Lab 5:  Determination of Antimicrobial Effects of Microbial Exracts

INTRODUCTION

There are a few groups of bacteria that can produce antimicrobial substances. An antimicrobial is an agent that kills microorganism or inhibits their growth. Antimicrobial medicines can be grouped according to the microorganisms they act primarily against. For example, antibacterials are used against bacteria and antifungals are used against fungi. They can also be classified according to their function. Organic acids, hydrogen peroxide, diacetyl and bacteriocins are included among these antimicrobial compounds. Since nowadays consumers demand “natural” and minimally processed” food therefore there is a rising of interest on naturally produced antimicrobial agents like bacteriocins.

      Bacteriocins are proteins or complexed proteins biologically active with antimicrobial action against other bacteria, principally closely related species. They are produced by bacteria and 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

      Bacteriocin production could be considered as an advantage for food and feed producers since, in sufficient amounts, these peptides can kill or inhibit pathogenic bacteria that compete for the same ecological niche or nutrient pool. This role is supported by the fact that many bacteriocins have a narrow host range, and is likely to be most effective against related bacteria with nutritive demands for the same scarce resources

      Lactic acid bacteria (LAB) are characterized as Gram-positive cocci or rods, non-aerobic but aerotolerant, able to ferment carbohydrates for energy and lactic acid production. LAB bacteriocins can work via different mechanisms to exert an antimicrobial effect, but the cell envelope is generally the target. The initial electrostatic attraction between the target cell membrane and the bacteriocin peptide is thought to be the driving force for subsequent events. 

Materials and Reagents

MRS broth

Sterile filter paper disk

Forceps

Sterile universal bottles

Cultures of LAB and spoilage/pathogenic organisms

Bench-top refrigerated centrifuge

Incubator 30oc and 37oc

UV/V is spectrophotometer

Distilled deionized water

Trypticase soy agar

Brain heart infusion agar

Yeast extract

PROCEDURE

Part 1: Determination Of Bacteriocin Activity Via Agar Diffusion Test

1. All the petri dishes are labelled according to the spoilage organisms and strains on LAB used.

2. Each plate was only used for one strain of spoilage organism and one strain of LAB. Plate was divided into 2 parts, each for on replicate.

3. Each group has 1 strain of LAB and 1 strain of spoilage/pathogenic organism.

4.10ml of trypticase soy-yeast extract agar (TSAYE) was loaded into the labeled petri ish and the agar was ensured to fully cover the entire surface of the plate. It is waited until it solidifies.

5. 2ml of broth containing the spoilage organism was innoculated into 10ml of brain hear infusion (BHI) agar and vortex.

6. The mixture was loaded on top of the TSAYE agar layer and ensurd that it covered the entire surface and waited to solidify.

7. The broth containing the lab cultures was centrifuge. The supernatent was used as a extracellular extracts.

8. A sterile filter paper disk is picked up aseptically with a sterile forcep and a disk is dipped into the extracellular extract.

9. The paper disk was placed on top of the solidifies BHI agar.

10. The plates were inoculated for 24-28 hours at 37oC.

11. Upon incubation, the inhibition zones were measured (in cm) and is recorded.

Part 2: Determination Of Bacteriocin Activity Via Optical Density

1. The broth was containing LAB cultures were centrifuge. The supernatent is used as extracellular extracts.

2. Each group has 1 strain of LAB and 1 strain of spoilage/pathogenic organism.

3. 5 ml of double-strength MRS was added with 1 ml of cultures containing spoilage/ pathogenic bacteria and the mixture was vortex.

4. A serial dilution of the extracellular extracts (diluted 0x, 2x, 10x, 50x, 100x) were 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 hours at 37ºC.

7. A control using 5 ml of double-strength MRS, 1 ml of cultures containing spoilage/ pathogenic bacteria, and 5 ml of sterile peptone was prepared. The mixtures were incubated for 12-15 hours at 37ºC.

8. A negative control for ‘auto zero’ via the spectrophotometer was prepared. 5 ml of double-strength MRS was added with 2 ml of distilled water. (Need not to be incubated)

9. Upon incubation, the optical density of the spoilage/ pathogenic bacteria at 600 nm was measured. The same was performed for the control as well.

10. 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.

11. 50% of the spoilage/ pathogenic bacteria growth were determined from the OD600 of the control.

