Lab 5 report prepared by Teoh Leong Sin

Name : Teoh Leong Sin

Matric no: 111433

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.

 

 Objective:

To determine the antimicrobial effects of the extracelluar extracts of selected LAB strains.

Results:

Part 1: Determination of bacteriocin activity via agar diffusion test

The sample result that shows that there is inhibition zone or presence of antimicrobial effect.

The sample result that shows that there is no inhibition zone or absence of antimicrobial effect.

Strains of Lab	Strains of spoilage/pathogenicbacteria	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	0
	K.pneumonia	(0.7+0.8)/2 = 0.75
Lactobacillus planterum	S.aureus	0
	P.aeruginosa	0
	K.pneumonia	(0.9+0.9)/2 = 0.9

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.pneunomia
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

Discussion:

1)An anti-microbial is a substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, and protozoans

2)The Lactic acid bacteria(LAB) comprise a clade of Gram-positive,acid-tolerant,generally non-sporulating, non-respiring rod or cocci that are associated by their common metabolic and physological characteristics.Lactic acid bacteria are rod-shaped bacilli or cocci characterizd by an increased tolerance to a lower pH range.This aspect partially enables LAB to outcomplete other bacteria in  a natural fermentation as they can withstand the increased acidity from organic acid production. LAB are amongst the most important groups of mircroorgnisms used in the food industry.

3)Part 1 determination of bacteriocin activity is mainly due to lactic acid and other acid production.

The agar diffusion test is means of measuring effect of an antimicrobial agent against bacteria grown in culture. A filter-paper disk, impregnated with the compound to be tested, is then placed on the surface of the agar. The compound diffuses from the filter paper into the agar. The concentration of the compound will be highest next to the disk, and will decrease as distance from the disk increases. If the compound is effective against bacteria at a certain concentration, no colonies will grow where the concentration in the agar is greater than or equal to the effective concentration. This is the zone of inhibition. Thus, the size of the zone is a measure of the compound's effectiveness: the larger the clear area around the filter disk, the more effective the compound.

4)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.

5)Spectrophotometer is a photometer that can measure the intensity features of spectrophotometers are spectral bandwidth and linear range of absorption or relectance measurement.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. 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. Typically the OD₆₀₀ is measured.

6)Strain of L. plantarum showed the strongest inhibition effects on K. pneumonia because its OD₆₀₀ were less than the OD₆₀₀ of the positive control among the three pathogenic bacteria.

Conclusion:

Bacteriocin was extracted by centrifuge method to determine for the antagonistic activity. LAB is a useful bacteria used to produce bacteriocin that can inhibit the growth of bacteria. Bacteriocins have been defined as proteinaceous substances exhibiting bactericidal activity against closely related species. Currently they are receiving increased attention because of their inhibitory activity against food spoilage and food-borne pathogenic bacteria . Commercial nisin preparations have been evaluate in food systems. It was now widely used as bio preservatives in the food industry due to their antibacterial properties. This allowed a more strict microbial control of a variety of commercial food products.

References:

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

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

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

http://www.pjbs.org/pjnonline/fin202.pdf

http://dictionary.reference.com/browse/arbitrary