Name : Kong Siao Thung
No. Matrice : 111372
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 ofListeria monocytogens, Staphyloccus aureus, Enterococcus faecalis
and Clostridium tyrobutyricum.
Objective
To determine the antimicrobial effects of
extracellular of selected LAB strains.
Results
Part 1 Determination of bacteriocin
activity via agar diffusion test
Strains of LAB
|
Strains of spoilage/ pathogenic
bacteria
|
Inhibition zone (cm)
|
Lactobacillus Plantarum
|
S. aureus
|
-
|
K. pneumonia
|
(1.40+0.79)/2= 1.095
|
|
P. aeruginosa
|
(0.70+0.81)/2=0.755
|
|
Lactobacillus Brevis
|
S. aureus
|
-
|
K. pneumonia
|
(1.40+0)/2=0.70
|
|
P. aeruginosa
|
(1.10+0)/2=0.55
|
|
Lactobacillus Casei
|
S. aureus
|
-
|
K. pneumonia
|
(1.00+0.90)/2=0.95
|
|
P. aeruginosa
|
(0+1.03)/2= 0.515
|
Lactobacillus Plantarum (S. aureus)
Lactobacillus Plantarum (K. pneumonia)
Lactobacillus Plantarum (P. aeruginosa)
Lactobacillus Brevis (S. aureus)
Lactobacillus Brevis (K. pneumonia)
Lactobacillus Brevis (P. aeruginosa)
Lactobacillus Casei (S. aureus)
Lactobacillus Casei (K. pneumonia)
Lactobacillus Casei (P. aeruginosa)
Discussions
Lactobacillus Plantarum,
Lactobacillus Brevis and Lactobacillus Casei
can produce antimicrobial
substances with the capacity to inhibit the growth of S. aureus, K. pneumonia
and P. aeruginosa. Antimicrobial is a substance that kill or inhibit the growth
of microorganisms such as bacteria, fungi,
or protozoas. The agar diffusion test is used to test the susceptibility
of the S. aueres, K. pneumonia, and P. aeruginosa against the antimicrobial
substances produced by the three LAB(lactic acid bacteria). However, the
composition of the agar, its depth in dishes, the quality of the paper discs,
their contents of antibiotic, the density of inoculated bacteria, the time of
incubation and other factors should be standardized.
According to the result obtained, there are no
inhibition zone formed in the diffusion test of Lactobacillus Plantarum,
Lactobacillus Brevis and Lactobacillus Casei against S. aueres. This may due to
the improperly inoculation of the plate or other factors such as contamination,
improperly placing of the discs etc. Each discs must be pressed down with
forceps to ensure complete contact with the agar surface or irregular zone may
be affected. If the plate was properly inoculated and all other conditions were
correct, the zones of inhibition should be uniformly circular and there will be
a confluent lawn of growth.
Besides that, the inhibition zones of P. aeruginosa
is smaller than K. pneumonia according to the result. This mean that the
susceptibility of P. aeruginosa is
better than K. pneumonia. The antimicrobial substances produced by
Lactobacillus Plantarum, Lacobacillus Brevis and Lactobacillus Casei have less
effect on P. aeruginosa.
Part II Determination of
bacteriocin activity via optical density
Serial
dilution of extracellular extract
Strain
of LAB : Lactobacillus Plantarum
Dilutions
|
OD600
of spoilage/pathogenic bacteria
|
||
Strain 1:
S. aureus
|
Strain 2: P.aeruginosa
|
Strain 3: K.
pneumonia
|
|
0x
|
0
|
0
|
0
|
2x
|
0.790
|
0.836
|
0.812
|
10x
|
0.931
|
1.125
|
1.086
|
50x
|
0.652
|
0.495
|
0.463
|
100x
|
0.455
|
0.462
|
0.449
|
Equation
|
y= -0.004x+0.878
|
y= -0.005x+0.959
|
Y= -0.005x+0.925
|
OD600 of control
|
0.508
|
0.129
|
1.156
|
50% of OD600
|
0.254
|
0.0645
|
0.578
|
AU/ml
|
156
|
178.9
|
69.4
|
Discussions
Spectrophotometry
involves the use of a spectrophotometer. A spectrophotometer is a photometer
that can measure intensity as a function of the light source wavelength.
Spectrophotometers are commonly used for the measurement of transmittance or reflectance
of solutions, transparent or opaque solids, usch as polished glass, or gases.
The
optical density of a material is a logarithmic ratio of the radiation falling
upon a material, to the radiation transmitted through a material. The graphs obtained
showed that the serial dilutions are inversely proportional to the OD
600 of S. auerus, P.aeruginosa,
and K.pneumonia. The highest absorbance reading in the bacteria strain is in
the 10x serial dilution. This indicates that when the Lactobacillus Plantarum
is in 10x serial dilution, the antimicrobial effect towards the three bacteria,
S. aerus, P.aeruginosa, and K. pneumonia is the largest.
The
result obtained might not be accurate due to the improperly preparation of the
serial dilution solution. This causes the result obtained is not like what its
suppose to be.
Conclusions
There are antimicrobial effects of LAB on the three
pathogenic bacteria, namely S.auerus, K. pneumonia and P.aeruginosa. 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.
References
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