LAB 5: DETERMINATION OF ANTIMICROBIAL EFFECTS OF MICROBIAL EXTRACTS
Introduction
Antimicrobial means that the organisms that can kills or inhibits the growth of pathogenic and spoilage microorganisms. The antimicrobial can be produced by certain groups of bacteria. The main classes of antimicrobial agents are disinfectants ("nonselective antimicrobials" such as bleach), which kill a wide range of microbes on non-living surfaces to prevent the spread of illness, antiseptics (which are applied to living tissue and help reduce infection during surgery), and antibiotics (which destroy microorganisms within the body). Antimicrobial compounds include organic acid, hydrogen peroxide, diacetyl and bacteriocins. Owing to fulfil the desire of the major customer who are prefer more nature and less processed food, the requirement of the bacteriocins is become more higher.
Bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain(s). They are phenomenologically analogous to yeast and paramecium killing factors, and are structurally, functionally, and ecologically diverse. Many lactic acid bacteria (LAB) produce a high diversity of different bacteriocins. Though these bacteriocins are produced by LAB found in numerous fermented and non-fermented foods, nisin is currently the only bacteriocins widely used as a food preservative. 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.
Materials and reagents
MRS broth
Forceps
Sterile filter paper disk(50mm x 50mm)
Sterile universal bottles
Cultures of LAB and spoilage/pathogenic organisms
Bench-top refrigerated centrifuge
Incubator 30 and 37
UV/V is spectrophotometer
Distilled deionized water
Trypticase soy agar
Brain heart infusion agar
Yeast extract
Procedures
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.
Part 2: Determination of Bacteria Activity via Optical Density
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
Table 1.1
Average Diameter of Inhibition Zone (cm)
| ||
Group Member
|
LAB 1
|
LAB 2
|
Kenneth
|
0.76
|
0.89
|
Jia Yin
|
0.85
|
0.93
|
Fazliyana
|
0.87
|
0.86
|
Average:
|
0.83
|
0.89
|
We can concluded that the inhibition zone for LAB 1 is 0.83cm and LAB 2 is 0.89cm in diameter.
Part 2
Serial dilution of extracellular extract
Dilutions
|
Optical Density
|
0x
|
0.958
|
2x
|
0.674
|
10x
|
0.627
|
50x
|
0.596
|
100x
|
0.397
|
Equation
|
y=-0.005875x+0.68575
|
Optical Density of Control
|
0.266
|
50% of Optical Density of Control (IC50)
|
0.133
|
DISCUSSION
In lab 5, we have two parts that we need to discuss. Firstly for part 1, we want to determine bacteria activity via agar diffusion test. 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. We called it as the zone of inhibition. The concentration of the compound will be highest next to the disk, and will decrease as distance from the disk increases. The larger the clear area around the filter disk, the more effective the compound. Thus, the size of the zone of inhibition is a measure of the compound's effectiveness.
Now the studies reveal the scope for bacteriocins, not only as preservatives but also as an antibiotic for many diseases and infections. Bacteriocin activity was determined in an agar well diffusion assay with aim to test the ability of the polyclonal antiserum to neutralize bacteriocin activity, serial dilutions of bacteriocin were mixed with an equal volume of undiluted antiserum in each well prior to adding the overlay. Preimmune serum and sterile deionized water were mixed with bacteriocin in the control wells. All tests were run in duplicate.
This is an area around a paper disk or colony of bacteria (LAB) or mold where no other organisms are growing. If you are testing antibiotic sensitivity for example, you can impregnate paper disks with antibiotic and then put them on an agar plate of growing bacteria. The antibiotic then diffuses into the agar away from the disk. If the bacteria are sensitive to the antibiotic, they will not grow near the disk. The size of the zone is proportional to how sensitive the organism is. If the organism is resistant to the antibiotic, it will grow right up to the disk.
For part 2, we will determine the bacteria activity via optical density. We need to use spectrophotometer to prepare a negative-control for ‘auto-zero’. A spectrophotometer is an instrument used to measure properties of light over a specific portion of the electromagnetic spectrum, typically used in spectroscopic analysis to identify materials. The variable measured is most often the light's intensity but could also be the polarization state. The independent variable is usually the wavelength of the light or a unit directly proportional to the photon energy, such as wave number or electron volts, which has a reciprocal relationship to wavelength. A spectrometer is used in spectroscopy for producing spectral lines and measuring their wavelengths and intensities. Spectrometer is a term that is applied to instruments that operate over a very wide range of wavelengths, from gamma rays and X-rays into the far infrared. If the instrument is designed to measure the spectrum in absolute units rather than relative units, then it is typically called a spectrophotometer. The majority of spectrophotometer are used in spectral regions near the visible spectrum. In general, any particular instrument will operate over a small portion of this total range because of the different techniques used to measure different portions of the spectrum. Below optical frequencies (that is, at microwave and radio frequencies), the spectrum analyzer is a closely related electronic device.
An example of an experiment in which spectrophotometry is used is the determination of the equilibrium constant of a solution. A certain chemical reaction within a solution may occur in a forward and reverse direction where reactants form products and products break down into reactants. At some point, this chemical reaction will reach a point of balance called an equilibrium point. In order to determine the respective concentrations of reactants and products at this point, the light transmittance of the solution can be tested using spectrophotometry. The amount of light that passes through the solution is indicative of the concentration of certain chemicals that do not allow light to pass through. The use of spectrophotometers spans various scientific fields, such as physics, materials science, chemistry, biochemistry, and molecular biology. They are widely used in many industries including semiconductors, laser and optical manufacturing, printing and forensic examination, as well in laboratories for the study of chemical substances.
CONCLUSION
In conclusion, the results show that lactic acid bacteria (LAB) may act as a bio preservatives. Antimicrobial compounds produced by LAB have provided these organisms with a competitive advantage over other microorganisms. From the experiment, the LAB successfully shows its bio preservatives properties on both gram-positive and gram-negative bacteria which are Escherichia coli and Staphylococcus aureus. Bacteriocins are agents that could act on the microbial cell through different ways when compared to conventional chemical food preservatives, provoking the formation of an inhospitable environment to microbial survival. In addition, these molecules present characteristics of resistance to heat, acidity, low water activity and oscillations of temperature. However, there is the necessity to develop studies involving the establishment of the some bacteriocins characteristics such as antimicrobial spectrum, isolation, toxicity and stability use as control agents to the growth and microbial survival in food. Lactic acid bacteria and their products are more effective and flexible in several applications. Most inhibitory substances produced by lactic acid bacteria are safe and effective natural inhibitors of pathogenic and food spoilage bacteria in various food.
REFERENCES
https://en.wikipedia.org/wiki/Antimicrobial
http://www.ncbi.nlm.nih.gov/pubmed/11764886
https://en.wikipedia.org/wiki/Bacteriocin
http://labreport102.blogspot.my/
