LAB 5 - Kong Siao Thung

Name : Kong Siao Thung

No. Matrice : 111372

LAB 5: Determination of Antimicrobial Effects of Microbial Extracts 

Introduction

       Certain groups of bacteria can produce antimicrobial substances with the capacity to inhibit the growth of pathogenic and spoilage microorganisms. 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 synthesized 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 received 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 ofListeria monocytogens, Staphyloccus aureus, Enterococcus faecalis and Clostridium tyrobutyricum.

Objective

To determine the antimicrobial effects of extracellular of selected LAB strains.

Results

Part 1 Determination of bacteriocin activity via agar diffusion test

Strains of LAB	Strains of spoilage/ pathogenic bacteria	Inhibition zone (cm)
Lactobacillus Plantarum	S. aureus	-
	K. pneumonia	(1.40+0.79)/2= 1.095
	P. aeruginosa	(0.70+0.81)/2=0.755
Lactobacillus Brevis	S. aureus	-
	K. pneumonia	(1.40+0)/2=0.70
	P. aeruginosa	(1.10+0)/2=0.55
Lactobacillus Casei	S. aureus	-
	K. pneumonia	(1.00+0.90)/2=0.95
	P. aeruginosa	(0+1.03)/2= 0.515

Lactobacillus Plantarum (S. aureus)

Lactobacillus Plantarum (K. pneumonia)

Lactobacillus Plantarum (P. aeruginosa)

Lactobacillus Brevis (S. aureus)

Lactobacillus Brevis (K. pneumonia)

Lactobacillus Brevis (P. aeruginosa)

Lactobacillus Casei (S. aureus)

Lactobacillus Casei (K. pneumonia)

Lactobacillus Casei (P. aeruginosa)

Discussions

Lactobacillus Plantarum, Lactobacillus Brevis and Lactobacillus Casei can produce antimicrobial substances with the capacity to inhibit the growth of S. aureus, K. pneumonia and P. aeruginosa. Antimicrobial is a substance that kill or inhibit the growth of microorganisms such as bacteria, fungi,  or protozoas. The agar diffusion test is used to test the susceptibility of the S. aueres, K. pneumonia, and P. aeruginosa against the antimicrobial substances produced by the three LAB(lactic acid bacteria). However, the composition of the agar, its depth in dishes, the quality of the paper discs, their contents of antibiotic, the density of inoculated bacteria, the time of incubation and other factors should be standardized.

According to the result obtained, there are no inhibition zone formed in the diffusion test of Lactobacillus Plantarum, Lactobacillus Brevis and Lactobacillus Casei against S. aueres. This may due to the improperly inoculation of the plate or other factors such as contamination, improperly placing of the discs etc. Each discs must be pressed down with forceps to ensure complete contact with the agar surface or irregular zone may be affected. If the plate was properly inoculated and all other conditions were correct, the zones of inhibition should be uniformly circular and there will be a confluent lawn of growth.

Besides that, the inhibition zones of P. aeruginosa is smaller than K. pneumonia according to the result. This mean that the susceptibility of  P. aeruginosa is better than K. pneumonia. The antimicrobial substances produced by Lactobacillus Plantarum, Lacobacillus Brevis and Lactobacillus Casei have less effect on P. aeruginosa.  

Part II  Determination of bacteriocin activity via optical density

Serial dilution of extracellular extract

 Strain of LAB : Lactobacillus Plantarum

Dilutions	OD600 of spoilage/pathogenic bacteria
	Strain 1: S. aureus	Strain 2: P.aeruginosa	Strain 3: K. pneumonia
0x	0	0	0
2x	0.790	0.836	0.812
10x	0.931	1.125	1.086
50x	0.652	0.495	0.463
100x	0.455	0.462	0.449
Equation	y= -0.004x+0.878	y= -0.005x+0.959	Y= -0.005x+0.925
OD600 of control	0.508	0.129	1.156
50% of OD600	0.254	0.0645	0.578
AU/ml	156	178.9	69.4

Discussions 

Spectrophotometry involves the use of a spectrophotometer. A spectrophotometer is a photometer that can measure intensity as a function of the light source wavelength. Spectrophotometers are commonly used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids, usch as polished glass, or gases.

The optical density of a material is a logarithmic ratio of the radiation falling upon a material, to the radiation transmitted through a material. The graphs obtained showed that the serial dilutions are inversely proportional to the OD ₆₀₀  of S. auerus, P.aeruginosa, and K.pneumonia. The highest absorbance reading in the bacteria strain is in the 10x serial dilution. This indicates that when the Lactobacillus Plantarum is in 10x serial dilution, the antimicrobial effect towards the three bacteria, S. aerus, P.aeruginosa, and K. pneumonia is the largest.

The result obtained might not be accurate due to the improperly preparation of the serial dilution solution. This causes the result obtained is not like what its suppose to be.

Conclusions

There are antimicrobial effects of LAB on the three pathogenic bacteria, namely S.auerus, K. pneumonia and P.aeruginosa. Although the agar diffusion assay is the most widely used method in routine measurements of bacteriocin activity, turbidometry (spectrophotometer) offers a simpler, faster and more reliable alternative since diffusion related problems are eliminated, the degree of human intervention and judgment is low, and very low bacteriocin concentrations can be quantified.

