Lab 4271

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 is naturally produced antimicrobial agents, such as bacteriocins, is on the rise, since nowdays consumers demand “natural” and “minimally processed” food.

Bacteriocins comprise a large and diverse group of ribosomally synthesised antimicrobial proteins or peptides. Although bactreriocins 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 monocytogenes, Staphylococcus aureus, Enterococcus faecalis and Clostridium tyrobutyricium.

OBJECTIVE

To determine the antimicrobial effects of extracellular extracts of selected LAB strains

RESULT

PART 1: Determination of bacterion activity via agar diffusion test

L.plantarum

L.brevis

L.casei

TABLE PART 1 : Determination of Bacteriocin Activity Via Agar Diffusion Test

STRAINS OF LAB	STRAINS OF SPOILAGE / PATHOGENIC BACTERIA	INHIBITION ZONE (cm)
L.plantarum	S.aureus	0.60
	K.pneumoniae	1.15
	P.aeruginosa	0.00
L.brevis	S.aureus	0.00
	K.pneumoniae	0.70
	P.aeruginosa	0.80
L.casei	S.aureus	0.00
	K.pneumoniae	1.00
	P.aeruginosa	0.65

PART 2 : Determination of bacteriocin activity via optical density.

Strain of lab : L.plantarum

DILUTION	OD₆₀₀of spoilage / pathogenic bacteria
DILUTION	STRAIN 1: P.aeruginosa	STRAIN 2: S.aureus	STRAIN 3: K.pneumoniae
0x	-	-	-
2x	1.025	0.787	0.871
10x	0.733	0.772	0.595
50x	0.755	0.560	0.506
100x	0.260	0.321	0.237
EQUATION	Y= -0.2273X + 1.4888	Y= -0.161X + 1.1735	Y= -0.1991X + 1.2491
POSITIVE CONTROL(Z)	0.432	0.270	0.829
50% of POSITIVE CONTROL (Z/2)	0.216	0.135	0.4215
AU/mL	5.5996	6.4503	4.1919

STRAIN 1 :P.aeruginosa

STRAIN 2 : S.aureus

STRAIN 3 : K.pneumoniae

DISCUSSION

Part 1.

Determination of bacterion activity via agar diffusion test
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 of inhibition is a measure of the compound's effectiveness: the larger the clear area around the filter disk, the more effective the compound.

Part 2.

Determination of bacterion activity via optical density

There is one step that we need to prepare a negative-control for ‘auto-zero’ via spectrophotometer Spectrophotometry involves the use of a spectrophotometer. A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function of the light source wavelength. Important features of spectrophotometers are spectral bandwidth and linear range of absorption or reflectance measurement. Spectrophotometers are commonly used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids, such as polished glass, or gases. However they can also be designed to measure the diffusivity on any of the listed light ranges that usually cover around 200nm - 2500nm using different controls and calibrations.Within these ranges of light, calibrations are needed on the machine using standards that vary in type depending on the wavelength of the photometric determination.

An example of an experiment in which spectrophotometry is used is the determination of the equilibrium constant of a solution. A certain chemical reaction within a solution may occur in a forward and reverse direction where reactants form products and products break down into reactants. At some point, this chemical reaction will reach a point of balance called an equilibrium point. In order to determine the respective concentrations of reactants and products at this point, the light transmittance of the solution can be tested using spectrophotometry. The amount of light that passes through the solution is indicative of the concentration of certain chemicals that do not allow light to pass through.

Conclusion

Bacteriocins were used as food biopreservatives and could be lead to the replacement of synthethic chemical preservatives, which have their antimicrobial action reduced due the continued appearance of multiresistant microbial lineages.

The increasing occurrence of classic and or emerging food borne disease and it is possibly related to the indiscriminate use of chemicals preservatives favoring the selection of microbial lineages more and more resistant and therefore of difficult control.

Bacteriocins are agents that can act on the microbial cell through different ways when compared to conventional chemical food preservatives, provoking the formation of an inhospitable environment to microbial survival. These molecules present characteristics of resistance to heat, acidity, low water activity and oscillations of temperature. There is the necessity to develop studies involving the establishment of the some bacteriocins characteristics such as antimicrobial spectrum, isolation, toxicity and stability use as control agents to the growth and microbial survival in food.

Lactic acid bacteria and their products are more effective and flexible in several applications. Most inhibitory substances produced by lactic acid bacteria are safe and effective natural inhibitors of pathogenic and food spoilage bacteria in various food.

Reference

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

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

http://medical-dictionary.thefreedictionary.com/bacteriocin