Introduction
An antimicrobial is an agent that kills microorganisms or inhibits the growth of the microorganisms. Use of substances with antimicrobial properties is known to have been common practice for at least 2000 years. Ancient Egyptians and ancient Greeks used specific molds and plant extracts to treat infection. Antimicrobial substances can be produced by certain group or species of bacteria with the capacity to inhibit the growth of pathogenic and spoilage microorganisms. These microbial compounds include organic acids, hydrogen peroxide, diacetyl and bacteriocins.
Bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strains. They are typically considered to be narrow spectrum antibiotics, though this has been debated nowadays. They are phenomenologically analogous to yeast and paramecium killing factors, and are structurally, functionally, and ecologically diverse. Bacteriocins were first discovered by A. Gratia in 1925. He was involved in the process of searching for ways to kill bacteria, which also resulted in the development of antibiotics and the discovery of bacteriophage, all within a span of a few years. He called his first discovery a colicine because it killed E. coli.
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. For example the bacteriocins produced by Staphylococcus warneri are called as warnerin or warnericin. In fact, one of the oldest known so-called colicins was called colicin V and is now known as microcin V. It is much smaller and produced and secreted in a different manner than the classic colicins.
Alternative methods of classification include: method of killing (pore-forming, DNase, nuclease, murein production inhibition, etc.), genetics (large plasmids, small plasmids, chromosomal), molecular weight and chemistry (large protein, polypeptide, with/without sugar moiety, containing atypical amino acids like lanthionine) and method of production (ribosomal, post-ribosomal modifications, non-ribosomal).One method of classification fits the bacteriocins into Class I, Class IIa/b/c, and Class III.
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.
Objective
To determine the antimicrobial effects of extracellular extracts of selected LAB strains.
Material and reagents
MRS broth
Sterile filter paper disk (50mm x 50mm)
Forceps
Sterile universal bottles
Cultures of LAB and spoilage / pathogenic organisms
Bench-top refrigerated centrifuge
Incubator 30˚C and 37˚C
UV/V is spectrophotometer
Distilled deionized water
Trypticase soy agar
Brain heart infusion agar
Yeast extract
Procedure Part I: Determination of Bacteriocin Activity via Agar Diffusion Test
1. All petri dishes have been labelled according to the spoilage organisms and the strains of LAB used for the experiment.
2. Each plate was used for only one strain of spoilage organism and one strain of LAB. The plate was divided into 4 parts, 2 parts for one member of the group and each part for one replicate as shown in figure 1.1.
3. Each group had only one strain of LAB and one strain of spoilage/pathogenic organism for this experiment.
4. 10ml of trypticase soy-yeast extract agar (TSAYE) was loaded into the labelled petri dishes and the agar was made sure that it covered the entire surface of the plate. The petri dishes were then left aside until the agar solidify.
5. 2ml of the broth containing the spoilage organism, E. coli was inoculated into 10ml of brain heart infusion agar (BHI) and vortexed.
6. The mixture was loaded on top of the layer of solidified TSAYE agar, making sure that the mixture covered the entire surface and then left aside for solidification.
7. The broth containing LAB cultures was centrifuged. The supernatant was used as the
extracellular extracts.
8. A sterile filter paper disk was picked up aseptically with a pair of sterile forceps and the disk of filter paper was dipped into the extracellular extract. The excess extract has been drained off before proceeding.
9. The paper disk was placed on top of the solidified BHI agar which contained spoilage
organism.
10. All the plate prepared were incubated at 37 °C for 24 to 48 hours.
11. The inhibition zones were measured (in cm) and the result was recorded.
Procedure part 2
1. The broth containing LAB culture (Lactobacillus plantarum) is centrifuged and the supernatant is used as extracellular extract.
2. A serial dilution of extracellular extracts (0x, 2x, 10x, 50x and 100x) are prepared with total of 5 ml for each serial dilution. A control medium is also prepared. The extract are diluted with double-strength MRS broth with volume as shown,
Dilution
Mixture
|
0x
|
2x
|
10x
|
50x
|
100x
|
Control
|
Extracellular Extract (ml)
|
5.0
|
2.5
|
0.5
|
0.1
|
0.05
|
0
|
MRS Broth (ml)
|
0
|
2.5
|
4.5
|
4.9
|
4.95
|
5.0
|
Total (ml)
|
5.0
|
5.0
|
5.0
|
5.0
|
5.0
|
5.0
|
3. Each dilution is added with another 5 ml of double strength MRS broth and 1 ml of culture
containing spoilage pathogen (E. coli).
4. Each mixture are vortex to ensure uniform distribution of the mixture.
5. Each mixture are incubated at 37°C for 24 hours.
6. A negative-control for “auto-zero” are prepared via the spectrophotometer by adding 200 μl of double strength MRS broth to the first chamber in the first column of a 8 X 12 chamber well.
7. 200 μl of mixture are transferred to the first column of the well ( control at the second row, 0x at third row, 2x at fourth row, 10x at fifth row, 50x at sixth row and 100x at seventh row)
8. 100 μl of distilled water are placed in all remaining chamber.
