Lab 5 report written by Tan Hwee Feng

Name: Tan Hwee Feng
Matric no: 111427

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 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

The sample shows the presence of antimicrobial effect

The sample shows the absence of antimicrobial effect

 

Strains of LAB	Strains of spoilage/pathogenic bacteria	Inhibition zone (cm)
Lactobacillus casei	S. aureus	(0.6+0.6)/2=0.60
	P. aeruginosa	(0.9+1.3)/2=1.10
	K. pneumonia	(1.0+0.3)/2=1.15
Lactobacillus brevis	S. aureus	0
	P. aeruginosa	0
	K. pneumonia	(0.8+0.7)/2=0.75
Lactobacillus planterum	S. aureus	0
	P. aeruginosa	0
	K. pneumonia	(0.9+0.9)/2=0.90

Part 2: Determination of  bacteriocin activity via optical density

Dilutions	OD600 of spoilage/pathogenic bacteria
	Strain 1 : S. aureus	Strain 2 : P. aeruginosa	Strain 3 : K. pneumonia
0x	-	-	-
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

The x-axis indicates serial dilutions of 2x, 10x, 50x and 100x

Discussion

1) Lactic Acid Bacteria (LAB) produce various substances that exhibit antagonistic activity against other pathogenic microorganisms such as fungi, bacteria or protozoan.
2) Antimicrobial effect of LAB is mainly due to lactic acid and other organic acid production.
3)  Lactobacilli are important organisms recognized for their fermentative ability as well as their health and nutritional benefits.They produce various compounds such as organic acid, diacetyl, hydrogen peroxide, and bacteriocin protein during fermentation.
4) Bacteriocins are proteinaceous antibacterial compounds and exhibit bactericidal activity.

Part 1: Agar diffusion test

1) Method of measuring the effect of an LAB against pathogenic microorganisms grown in culture.

2) The agar diffusion test consists in placing sterile filter paper disks (5cm) on a layer of petri dish (contains TSAYE in the bottom and brain heart infusion agar+pathogenic microorganisms on the second layer) incubate at 37 degree C for 24-48 hour. After that, examine the inhibition zones (average radius-cm) around filter paper disks.
3) Three types of LAB have been used in the experiments are Lactobacillus casei, Lactobacillus brevis, Lactobacillus plantirum. Three types of pathogenic microorganisms have been used are S.aureus, P.aeruginosa, K.pneumonia.


 Part 2: Optical density

1) A measure of the concentration of bacteria in a suspension.
2) Optical density of extracellular extract of LAB contained MRS medium and pathogenic microorganisms was determined with the help of spectrophotometer at 600nm.
3) One type of LAB has been used in the experiment is Lactobacillus plantirum.  Three types of pathogenic microorganisms have been used are S.aureus, P.aeruginosa, K.pneumonia.
4) Positive control(5ml MRS medium+1ml pathogen+5ml peptone without contain extracellular extract of LAB) is prepared and incubate at 37 degree C for 12-15 hour. When OD of sample is smaller than OD of positive control indicate presence of antimicrobial effect.
5) Negative control( 5ml of MRS+2ml of water) for 'auto-zero' via spectrophotometer.
6) 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.
x indicate AU/mL

Conclusion

Bacteriocin was extracted by centrifuge method is to determine for its antagonistic activity.Lactic Acid Bacteria (lactobacilli) will produce various substances such as bacteriocin to inhibit the growth of pathogenic microorganisms

References


1) http://www.udea.edu.co/portal/page/portal/bibliotecaSedesDependencias/
    unidadesAcademicas/CorporacionAcademicaAmbiental/BilbiotecaDiseno/Archivos/Potential.pdf
2) http://www.udea.edu.co/portal/page/portal/bibliotecaSedesDependencias/
    unidadesAcademicas/CorporacionAcademicaAmbiental/BilbiotecaDiseno/Archivos/Potential.pdf
3) http://www.ajol.info/index.php/ajb/article/viewFile/14770/58576
4) http://www.ajol.info/index.php/ajb/article/viewFile/14770/58576