Saturday, 5 May 2012

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


No comments:

Post a Comment