Matric's card no: 111431
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.
Lactobacillus casei, Lactobacillus brevis and Lactobacillus plantarum are the
examples of lactic acid bacteria.
L casei, is a lactic acid producing
bacteria that occurs naturally in the human gut and mouth. It is a
transient bacterium that usually lives for about ten days at a time in the
intestines but it is resistant to gastric acid, so unlike some beneficial bacteria,
it can survive quite happily in the intestinal tract. Lactobacillus casei is considered a probiotic safe for
consumption. Lactobacillus casei is typically the dominant species of
nonstarter lactic acid bacteria (NSLAB) present in ripening Cheddar cheese, and,
recently, the complete genome sequence of L.
casei ATCC 334 has
become available. L. casei is also the dominant species in
naturally fermented Sicilian green
olives. Whereas, Lactobacillus
brevis can be found in
many different environments and in fermented foods such as sauerkraut and pickles. It is also
one of the most common causes of beer spoilage. Ingestion has been shown to improve human immune function, and it has been patented several times. Lactobacillus plantarum is
a widespread member of the genus Lactobacillus, commonly found in many fermented food products
as well as anaerobic plant matter. It is also present in saliva (from which it
was first isolated). It has the ability to liquefy gelatin. L.plantarum has one of the largest genomes known among the lactic acid bacteria and is a very
flexible and versatile species. The ability of L. plantarum to produce antimicrobial substances
helps them survive in the gastro-intestinal tract of humans. The antimicrobial
substances produced have shown significant effect on Gram-positive and
Gram-negative bacteria.
There are two methods have been introduced to determine the bacteriocin
activity, namely diffusion test and optical density method. 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. Whereas, 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, we 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.
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.
The sample result that shows that there is no inhibition zone.
Strains of LAB
|
Strains of spoilage / pathogenic bacteria
|
Inhibition zone (cm)
|
L. casei
|
S. aureus
|
(0.60 + 0.60)/2 = 0.60
|
K. pneumonia
|
(1.00 + 1.30)/2 = 1.15
|
|
P. aeruginosa
|
(1.30 + 0.90)/2 = 1.10
|
|
L. brevis
|
S. aureus
|
No inhibition zone.
|
K. pneumonia
|
(0.70 + 0.80)/2 = 0.75
|
|
P. aeruginosa
|
No inhibition zone.
|
|
L. plantarum
|
S. aureus
|
No inhibition zone.
|
K. pneumonia
|
(0.90 + 0.90)/2 = 0.90
|
|
P. aeruginosa
|
No inhibition zone.
|
Discussions:
Part 1: Determination of the bacteriocin activity via agar
diffusion method.
1.)
From
the results of this experiment, we found that, among the three strains of LAB
used, the bacteriocin from L. casei
was the most effective against towards the three pathogenic bacteria, namely S.
aureus, K.pneumonia and P.aeruginosa.
Whereas, the bacteriocin from L.brevis
and L. plantarum were effective
against K. pneumonia only. This is because the extracts of the lactic acid
bacteria gave zones of inhibition onto the indicator pathogen strains tested.
In the agar diffusion assay, a linear relationship existed between area of the
zone of inhibition.
2.) Generally bacteriocin
production is strongly dependent on pH, nutrient sources and incubation
temperature, and activity levels do not always correlate with cell mass or
growth rate of the producer strain. Higher bacteriocin levels are often
obtained at temperatures, nutrient sources and pH values lower than required
for optimal growth. Maximal bacteriocin production may be obtained by
supplementing the medium with growth limiting factors, such as sugars, nitrogen
sources, vitamins and potassium, phosphate, or by regulating the medium pH. The
culture medium pH and temperature might be the reasons of L. pneumonia and L. brevis failed to display their
antimicrobial activity in this experiment.
3.)
L. caseisecretes
a substance called “peptidoglycan”, which supports the natural defences of the
body and stimulates immune responses in the intestinal tract. L. casei has
demonstrated effectiveness in increasing circulating IgA (immunoglobulin A) in
infants infected with rotavirus and has shortened the duration of associated
diarrheal episodes. In a 2003 issue of the Journal of Nutrition
Health and Aging, fermented milk containing the probiotic L. casei had
a positive effect on lessening winter infections in the elderly. It is a highly
prolific organism and has strong resistance to digestive enzymes.
4.) L. plantarum has been studied
for the treatment of recurrent Clostridium difficile-associated diarrhea
and for Candida yeast infections. A particular strain called “299v”, derived
from sour dough and used to ferment sauerkraut and salami, has demonstrated
that it can improve the recovery of patients with enteric bacterial infections.
The adherence of this bacterium reinforces the barrier function of the
intestinal mucosa, thus preventing the attachment of the pathogenic bacteria.
Eradicating such pathogens as Staphylococcus
aureus from fermented food is one of the functions of L. plantarum too. Hence, the experiment
conducted shown that strains of L. plantarum
against the S. aureus might be due to
the pH factor.
5.)
L. brevis is a lactic acid-producing organism important in the
synthesis of vitamins D and K. Research studies have shown that L. brevis decreases intestinal permeability
(leaky gut syndrome), improves intestinal micro flora, and has a positive
effect on the intestinal immune system. A recent study showed that this
friendly bacterium also has a positive effect in eliminating the ulcer-causing
bacteria Heliobacter pylori.
Results:
Part 2:
Determination of bacteriocin activity via optical density
(Serial
dilution of extracellular extract)
Strain of
LAB: L. plantarum
Dilutions
|
OD600 of spoilage /
pathogenic bacteria
|
||
Strain 1: S. aureus
|
Strain 2: P. aeruginosa
|
Strain 3: K. pneumonia
|
|
0x
|
-
|
-
|
-
|
2x
|
0.699
|
0.854
|
0.828
|
10 x
|
0.698
|
0.990
|
0.943
|
50 x
|
0.590
|
0.780
|
0.625
|
100 x
|
0.168
|
0.553
|
0.512
|
Equation
|
Y = -0.0054x+0.7572
|
Y = -0.0038x+0.9468
|
Y = -0.004x+0.8894
|
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
|
1) S.aureus
2) P.aeruginosa
3) K.pneumonia
Discussions:
1.)
The optical density (OD) or called as scattering intensity is
proportional to the cell density. Thus, we can determine a proportionality
factor and get an equation. This factor allows us to calculate the cell density
from any measured OD. A spectrophotometer can be set at a wavelength of 420 –
660 nm. In this experiment, the OD600 is measured. Typically, the
wavelength must be standardized and may need to be adjusted specifically to the
material being tested. Different vegetative cells and bacterial spores may not
have the same maximal absorbance wavelength. For instance, the wavelength in
this experiment was set as 600nm.
2.)
The
positive control which showed the growth of bacteria without extracellular
extract of lactic acid bacteria has been set up for each pathogenic bacteria.
The OD600 of
the positive control was then measured in order for us to investigate whether
there is inhibition of pathogenic bacteria activity by comparing the OD600
of the samples. If the OD600 of the sample is less than OD600 of the
positive control, there will be inhibition of spoilage bacteria by the strains
of lactic acid bacteria, like L. plantarum
used in this experiment.
3.)
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:
In conclusion, the results from the agar diffusion method corresponded
with the results from the optical density method. Both of the results showed
that the strain of L. plantarum against the K. pneumonia, a
pathogenic bacteria effectively. In addition, the results clearly suggest the potential
usefulness of the bacteriocins produced by three lactic acid bacteria (LAB) as
bio preservatives against K. pneumonia effectively. Generally,
the L. casei might be the best bio
preservative since it showed against the three strains of pathogenic bacteria
in part one of the experiment.
References:
