LAB 5 DETERMINATION OF ANTIMICROBIAL EFFECTS OF MICROBIAL EXTRACTS 

Introduction.

An antimicrobial is an agent that kills microorganisms or inhibits their growth. Antimicrobial medicines can be grouped according to the microorganisms they act primarily against. For example, antibacterials (commonly known as antibiotics) are used against bacteria and antifungals are used against fungi. They can also be classed according to their function. Antimicrobials that kill microbes are called microbicidal; those that merely inhibit their growth are called microbiostatic. Disinfectants such as bleach are non-selective antimicrobials.

Bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain . They are typically considered to be narrow spectrum antibiotics, though this has been debated.  They are phenomenologically analogous to yeast and paramecium killing factors, and are structurally, functionally, and ecologically diverse.

In fermented foods, lactic acid bacteria (LAB) display numerous antimicrobial activities. This is mainly due to the production of organic acids, but also of other compounds, such as bacteriocins and antifungal peptides. Several bacteriocins with industrial potential have been  purified  and  characterized. These bacteriocins have been reported to inhibit the growth of many pathogens. In this experiment, we are going to examine and discuss the effects of LAB strains on Escherichia coli (E.coli)  and Staphylococcus aureus (S.aureus).

Bacteriocins are categorized in several ways, including producing strain, common resistance mechanisms, and mechanism of killing. There are several large categories of bacteriocin which are only phenomenologically related. These include the bacteriocins from gram-positive bacteria, the colicins, the microcins, and the bacteriocins from Archaea. The bacteriocins from E. coli are called colicins (formerly called 'colicines,' meaning 'coli killers'). They are the longest studied bacteriocins. They are a diverse group of bacteriocins and do not include all the bacteriocins produced by E. coli. For example the bacteriocins produced by Staphylococcus warneri are called as warnerin or warnericin

E. coli is a Gram-negative rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms).It normally lives inside your intestines, where it helps your body break down and digest the food you eat. Unfortunately, certain strains of E. coli can get from the intestines into the blood. This is a rare illness, but it can cause a very serious infection.

Objective

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

Materials and reagents:

·         MRS broth

·         Sterile filter paper

·         Forceps

·         Sterile universal bottles

·         Cultures of LAB and spoilage/pathogenic organisms

·         Bench-top refrigerated centrifuge.

·         Incubator 30 and 37 degree celcius .

·         UV/V is spectrophotometer.

·         Distilled deionized water

·         Trypticase soy agar

·         Brain heart infusion agar

·         Yeast extract

Part 1 Determination of bacteriocin activity via agar diffusion test

1. All the petri dishes are labelles according to the spoilage organisms and starins of LAB used .
2. One strain of spoilage organism and one strain of LAB will be used in each plate. The plate is divided into 2, each side for one replicate.
3. Each group will have 3 strains of LAB and 3 trains of spoilage/pathogenic organisms.
4. 1. 
5. 10 ml of tryticase soy-yeast extract agar (TSAYE) is loaded into the labeled petri
6. dishes and the agar is ensured to cover the entire surface of the plate. The agar is waited until it became solidified.
7. 2 ml of the broth containing the spoilage organism is inoculated into 10 ml of the brain hear infusion (BHI) agar and then it is being vortex.
8. The mixture is loaded on the top of the TSAYE agar layer and is ensured that it covers the entire surface. The gar is waited until it became solidified. 
9. The broth containing LAB cultures is centrifuged. The supernatant will be used as extracellular extracts
10. A sterile filter paper disk is aseptically picked up with sterile forceps and is dipped into the extracellular extract. The excess extract is ensured to has drained off.
11. The paper disk is placed on the top of the solidified BHI agar.
12. Next, the plates is incubated for 24 – 28 hours at 37˚C.
13. The inhibiton zones is measures in cm and the readings is recorded upon incubation.

Part 2: Determination of bacteria activity via optical density

1. Broth containing LAB culture is centrifuged. The supernatant will be used as extracellular extracts.
2. 3 strains of LAB and 3 strains of spoilage or pathogenic organisms are obtained.
3. 5 ml of double-strength MRS is added with 1 ml of cultures containing spoilage or photogenic bacteria. The mixture is then vortex.
4. A serial dilution of the extracellular extracts is prepared. ( diluted 0x, 2x, 10x, 50x, 100x)
5. 5 ml of each extracellular extracts dilution is added into the mixture as prepared in step (3).
6. The mixture is incubated for 12-15 hours at 37 °c.
7. A control using 5 ml of double-strength MRS, 1 ml of cultures containing spoilage or pathogenic bacteria ,and 5 ml of sterile peptone is prepared. The mixture is incubated for 12-15 hours at 37 °c.
8. A negative control for auto-zero is prepared via the spectrophotometer. 5 ml of double-strength MRS is added with 2 ml of distilled water. (Need not incubate)
9. Upon incubation, the optical density of the spoilage or pathogenic bacteria is measured at 600nm. The same is performed for control as well.
10. One arbitrary unit (AU) is defined as the dilution factor of the extracellular extract that inhibited 50% of the spoilage or pathogenic bacteria growth and expressed as AU/ml.
11. 50% of the spoilage or pathogenic bacteria growth are determined from the OD₆₀₀ of the control.

