Meat Model System Development for Antibacterial Activity Determination

Graciela Vignolo and Patricia Castellano

1. Introduction

Bacteriocins are antibacterial substances produced by many different bacterial species. Although the bacteriocins form a heterogenous group with respect to production of bacteria, antibacterial spectrum, mode of action, and chemical properties, they are by definition proteinaceous compounds that are bactericidal toward organisms taxo-nomically close to the producer (1). The ability of lactic acid bacteria (LAB) to compete and finally dominate in mixed fermentations has been attributed to the production of several antimicrobial metabolites such as organic acids, carbon dioxide, hydrogen peroxide, diacetyl, and bacteriocins (2,3). The antimicrobial activities of the LAB have long been known, but their bacteriocins have received limited attention until recently (4). Numerous strains of lactic acid bacteria associated with food systems are capable of producing bacteriocins, or antibacterial proteins with activity against foodborne pathogens and contaminants. Recently, considerable enphasis has been placed on the physicochemical, biochemical, and genetic characterization of these proteins.

Many methods for the detection of bacteriocin production as well as the determination of the potency of bacteriocin preparations have been described. All the usual techniques are based on the fact that bacteriocins can diffuse in solid or semisolid culture media, which are subsequently inoculated with a suitable indicator strain. This method has long been shown to have a good perfomance in the characterization of new bacte-riocins.

Although results obtained from broth systems show that bacteriocins inhibit target organisms, applied studies must be performed to confirm their effectiveness in food. As many lactic acid bacteria associated with meat products were described to be important natural bacteriocin producers, it has been necessary to assay their inhibitory efficacy in meat or meat products. Because of the complexity of these kinds of foods a simplified meat-model system was developed to determine the inhibitory activity of a bacteriocin.

From: Methods in Molecular Biology, vol. 268: Public Health Microbiology: Methods and Protocols Edited by: J. F. T. Spencer and A. L. Ragout de Spencer © Humana Press Inc., Totowa, NJ

2. Materials

2.1. Meat Slurry

1. Stomacher® 400 Circulator (Seward, London, UK) and stomacher bags.

2. Fresh lean beef/pork muscle aseptically minced after 24 h of exsanguinization and stored at -30°C.

3. Distilled water (dH2O).

2.2. Preparation of the Bacteriocin or the Bacteriocinogenic Strain

1. LAB bacteriocin producer strain.

2. de Man-Rogosa-Sharpe (MRS) broth (5): 10 g/L peptone, 10 g/L meat extract, 5 g/L yeast extract, 20 g/L glucose, 5 g/L sodium acetate, 1.08 g/L Tween-80, 2 g/L ammonium citrate, 2 g/L K2HPO4, MgSO4-7H2O (0.2g/L), 0.05 g/L MnSO4-4H2O, pH 6.5. Sterilize in an autoclave at 121°C for 15 min.

3. Active lactic acid bacteria culture grown in MRS broth for about 16 h at 30°C.

5. Centrifuge.

6. TurboVap LV evaporator (Zymark).

2.3. Bacteriocin Activity Determination

1. Distilled and sterile water.

4. Sterile hollow punch, 0.5 cm in diameter.

5. Indicator or sensitive strain.

2.4. Inoculation of the Meat Slurry

1. Meat slurry previously prepared.

2. An active culture of the sensitive strain (required volume to reach the desirable colony-forming units [CFU]/mL in the slurry) (see Note 1).

3. Concentrated bacteriocin (required volume to reach the desirable activity in the slurry) or bacteriocinogenic strain (required volume to reach the desirable CFU/mL in the slurry) (see Note 2).

4. Stomacher 400 blender to homogenize.

5. Incubator.

3. Methods

3.1. Meat Slurry Preparation

1. Mince aseptically fresh lean beef/pork muscle, obtained from a local abattoir. Store at -30°C (see Note 3).

2. Mix 10-g portions of minced meat with dH2O in a stomacher bag. The final volume will be adjusted by separate addition of the cell suspension of the bacteriocin producer strain or the concentrated bacteriocin and the cell suspension of the indicator strain.

