→ Introduction
Certain group of bacteria can produce antimicbial substances with the capacity to inhibit the growth of pathogenic and spoilage micro-organisms. 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 synthesised 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 receive 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 monocytogenes, Staphylococcus aureus, Enterococcus faecalis and Clostridium tyrobutyricum.
Part 1- Determination of bacteriocin activity via agar diffusion test.
Kirby-Bauer disk-diffusion method is the method of measuring the effect of an animicrobial agent against bacteria grown in culture. 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.
Part 2- Determination of bacteriocin activity via optical density.
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 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 the OD600 is used in the experiment.
Kirby-Bauer disk-diffusion method is the method of measuring the effect of an animicrobial agent against bacteria grown in culture. 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.
Part 2- Determination of bacteriocin activity via optical density.
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 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 the OD600 is used in the experiment.
→ Objective
To determine the antimicrobial effect of extracellular extracts of selected LAB strains.
→ Results
Part 1- Determination of bacteriocin activity via agar diffusion test
Calculations
Inhibition zone :
Serial dilutions of extracellular extract
Y axis: Abs600 or OD600 X axis: Serial dilutions of extracellular extract
m and c: Constants
One arbitrary unit (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
Control : Abs600 = Z. Thus, 50% of Z = Z/2
Y = mx + c. Therefore, x = (Y – c)/m
When Y = Z/2, thus, x= (Z /2-c)
Strains of LAB
|
Strains of spoilage/pathogenic bacteria
|
Inhibition zone (cm)
|
Lp
|
Kp
|
0.7
|
Sa
|
-
| |
Pa
|
0.6
| |
Lb
|
Kp
|
0.7
|
Sa
|
-
| |
Pa
|
0.8
| |
Lc
|
Kp
|
0.6
|
Sa
|
-
| |
Pa
|
0.7
|
Strain of LAB
|
Strains of spoilage/pathogenic bacteria
|
Inhibition zone (cm)
|
LAB species
|
Sa
|
-
|
Kp
|
1.0
| |
Pa
|
1.05
|
Part 2- Determination of bacteriocin activity via optical density
Serial dilution of extracellular extract
Dilutions
|
OD600 of spoilage/pathogenic bacteria
| ||
Strain 1:Sa
|
Strain 2:Kp
|
Strain 3:Pa
| |
0x
|
-
|
-
|
-
|
2x
|
0.735
|
0.637
|
0.710
|
10x
|
1.032
|
0.691
|
0.076
|
50x
|
0.621
|
0.641
|
0.804
|
100x
|
0.502
|
0.765
|
0.528
|
Equation
|
y=-0.111x + 1.0
|
y=0.033x + 0.6
|
y=0.018x + 0.484
|
OD600 of control
|
0.270
|
0.829
|
0.432
|
50% of OD600
|
0.135
|
0.415
|
0.216
|
AU/mL
|
7.793
|
5.606
|
14.889
|
Sa
Dilutions
|
Abstract
|
0x
|
0.000
|
2
|
0.735
|
10x
|
1.032
|
50x
|
0.621
|
100x
|
0.502
|
Kp
Dilution
|
Abstract
|
0x
|
0
|
10x
|
0.637
|
20x
|
0.691
|
50x
|
0.641
|
100x
|
0.765
|
Pa
Dilutions
|
Abstract
|
0x
|
0
|
10x
|
0.710
|
20x
|
0.076
|
50x
|
0.804
|
100x
|
0.528
|
→ Discussion
Part 1
Bacteriocins are antimicrobial peptides or proteins produced by
lactic acid bacteria (LAB).Diameters
of inhibition zones produced in plates by the various test-microorganisms
exposed to equal nisin concentrations showed wide variation. 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 carbohydratefermentation.
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,
andStreptococcus as
well as the more peripheral Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus, Vagococcus, and Weisella; these belong
to the order Lactobacillales. The extract of LAB give zone
of inhibition into the indicator pathogen strains tested. Nutrient that mixed
with phatogenic bacteria and Lab extract is place on it, Then it is incubate
for 24 to 48 hours at 37 degree celcius. The diameter of inhibition theen is
measured in cm. The size of inhibition indicate the difference of bacteriocin
activity between LAB.
Part 2
During this experiment, we use spectrophotometer to measure the concentration of bacteria in suspension. Light entering a cloudy
solution will be absorbed. A clear
solution will allow almost all of the light through. The amount of absorbance can be determined by
using a spectrophotometer, which measures what fraction of the light passes
through a given solution and indicates on the absorbance display the amount of
light absorbed compared to that absorbed by a clear solution. Inside, a light shines through a filter (which
can be adjusted by controlling the wavelength of light), then through the
sample and onto a light-sensitive phototube.This produces an electrical
current.The absorbance meter measures how much light has been blocked by the sample
and thereby prevented from striking the phototube. A clear tube of water or other clear solution
is the BLANK and has zero absorbance.
The amount of substance in the solution is directly proportional to the
absorbance reading. A graph of
absorbance vs. concentration will produce a straight line.Optical density extracellular extract of LAB contained MRS medium and pathogenic microorganism was detemined with spectrophotometer 600 nm. One type of LAB used in thie experiment is Lactobacillus plantirum. Three types of pathogenic microorganisms have been used are S.aureus, P.aeruginosa, and K.pneumonia.Negative-control is prepared for auto-zero via spectrophotometer while positive-control which show higher optical density than the other sample show atimicrobial effect.
→ Conclusion
In the conclusion, Lactic acid bacteria (LAB) in fermented foods produce a large
variety of compounds which give these products their characteristic flavor and
color apart from improving its safety and quality. Bacteriocins are categorized
in several ways, including producing strain, common resistance mechanisms, and
mechanism of killing. There are several large categories of bacteriocin which
are only phenomenologically related. These include the bacteriocins from
gram-positive bacteria, the colicins, the microcins,
and the bacteriocins from Archaea. The bacteriocins from E. coli are
called colicins (formerly
called 'colicines,' meaning 'coli killers'). They are the longest studied
bacteriocins. They are a diverse group of bacteriocins and do not include all
the bacteriocins produced by E. coli.
→ Reference
