Matric No : 111356
Lab 5:Determination of Antimicrobial Effect of Microbial Extracts
Introduction
Certain groups of bacteria can produce antimicrobial substances with the capacity to inhibit the growth of pathogenic and spoilage micro-organisms.Organic acids,hydrigen peroxide,diacetly 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 synthesised antimicrobial protein 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 biochemal 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
Result:
Part 1:Determination of bacteriocin activity via agar diffusion test
There are inhibition zone between L.Casei and K. peunomia
Presence of antimicrobial effects
Discussion:
1)Bacteriocins are protein produced by bacteria to inhibit the growth of similar or closely related bacterial strains.
2)Bacteriocin inhibit and kill the bacterial by three way,that are:
Part 2:Determination of bacteriocin activity via optical density
Strain of Lab1 : Lactobaciilus plantarum
Presence of antimicrobial effects
No inbition zone between L. brevis and S. aereus
Absence of antimicrobial effects
Absence of antimicrobial effects
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
|
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
|
Discussion:
1)Bacteriocins are protein produced by bacteria to inhibit the growth of similar or closely related bacterial strains.
2)Bacteriocin inhibit and kill the bacterial by three way,that are:
- Destruction of membrane of the bacterial by forming pores in the membrane.
- Endonucleolytic activity by breakdown the chromosomal DNA as well as RNA.
- Inhibition of protein synthesis but does not lyses the cell.
4)There are two reason causes the absent of the inhibition zone between the three LAB strains and S.aureus
i.The S.aureus that used is unsuccesfully cultured.There are no live S.aureus in the specimen pathogenic bacterial.
ii.During the process of adding trypticase soy-yeast extract agar(TSAYE),the temparature might be too high and finally kill the S.aureus .
Part 2:Determination of bacteriocin activity via optical density
Serial dilution of
extracellular extract
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
|
0.508/2=0.254
|
0.129/2=0.0645
|
1.156/2=0.578
|
AU/ml
|
156
|
178.9
|
69.4
|
Discussion:
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.
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)/mWhen y = Z/2, Thus x = (Z/2 - c)/m3) 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 OD600.
4)From the result ,the graph can be seen that the optical density of three strains of LAB decrease when the serial dilution increase.This might caused by using the the distilled water in the process of serial dilution .As we know that the colour of distilled water is colourless. Lactobacillus plantarum with 100x dilution is approaching colourless,therefore optical density spectrophotometer cannot detect the optical colour density.
5) Peptone is suggested to use because the colour of peptone is similar with the colour of Lactobacillus plantarum culture .Therefore,it will not greatly affect the result.
Conclusion:
Bacteriocins produced by Lactic acid bacteria (LAB) as bio preservatives against S.aureus,K. pneumonia and P. aeruginosa. Both acidification and the production of hydrogen peroxide by LAB were ruled out as the source of the inhibition.LAB competed with spoilage microorganisms, such as certain Gram-negative bacteria for nutrients or space with. Moreover, the shelf-life of food products could extend the production of organic acids, hydrogen peroxide, low molecular weight metabolites (such as diacetyl and bacteriocins) due to their inhibiting effect on the growth of spoilage and pathogenic bacteria.
References:
http://www.academicjournals.org/ajfs/PDF/Pdf2009/Jan/Khalil%20et%20al.pdf
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