RESULTS

Part 1: Determination Of Bacteriocin Activity Via Agar Diffusion Test

Presence of inhibition zone

Absence of inhibition zone

Strain of lab	Strain of spoilage/pathogenic bacteria	Name	Inhibition  Zone (cm)	Average
Lactobacillus fermentum	S.Aureus	sample 1	1 and 1.1	1.05
Lactobacillus fermentum	S.Aureus	sample 2	1 and 0.9	0.9
Lactobacillus fermentum	S.Aureus	sample 3	0.6 and 0.7	0.65
Lactobacillus fermentum	S.Aureus	sample 4	0.7 and 0.7	0.7
Lactobacillus fermentum	S.Aureus	sample 5	No inhibition zone
Lactobacillus fermentum	S.Aureus	sample 6	No inhibition zone

Part 2: Determination of Bacteriocin Activity Via Optical Density

Dilution	OD600 of Spoilage/ Pathogenic Bacteria
0x	0.269
2x	0.397
10x	0.448
50x	0.174
100x	0.123
Equation	y= -0.0026x + 0.3678
OD600 of Contol	0.107
50% of OD600	0.0535
AU/ml	100/11= 9.091

 

DISCUSSION

Part 1: Determination Of Bacteriocin Activity Via Agar Diffusion Test

1. Bacteriocins are bactericidal, antibiotic-like substances, apparently protein in nature, which are produced by many bacteria and have a killing action on strains of the same or closely related species.

2.The larger the inhibition zone(no bacteria growing area) on the agar medium,means that the bacteriocin is effective on the pathogenic bacteria and vise versa.

3. Type of the LAB of bacteriocin  that being used is Lactobacillus fermentum and the bacteria that we used is S.Aureus.

4.The bacteriocin will cause the destruction of the membrane potential by forming the pores on the pathogenic bacteria. It will cause the destruction of the membrane potential by forming the pores on the pathogenic bacteria.It will inhibits the nucleolytic activity of the pathogenic bacteria strains which breaks down the DNA chromosomes as well as RNA.Then the bacteriocin will inhibits the protein synthesis of the pathogenic bacterias but does not kills them.

5.As for the none inhibition zone exist result are because not enough Lactobacillus fermentum are being aplied  around the pathogenic bacteria.This is because without adequate numbers of Lactobacillus fermentum, the point of critical mass which is needed cannot occur and the bacteria will be unable to have the desired impact on the symptoms being treated.

Part 2: Determination Of Bacteriocin Activity Via Optical Density

1.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, you 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. Typically the OD600 is measured.

2. One arbitrary (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

3. As the serial of dilution increse, the optical density will increase to indicate that the Lab which is Lactobacillus Plantarum has strong microbial effec on the pathogenic bacteria which is E.coli.

4. The control value of the experiment is 0.209 and the value of the absorbant is 0.102.

5. From the result,the graph shows that as the serial dilution increases, the optical density decreases. This shows that there is negative inhibition of the pathogenic bacteria. This might be caused by the using of distilled water during the process of serial dilution. As we know, the distilled water is colourless. When the Lactobacillus Plantarum is diluted with distilled water, the optical colour density will become very much lower compared to the normal colour density of Lactobacillus Plantarum culture. So, the results obtained is wrong. 

6. Therefore, the peptone is suggested to replace with the distilled water in the serial dilution of   Lactobacillus Plantarum culture as the colour of peptone is quite similar with the culture. 

CONCLUSION

LAB is a useful bacteria used to produce bacteriocin that can inhibit the growth of bacteria.The use of strains that produce multiple bacteriocins could be advantageous to limit the potential emergence of bacteriocin-resistant populations An important aspect to take into consideration in relation to the commercial use of bacteriocins is the tolerance or resistance of certain pathogenic bacterial species that are normally sensitive since it may compromise the antibacterial efficiency of these compounds

REFERENCES

http://www.academia.edu/3745035/Bacteriocins_Nature_Function_and_Structure

http://www.scielo.br/scielo.php?pid=s1516-89132007000300018&script=sci_arttext

http://nootriment.com/lactobacillus-fermentum/

http://people.hofstra.edu/beverly_clendening/adv_molecular_biology/Protocols/Measuring_Optical_Dens.html