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 synthesised antimicrobial proteins or peptides. Although bacteriocins can be found in numerous Gram-positive ad 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 Escherichia Coli, Staphylococcus Aureus, and Salmonella.

Objective:

To determine the anitmicrobial effects of extracellular extracts of selected LAB strains.

Materials and reagents:

MRS broth
Sterile filter paper disk (50 mm X 50 mm)
Forceps
Sterile universal bottles
Cultures of LAB and spoilage/pathognic organisms
Bench-top refrigerated centrifuge
Incubator 30^oC and 37^oC
UV/Vis spectrophotometer
Distilled deionized water
Trypticase soy agar
Brain heart infusion agar
Yeast extract

Procedure:

Part I. Determination of baceriocin activity via agar diffusion test:
          1. Label all the petri dishes according to the spoilage organisms and strains of LAB used.
          2. Each plate will be used for one strain of spoilage organism and one strain of LAB. Divide the plate
              into 2, each side for one replicate.
          3. Each group will have a strains of LAB and 3 strains of spoilage/pathogenic organisms.
          4. Load 10 mL of trypticase soy-yeast extract agar (TSAYE) into the labeled petri dishes and ensure
              that the agar covers the entire surface of the plate. Wait for it to solidify.
          5. Innoculate 2 mL of the broth containing the spoilage organism into 10 mL of brain heart infusion
              (BHI) agar and vortex.
          6. Load the mixture on top of the TSAYE agar layer, ensure that it covers the entire surface, and wait
              for it to solidify.
          7. Centrifuge the broth containing LAB cultures. The supernatant will be used as extracellular extracts.
          8. Aseptically pick up a sterile filter paper disk with your sterile forceps and dip the disk into the
              extracellular extract.
          9. Place the paper disk on top of the solidified BHI agar.
          10. Incubate the plates for 24 - 48 h at 37^oC
          11. Upon incubation, measure the inhibition zones (in cm) and record your readings.

Part II. Determination of bacteriocin activity via optical density:
          1. Centrifuge the broth containing LAB cultures. The supernatant will be used as extracellular extracts.
          2. Each group will have a strain of LAB and 3 strains of spoilage/pathogenic organisms.
          3. Add 5 mL of double-strength MRS with 1 mL of cultures containing spoilage/pathogenic bacteria
              and vortex the mixture.
          4. Prepare a sterile dilution of the extracellular extracts. (Diluted 0x, 2x, 10x,50x, 100x)
          5. Add 5 mL of each extracellular extracts dilution into mixture as prepared in step (3).
          6. Incubate the mixtures for 12 - 15 h at 37^oC.
          7. Prepare a control using 5 mL of double-strength MRS, 1 mL of cultures containing
              spoilage/pathogenic bacteria, and 5 mL of sterile peptone. Incubate the mixtures for 12 - 15 h at
              37^oC.
          8. Prepare a negative-control for 'auto-zero' via the spectrophotometer. Add 5 mL of double-strength
              MRS with 2 mL of distilled water. (Need not incubate)
          9. Upon incubation, measure the optical density of the spoilage/pathogenic bacteria at 600 nm.
              Perform the same for the control as well.
          10. One arbitrary unit (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.
          11. 50% of the spoilage/pathogenic bacteria growth are determined from the OD600 of the control.

Calculations:

Inhibition zone:

Strains of spoilage / pathogenic bacteria	Diameter 1 (cm)	Diameter 2 (cm)	Average diameter (cm)
E. coli	1.0	0.9	1.0
Salmonella	0.9	0.8	0.9
S. aureus	1.4	0.9	1.2

Arbitrary unit (AU/mL):

x: Serial dilutions of extracellular extract

y: Abs₆₀₀ or OD₆₀₀
m and c: constants

One arbitrary unit (AU) is defined as the dilution factor of the extracellular etract 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

Graph :

Discussion:

Part 1 :

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

   1.  Before we pour the 2mL of the broth containing the spoilage organism inoculate into 10mL of 

         brain hear infusion ( BHI ) agar,vortex centrifugation is needed. Avoid direct poured of the 

        mixture into the Petri dish that contain TSAYE agar and mixed it with  "8" number motion .

   2.  Do not cover the petri dish immediately to prevent the trapping of water vapours which

        released from hot tryticase soy-yeast extract agar ( TSAYE ) agar to prevent the formation of

        water droplets in order to maintain the growth rate of bacteriocins.

   3. The Escherichia Coli and Salmonella were not inhibited by the LAB strain successfully. The

        specimen of pathogenic bacteria provided contain live Escherichia Coli and

        Salmonella.

  

     Part 2 :

      

          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 420 nm- 660 nm 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.

      

      1. The results showed that the control sample value, OD600 is less than the extracted
          values because lactic acid and other types of acid were existed in the extracted sample.
          Thus, it caused the growth of the bacteria in it while peptone which found in the control
          sample only has nutrient and without any other promoting factors.

Conclusion:

 In conclusion, the diffusion of agar method showed that the strain of LAB against the S.aureus , a
 pathogenic bacteria most effectively. Moreover, the S.aureus in the second part of the experiment also
 showed the best inhibition but the LAB does not showed the strongest inhibition effects on the S.aureus
 pathogenic bacteria due to the technical error in the preparation of the sample of OD600.


Reference:

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

2. http://en.wikipedia.org/wiki/Staphylococcus_aureus

3.http://www.udea.edu.co/portal/page/portal/bibliotecaSedesDependencias/unidadesAcademicas/Corpor

   acionAcademicaAmbiental/BilbiotecaDiseno/Archivos/Potential.pdf