Lab 5: Determination of antimicrobial effects of microbial extracts

Introduction
Human being nowadays are more concern about the naturality of the product and try to consume natural made but not artificial made product, thus antimicrobial substance are now widely investigate and used in the daily products especially food.

Antimicrobial is any substance of natural, semisynthetic or synthetic origin that kills or inhibits the growth of microorganisms but causes little or no damage to the host. It is produced by bacteria and one of its example is bacteriocins that widely used in food industry. The bacteria that we used in this experiment is LAB1 and LAB2 to produce bnjacteriocins as its extracellular extracts. Bacteriocins are proteins or complexed proteins biologically active with antimicrobial action against pathogen. They are normally not termed antibiotics in order to avoid confusion and concern with therapeutic antibiotics, which can potentially illicit allergic reactions in humans and other medical problems. 

Objective
To determine the antimicrobial effects of extracellular extracts of selected LAB strains

Materials 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/Vis 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 the petri dishes were labelled according the spoilage organisms and strains of LAB used
2. Each plate was used for one strain of spoilage organism and one strain of LAB. The plate was divided into 2, each side for one replicate.
3. Each group was given 2 strains of LAB and 2 strains of pathogenic organisms.
4. 10 ml of trypticase soy-yeast extract agar (TSAYE) was loaded into the labelled petri dishes and ensure that the agar covers the entire surface of the plate. Wait for it to solidify.
5. 2 ml of the broth containing the spoilage organism was inoculated into 10 ml of Brain Heart Infusion (BHI) agar and vortex.
6. The mixture was loaded on top of the TSAYE agar layer, ensure that it covers the entire surface, and wait for it to solidify.
7. The broth containing LAB cultures was centrifuged. The supernatant was used as extracellular extracts.
8. A sterile filter paper disk was aseptically picked up with sterile forceps and  the disk was dipped into the extracellular extract. (Be sure that the excess extract has drained off).
9. The paper disk was placed on top of the solidified BHI agar.
10. The plates were incubated for 24-48 h at 37 °C.
11. Upon incubation, the inhibition zones were measured (in cm) and the reading was recorded in the table 1.

Part II. Determination of bacteriocin activity via optical density
1. The broth containing LAB cultures was centrifuged. The supernatant was used as extracellular extracts.
2. Each group was given 2 strains of LAB and 2 strains of pathogenic organisms.
3. 5 ml of double-strength MRS was added with 1 ml of cultures containing pathogenic bacteria and the mixture was vortexed.
4. A serial dilution of the extracellular extracts (diluted 0x, 2x, 10x, 50x, 100x) was prepared.
5. 5 ml of each extracellular extracts dilution was added into mixture as prepared in step (3).
6. The mixtures were incubated for 12-15 hr at 37 °C.
7. A control using 10 ml of double-strength MRS and 1 ml of cultures containing pathogenic bacteria was prepared and the mixtures was incubated for 12-15 hr at 37 °C as well.
8. Upon incubation, measure the optical density of the spoilage/pathogenic bacteria at 600 nm. Perform the same for the control as well.
9. 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.
10. 50% of the spoilage/pathogenic bacteria growth were determined from the OD600 of the control.

Calculations
Inhibition zone:
 Get the average of the 2 inhibition zone.

Arbitrary unit:
y:   Abs600 or OD600                
x:   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;
Thus, x = (y - c)/m
When y = Z/2, thus x = (Z/2 – c)/m

Result 
Part I  Determination of bacteriocin activity via agar diffusion test

Figure 1 shows samples of LAB 1 with Escherichia coli 

Figure 2 shows samples of LAB 1 with Salmonella

Figure 3 shows samples of LAB 2 with Escherichia coli 

Figure 4 shows the samples of LAB 2 with Salmonella

Strains of LAB	Strains of spoilage/pathogenic bacteria	Inhibition zone (cm)		Average (cm)
LAB 1	Escherichia coli	0.8	1.0	0.9
	Salmonella	1.1	0.9	1.0
LAB 2	Escherichia coli	0	0	0
	Salmonella	1.3	1.1	1.2

Table 1: The Result of Determination of Bacteriocin Activity via Agar Diffusion Test














Part II.   Determination of bacteriocin activity via optical density

Serial dilution of extracellular extract

Strain of LAB 2

Dilutions	OD600 of pathogenic bacteria
	Strain 1: E. coli	Strain 2: Salmonella
0x	0.219	0.218
2x	0.586	0.657
10x	0.927	0.764
50x	1.035	0.750
100x	0.968	0.734
Equation	y = 0.1947x + 0.1629	y = 0.1125x + 0.2871
OD600 of control	0.05	0.054
50% of OD600	0.067	0.051
AU/ml	-0.4926	-2.0987

Table 2: OD600 of pathogenic bacteria with different dilution

Graph 1: Graph of Abs600 of E. coli versus serial dilution of extracellular extract.

Graph 2: Graph of Abs 600 of Salmonella versus serial dilution of extracellular extract

Discussion :
Part I  Determination of bacteriocin activity via agar diffusion test

1. Lactic acid bacteria (LAB) is a clade of Gram-positive, low-GC, acid-tolerant, generally non-sporulating, non-respiring, either rod-shaped or coccus-shaped bacteria that share common metabolic and physiological characteristics. LAB produce bacteriocin which are proteinaceous toxins that inhibit the growth of its similar or closely related bacterial strain.
2. Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms. Salmonella species are Gram-negative, flagellated facultatively anaerobic bacilli characterized by O, H, and Vi antigens.
3. The agar diffusion test is used to measure the effect of LAB against the growth of Escherichia coli and Salmonella in the culture. The two bacteria are swabbed uniformly on the culture plate respectively. The filter paper disks are dipped with one of the LAB culture is then placed on the surface of agar. The LAB diffuse into the agar from the filter paper. If the LAB is effective against the bacteria on the agar, zone of inhibition will present. It is the clear region that can found around the filter paper on the agar. The diameter of the zone of inhibition is a measure of the effectiveness of LAB. The larger the diameter of zone inhibition, the greater the effectiveness of bacteriocin in LAB. 
4. The average diameter of zone of inhibition obtained from the Escherichia coli is (0.9+0)/2=0.45cm while the average diameter of zone of inhibition obtained from the Salmonella is (1.0+1.2)/2=1.1cm. Since Salmonella has the greater diameter of inhibition zone, the effectiveness of bacteriocin is greater.

Part 2: Determination of bacteriocin activity via optical density

1. Optical density measurement of bacterial culture is a common technique used in microbiology. Spectrophotometer is an instrument used to measure Optical density. It also can be used to measure the concentration of bacteria in a suspension. When visible light passes through a cell suspension, the light is scattered. Spectrophotometer can be measured the amount of light scattered by the cell suspension. The greater scatter indicates that the presence of more bacteria or other material. 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 that each compound absorbs or transmits light over a certain range of wavelength. The amount of a known chemical substance can be measure by measuring the intensity of light detected. 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. According 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. Arbitrary unit (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 and Salmonella 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. 

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 resistance to heat, acidity, low water activity and oscillations of microorganisms. In addition, these molecules present characteristics of effective natural inhibitors of pathogenic and food spoilage bacteria in temperature. Lactic acid bacteria produced inhibitory substances are safe and various food.