Matric no: 111410
Introdution :
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 produce antimicrobial agents, such as bacteriocins, is on the rise, since nowadys 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 monocyotogenes, Staphylococcus aureus, Enterococcus faecalis and Clostridium tyrobutyricum.
Objective:
To determine the antimicrobial effects of extracellular extracts 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 casei
|
S. aureus
|
(0.6+0.6)/2=0.6
|
P. aeruginosa
|
(1.3+0.9)/2=1.15
|
|
K. pneumonia
|
(1.0+1.3)/2=1.1
|
|
Lactobacillus brevis
|
S. aureus
|
0
|
P. aeruginosa
|
||
K. pneumonia
|
(0.7+0.8)/2=0.75
|
|
Lactobacillus planterum
|
S. aureus
|
0
|
P. aeruginosa
|
0
|
|
K. pneumonia
|
Example:
The picture shows the presence of antimicrobial effects of extracellular extracts of selected LAB strains.
The picture shows the absence of antimicrobial effects of extracellular extracts of selected LAB strain.
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
| |
2x
|
0.699
|
0.854
|
0.828
|
10x
|
0.698
|
0.990
|
0.943
|
50x
|
0.590
|
0.780
|
0.625
|
100x
|
0.168
|
0.553
|
0.512
|
Equation
|
y = -0.0054x + 0.7572
|
y = -0.0038x + 0.9468
|
y = -0.004x + 0.8849
|
OD600 of control
|
0.508
|
0.129
|
1.156
|
50% of OD600
|
0.254
|
0.0645
|
0.578
|
AU/ml
|
93.19
|
232.18
|
77.85
|
Example of graph :
Graph 1 : S.aureus
1.An antimicrobial is a substance that kills or inhibits the growth of microorganisms such as
bacteria, fungi, or protozoans.
2.Lactic acid bacteria (LAB) are an important group of industrial starter cultures, applied in the
production of fermented foods. They contribute to the enhancement of the organoleptic attributes
of these foods, as well as to their preservation and microbial safety.Their antimicrobial activity is
due to the production of organic acids (in particular, lactic acid and acetic acid), carbon dioxide,
ethanol, hydrogen peroxide, and diacetyl. The inhibition, however, can also be caused by
bacteriocins that are low-molecular-mass peptides, or proteins, with a bactericidal or bacteriostatic
mode of action, in particular against closely related species.
3.Bacteriocins from LAB may be subdivided into three classes. One class of bacteriocins is formed
by the lantibiotics. These are small and heat-stable peptides that contain thioether amino acids like
lanthionine. The next class of LAB bacteriocins consists of small, heat-stable and hydrophobic
peptides with an antilisterial activity. Another class of bacteriocins consists of large, heat-labile
and hydrophilic proteins.
4.Explain the mehod used to test the bacteriocin activity:
-Agar diffusion :
(a) Agar diffusion method is a means of measuring the effect of an antimicrobial agent against
bacteria grown in culture.
(b) Nutrient is mixed with the pathogenic bacteria in an agar layer where the LAB's extracellular
extract (in the sterile filter paper disk) is placed on it which is then incubate for 24-48 hours at
37ºC and the diameter of inhibition zones (in cm) is measured.
(c) The size of the inhibition zones indicate the difference of bacteriocin activity between LAB.
-Optical density :
(a) 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.
(b) Difference dilution of LAB is added to equal amount of MRS solution with same amount of
pathogenic bacteria and is then incubate for 24-48 hours at 37ºC. The measured optical density
of the spoilage/pathogenic bacteria at 600nm show the bacteriocins activity.
(c)A negative-control (MRS with 2ml of distilled water)is prepared for "auto-zero" via
spectrophotometer.
(d)A positive-control (MRS and pathogenic bacteria grow without LAB's extracellular extract ) is
prepared. Only the sample with optical density lower than optical density of positive control
show antimicrobial effects.
(e)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
Conclusion :
Lactobacillus is a useful bacteria which produced substances such as bacteriocins that can kill or inhibit the growth of other bacteria, fungal or protozoan. Some of them can even inhibit the growth of variety species of pathogenic microorganism.
Refenrences :
1.http://en.wikipedia.org/wiki/Bacteriocin
2.http://www.ncbi.nlm.nih.gov/pubmed/17827969
3.http://en.wikipedia.org/wiki/Agar_diffusion_test
4.http://people.hofstra.edu/beverly_clendening/adv_molecular_biology/Protocols/Measuring_Optical_Dens.html
No comments:
Post a Comment