been used are S.aureus, P.aeruginosa, K.pneumonia.
5)Spectrophotometer is a photometer that can measure the intensity features of spectrophotometers are spectral bandwidth and linear range of absorption or relectance measurement.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. 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. Typically the OD600 is measured.
6)Strain of L. plantarum showed the strongest inhibition effects on K. pneumonia because its OD600 were less than the OD600 of the
positive control among the three pathogenic bacteria.
Conclusion:
Bacteriocin was extracted by centrifuge method to determine for the antagonistic activity. LAB is a useful bacteria used to produce bacteriocin that can inhibit the growth of bacteria. 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 .
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.
References:
http://en.wikipedia.org/wiki/Lactic_acid_bacteria
http://en.wikipedia.org/wiki/Spectrophotometry
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1622751/
http://www.pjbs.org/pjnonline/fin202.pdf
http://dictionary.reference.com/browse/arbitrary