References

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1622751/

http://en.wikipedia.org/wiki/Agar_diffusion_test

http://en.wikipedia.org/wiki/Zone_of_inhibition

http://www.microbelibrary.org/component/resource/laboratory-test/3189-kirby-bauer-disk-diffusion-susceptibility-test-protocol

http://jac.oxfordjournals.org/content/6/2/181.extract

http://en.wikipedia.org/wiki/Absorbance

LAB 5 : DETERMINATION OF ANTIMICROBIAL EFFECTS ON MICROBIAL EXTRACTS

Name : See Yen Shan
Matric number : 111415

LAB 5: Determination of Antimicrobial Effects of Microbial Extracts

Introduction

       Certain groups of bacteria can produce antimicrobial substances with the capacity to inhibit the growth of pathogenic and spoilage microorganisms. 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 synthesized 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 received 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 monocytogens, Staphyloccus aureus, Enterococcus faecalis and Clostridium tyrobutyricum.


Objectives
To determine the antimicrobial effects of extracellular of selected LAB strains.


Results


Part 1 Determination of bacterioacin activity via agar diffusion test

L.plantarum with S.aureus

L.plantarum with K.pneumonia

L.plantarum with P.aeruginosa 

L.brevis with S.aureus

L.brevis with K.pneumonia

L.brevis with P.aeruginosa

L.casei with S.aureus

L.casei with K.pneumonia

L.casei with P.aeruginosa

Strains of LAB	Strains of spoilage/ pathogenic bacteria	Inhibition zone (cm)
Lactobacillus Plantarum	S. aureus	No inhibition zone
	K. pneumonia	(1.40+0.79)/2 = 1.095
	P. aeruginosa	(0.70+0.81)/2 = 0.755
Lactobacillus Brevis	S. aureus	No inhibition zone
	K. pneumonia	(1.40+0)/2 = 0.700
	P. aeruginosa	(1.10+0)/2 = 0.550
Lactobacillus Casei	S. aureus	No inhibition zone
	K. pneumonia	(1.00+0.90)/2 = 0.950
	P. aeruginosa	(0+1.30)/2 =  0.650

Part 2 Determination of bacteriocin activity via optical density

Strain of LAB : Lactobacillus Plantarum

Dilutions	OD600 of spoilage/pathogenic bacteria
	Strain 1: S. aureus	Strain 2: P.aeruginosa	Strain 3: K. pneumonia
0x	-	-	-
2x	0.790	0.836	0.812
10x	0.931	1.125	1.086
50x	0.652	0.495	0.463
100x	0.455	0.462	0.449
Equation	y= -0.004x+0.878	y= -0.005x+0.959	Y= -0.005x+0.925
OD600 of control	0.508	0.129	1.156
50% of OD600	0.254	0.0645	0.578
AU/ml	156	178.9	69.4

Discussions


    LAB(Lactic acid bacterias) act as the bacteriocins to inhibits the growth of the strains of spoilage or pathogenic bacteria. Bacteriocin is a protein which is secreted by bacteria that kills or inhbits the strain of certain competing bacteria strain. Usually bacteriocin will only kills the closely-related bacteria strains. There is three main known ways of how bacteriocins inhibit the growth of pathogenic bacteria strains.

• The bacteriocin 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.
• The third way is the bacteriocin will inhibits the protein synthesis of the pathogenic bacterias but does not kills them.

Part 1 Determination of bacteriocin activity via agar diffusion test : 

    From the results, we can observed that the petri dishes that cultured with the Staphyloccus aureus pathogenic bacteria do not show any inhibition zone around the paper disks that are immersed with three types of LAB each. This might be due to two reasons :

• The Staphyloccus aureus is not culture successfully. The specimen of pathogenic bacteria provided does not contain live Staphyloccus aureus.
• During the process of adding the Staphyloccus aureus into the tripticase soy-yeast extract agar, the temperature of agar is too high, that eventually kills the pathogenic bacteria.

    Besides that, we can observe that the inhibition zone between Lactobacillus plantarum and three pathogenic bacteria strain is the biggest. The bacteriocins produced by the Lactobacillus plantarum have the strongest effect on inhibiting the pathogenic bacteria strains.

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 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.

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.

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

   

    Generally,the OD value is higher from the 100X dilution. It indicated that the LAB has stronger antimicrobial effect on the pathogenic bacteria compare to a more diluted solution.The  more diluted the extracellular extract, the greater the value of OD_600.

 From the results of our experiment, the graph shows us when the serial dilution is increasing, the optical density is decreasing. The outcome is totally different from what we expect. 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. 

   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. Then the result will not be affected.

Conclusions

        LAB is a useful bacteria used to produce bacteriocin that can inhibit the growth of bacteria. Currently, the usage of LAB is getting attention as it exerts a strong antagonistic activity against many microorganisms, including food spoilage organisms and pathogens. Production of the primary metabolite lactic acid and the resulting pH decrease is the main preserving factor in food fermentation.  

References


http://en.wikipedia.org/wiki/Bacteriocin
http://ejeafche.uvigo.es/component/option,com_docman/task,doc_view/gid,634/
http://en.wikipedia.org/wiki/Brain_heart_infusion_broth
http://en.wikipedia.org/wiki/Agar_diffusion_test
http://en.wikipedia.org/wiki/Absorbance
http://en.wikipedia.org/wiki/OD