9. The mixture are diluted by transferring 100 μl of the content in the first column to the chamber at the second column of the same row. The content are mix thoroughly before the process is repeated for other chamber. The remaining 100 μl at the last column are discarded.
10. The optical density of the E. coli in the mixture are measured with the spectrophotometer.
11. The data are recorded and the column with reading between 0.1 and 0.4 are selected.
12. The reading are subtracted with the reading of “auto-zero” before any subsequent calculation.
13. A graph is plotted and the value of IC50 is determined from the graph.
Results:
Part 1
The result is taken using the method as shown as below:
Figure 1.2: Method of Measuring the Inhibition Zone
Table 1.1: The Result of Determination of Bacteriocin Activity via Agar Diffusion Test
*** The conclusion is that the inhibition zone is 0.86cm in diameter. ***
RESULT Part 2
Column Selected: 1
Strain of LAB: Lactobacillus plantarum
Strain of spoilage bacteria: E. coli
“Blank” Reading: 0.097
Dilutions
|
Optical Density
|
0x
|
0.358
|
2x
|
0.179
|
10x
|
0.546
|
50x
|
0.143
|
100x
|
0.167
|
Equation
|
Y= -0.0021X + 0.3467
|
Optical Density of Control
|
0.567
|
50% of Optical Density of Control (IC50)
|
0.284
|
Arbitrary Unit (AU) of IC50
|
29.86
|
Correlation Index, R2 = 0.2742
Discussion:
Part I : Determination of bacteriocin activity via agar diffusion test
1. Bacteriocin are proteinaceous toxins produced by bacteria to inhibit the growth of microorganisms such as fungi, protozoans and similar or closely related bacterial strains by producing intraspecies antagonistic effects.
2. The production of bacteriocins, organic acids, free fatty acids, ammonia, diacetyl and hydrogen peroxide by lactic acid bacteria (LAB) promotes bacterial interference. These metabolites have been applied for many years to extend the shelf life of foods in food industry by enabling acidification that will inhibit the growth of spoilage agents. Besides that, proteinaceous bacteriocins produced by LAB supress the growth of pathogenic microorganisms.
3. Area around a paper disk or colony of lactic acid bacteria (LAB) where no other organisms are growing. The bacteriocin produced is then diffuses into the agar away from the disk. The Lactobacillus Fermentum is sensitive to the bacteriocin, and they will not grow near the disk. 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 bacteriocin. The size of the zone is proportional to how sensitive the pathogen is. If the pathogen is resistant to the bacteriocin, it will grow right up to the disk.
Part 2: Determination of bacteriocin activity via optical density
1.) Optical density measurement of bacterial culture is a common technique used in microbiology. Optical density is measured in a spectrophotometer. It can be used to measure of the concentration of bacteria in a suspension. When visible light passes through a cell suspension, the light is scattered. The greater scatter indicates that the presence of more bacteria or other material. The amount of light scattered can be measured in a spectrophotometer. Typically, the optical density at a particular wavelength that correlates with the different phases of bacterial growth can be determined when working with a particular type of cell. Generally, the cells that are in their mid-log phase of growth are being used. Typically the OD600 is measured.
2.) Spectrophotometry is a method to measure how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through sample solution. The basic principle is each compound absorbs or transmits light over a certain range of wavelength. A spectrophotometer is an instrument that measures the amount of photons or the intensity of light absorbed after it passes through sample solution. In general, a spectrophotometer consists of two devices, that are a spectrometer and a photometer. A spectrometer is a device that disperses and measures light while a photometer indicates the photoelectric detector that measures the intensity of light. However, the spectrophotometer can also be designed to measure the diffusivity on any of the listed light ranges that usually cover around 420 nm to 660 nm using different controls and calibrations. Within these ranges of light, calibrations are needed on the machine using standards that vary in type depending on the wavelength of the photometric determination. According to Beer-Lambert Law, amount of light absorbed by a medium is proportional to the concentration of the absorbing material or solute present. In this case, the concentration of a coloured solute in a solution could be determined in the lab by measuring the absorbency of light at a given wavelength.
3.) One arbitrary (AU) is known as the dilution factor of the extracellular extract that inhibited 50% of the spoilage or pathogenic bacteria growth and expressed as AU/mL.
Abs600 = Z. Thus, 50% of Z = Z/2
y = mx + c ; Thus, x = (y-c)/m
When y = Z/2, Thus x = (Z/2 - c)/m
4.) From the result obtained from the experiment, Escherichia coli shows a negative gradient graph. This shows that there has a negative inhibition from the bacteriocin produced by lactic acid bacteria (LAB) on the growth of the bacteria strains.
5.) From the data shown, there is an increasing on the growth of bacteria from 0x to 100x dilution. This is because of the presence of the promoting factor, that are some organic acids produced from the extracellular extracts and the peptones that are being added in different volumes.
6.) This shows the result that bacteriocin produced by Lactobacillus Plantarum does not have much inhibition effect on Escherichia coli which belongs to the class Gammaproteobacteria.
Conclusion :
In conclusion, Bacteriocin from Lactobacillus best works on E.coli.