3. Stomach the bags for 1 min to homogenize.

3.2. Obtaining the Bacteriocin From the Producer Strain

1. Centrifuge (14,000g x 5 min) the overnight culture of the LAB bacteriocin producer strain.

2. Remove the supernatant and neutralize to pH 7.0 with 5 N NaOH.

4. Concentrate the bacteriocin in the supernatant using an evaporator (TurboVap).

5. Store the concentrated bacteriocin at 5°C for no longer than 30 d.

3.3. Use of the LAB Bacteriocinogenic Strain

1. Inoculate the LAB bacteriocin producer in the appropriate medium and grow at the usual temperature for 16-18 h. Calculate the medium volume necessary to have a desired activity in the meat slurry.

3.4. Bacteriocin Activity Determination

1. Centrifuge an overnight culture of the bacteriocin producer strain (14,000g x 5 min).

2. Remove the supernatant and neutralize to pH 7.0 with 5 N NaOH.

3. Sterilize by filtration through a membrane of 0.45 ^m (see Note 4).

4. Prepare serial twofold dilutions of the bacteriocin in sterile dH2O (1:2, 1:4, 1:8, 1:«).

5. Prepare a Petri dish with 10 mL of MRS base agar. Let solidify and dry.

6. Mix gently 70 ^L of an overnight culture of the sensitive strain with 7 mL of molten MRS soft agar (45°C) and pour onto the base MRS layer. Slide the plate in circles on the bench top immediately to spread the top agar over the plate. Let solidify and dry.

7. Cut wells 0.5 cm in diameter with sterile hollow punches in the agar.

8. In each well, seed 30 ^L of undiluted bacteriocin and each dilution of it.

9. Incubate the plates (with the agar side up) overnight at 30°C.

10. Bacteriocin activity is detected by the presence of growth inhibition zones (halos) of the indicator strain around the wells.

11. Calculate bacteriocin activity expressed in arbitrary units (AU) per milliliter, as follows: AU/mL = 1/0.03 mL x maximal dilution, which inhibits growth of the indicator strain.

3.5. Antibacterial Activity Determination in the Meat Slurry

1. Prepare the meat slurry in stomacher bags.

2. Add the sensitive strain.

3. Add the concentrated bacteriocin or the bactriocinogenic strain.

4. Adjust the final volume to 20 mL with dH2O.

5. Mix the stomacher bags thoroughly.

6. Incubate the bags at the desired temperature.

7. Take samples at time intervals.

8. Determine CFU/mL of the sensitive strain.

9. Determine the AU/mL of the bacteriocin in the slurry (see Note 5).

4. Notes

1. The cell concentration of the sensitive strain has to be previously optimized. The cell lawn in the agar layer must show a marked inhibition zone (halo) around the seeded well.

2. The bacteriocin concentration must be calculated taking into acount the final volume of the slurry and the required AU/mL for the study.

3. Before mincing the beef/pork muscle, remove fat and connective tissue in aseptic conditions and cut the lean muscle into small cubes (3 cm).

4. Filtration through a membrane of 0.45 ^m will not produce detectable loss of activity. If the bacteriocin is sufficiently thermostable to withstand temperatures that kill the producer cells, it can be sterilized by autoclaving.

5. An aliquot of the slurry must be centrifuged to eliminate meat particles; the supernatant will be used to prepare the serial twofold dilutions.

References

1 Tagg, J. R., Dajani, A. S., and Wannamaker, L. W. (1976) Bacteriocins of gram-positive bacteria. Bacterid Rev. 40, 722-756.

2. Piard, J. C. and Desmazeaud, M. (1991) Inhibiting factors produced by lactic acid bacteria, 1. Oxygen metabolites and catabolism end-products. Lait 71, 525-541.

3. Piard, J. C. and Desmazeaud, M. (1992) Inhibiting factors produced by lactic acid bacteria. 2. Bacteriocins and other antibacterial substances. Lait 72, 113-142.

4 Klaenhammer, T. R. (1988) Bacteriocins of lactic acid bacteria. Biochimie 70, 337-349.

5. de Man, J., Rogosa, M., and Sharpe, M. (1960) A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23, 130-135.

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