Result:

Part  I. Determination of bacteriocin activity via agar diffusion test.

Strains of LAB	Strains of spoilage/ pathogenic bacteria	Inhibition zone (cm)
Lactobacillus fermentum	E.coli	(1.1+0.8)/2  = 0.95
	Salmonella	(1.0+ 1.0)/2  = 1.0
	S. Aureus	(1.0+0.8)/2  = 0.9

E. coli

S. aureus

Salmonella

Part II. Determination of bacteriocin activity via optical density.

Strain of LAB: Lactobacillus Fermentum

Dilutions	OD600 of spoilage/ pathogenic bacteria
	Strain 1 : E.coli	Strain 2: Salmonella	Strain 3: S. Aureus
0x	(3.504+3.156+3.276)/3= 3.31	(3.3+3.204+3.224)/3= 3.24	(2.892+2.904+3.032)/3= 2.94
2x	(3.548+3.704+3.848)/3= 3.70	(3.688+3.728+3.22)/3= 3.55	(3.352+3.268+3.324)/3= 3.33
10x	(3.456+2.872+3.036)/3= 3.12	(3.42+3.16+3.836)/3= 3.47	(3.076+3.024+3.004)/3= 3.03
50x	(2.824+2.748+3.14)/3= 2.90	(3.536+3.02+3.42)/3= 3.32	(3.867+3.648+3.877)/3= 3.79
100x	(3.068+3.084+2.72)/3= 2.96	(3.136+3.54+3.968)/3= 3.55	(4.101+4.132+3.976)/3= 4.07
Equation			Y= 0.0262X + 2.91
OD600 of control	(2.552+2.48+2.44)/3= 2.49	(2.83+2.64+2.54)/3= 2.67	(3.584+3.98+3.65)/3= 3.74
50% of OD600	1.245	1.335	1.87
AU/ml			-39.69

Discussion:

Escherichia coliis a Gram-negative, rod-shapedbacterium that is commonly found in the lower intestineof warm-blooded organisms (endotherms). Salmonella is a genus of rod–shaped, Gram-negative, non-spore-forming, predominantly motile bacteria and flagella that grade in all directions. Staphylococcus aureus is a bacterium that is a member of the Firmicutes, and is frequently found in the human respiratory tract and on the skin.It stains Gram positive and is non-moving small round shaped or non-motile cocci. It is found in grape-like (staphylo-) clusters.

Part 1. Determination of bacteriocin activity via agar diffusion test

            The agar diffusion test is used to measure the effect of an antimicrobial agent against bacteria grown in culture.The bacteria are swabbed uniformly across a culture plate. A filter-paper disk, impregnated with the compound to be tested, is then placed on the surface of the agar. 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.

            Lactobacillus fermentum is a Gram-positivespecies of bacterium in the genus Lactobacillus. The strain Lactobacillus fermentum ME-3 has been discovered and identified as an antimicrobial and antioxidative probiotic.

Lactobacillus fermentum had observed and it inhibited the growth of bacteria which are Escherichia coli, Salmonellaand Staphylococcus aureus. In this experiment, Salmonella has the largest inhibition zone and the Staphylococcus aureushas the smallest inhibition zone.Through the result, we can know that Staphylococcus aureushas the highest susceptibility. The antimicrobial produced by the Lactobacillus fermentumhas the least effective on the Staphylococcus aureus.Lactobacillus fermentumhas the highest effective on the Salmonellaas the inhibition zone produced is the largest.

Part 2. Determination of bacteriocin activity via optical density

        OD600 is an acronym indicating the absorbance, or optical density, of a sample measured at a wavelength of 600 nm. It is a common method for estimating the concentration of bacterial or other cells in a liquid.OD600 is preferable to UV spectroscopy when measuring the growth over time of a cell population because at this wavelength, the cells will not be killed as they would under too much UV light.

         Optical density, measured in a spectrophotometer, can be used as a measure of theconcentration 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.

Based on the table from the result, we can see that the Staphylococcus aureus has the highest value of OD600 control. This shows that Staphylococcus aureushas the strongest inhibition effect. In contrast, Escherichia coli has the lowest value of OD600 control.

Conclusion:

The best known examples of biopreservation involve bacteriocins. However, with the exception of nisin, bacteriocins have received limited use in the food industry. Peptides can be added to foods to improve consumer health.  This review critically discusses the use and potential of peptides and bacteriocins in food systems in terms of safety, quality, and improvement of human health.Antimicrobials are important tools that are integral to our complex food system. Antimicrobials provide for high quality or good physical condition of crops and good health of food animals entering the food chain.

Antibiotics are used to treat, prevent, and control disease among food animals and in some cases to improve feed utilization and, thus, growth rate. Nonantibiotic antimicrobial agents enable disinfection or sanitization of animal production premises, transport equipment, carcasses, slaughter facility equipment, and effective sanitation during food processing, and ensure food quality and safety.The availability of antibiotics to treat infectious diseases has radically improved human and animal well-being. Paradoxically, this very success threatens their future utility. Both the prudent and inappropriate use of antibiotics in human medicine, veterinary medicine, and animal husbandry create selective pressure that favors the emergence of antibioticresistant microbes. Coupled with specific  genetic resistance mechanisms, the selective pressure of antimicrobials may result in foodborne bacteria that are resistant to antimicrobials.