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Determination of bacteriocin activity with bioassays carried out on solid and liquid substrates: assessing the factor "indicator microorganism".

Papagianni M, Avramidis N, Filioussis G, Dasiou D, Ambrosiadis I - Microb. Cell Fact. (2006)

Bottom Line: Sensitivity limits and linearity of responses to bacteriocin varied significantly among different test-microorganisms in both applied methods, the lower sensitivity limits depending on both the test-microorganism and the applied method.The present work shows that in growth inhibition techniques used in bacteriocin quantification, the choice of the indicator microorganism is critical.Evaluation of sensitivity levels and type of produced responses showed that they can vary widely among different test-microorganisms and different applied methods, indicating that not all microorganisms can be used successfully as indicators and that measurements of growth inhibition in liquid media produce more reliable results.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Hygiene and Technology of Food of Animal Origin, Laboratory of Food Technology, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece. mp2000@vet.auth.gr

ABSTRACT

Background: Successful application of growth inhibition techniques for quantitative determination of bacteriocins relies on the sensitivity of the applied indicator microorganism to the bacteriocin to which is exposed. However, information on indicator microorganisms' performance and comparisons in bacteriocin determination with bioassays is almost non-existing in the literature. The aim of the present work was to evaluate the parameter "indicator microorganism" in bioassays carried out on solid -agar diffusion assay- and liquid -turbidometric assay- substrates, applied in the quantification of the most studied bacteriocin nisin.

Results: The performance of characterized microorganisms of known sources, belonging to the genera of Lactobacillus, Pediococcus, Micrococcus and Leuconostoc, has been assessed in this work in the assays of plate agar diffusion and turbidometry. Dose responses and sensitivities were examined and compared over a range of assay variables in standard bacteriocin solutions, fermentation broth filtrates and processed food samples. Measurements on inhibition zones produced on agar plates were made by means of digital image analysis. The data produced were analyzed statistically using the ANOVA technique and pairwise comparisons tests. Sensitivity limits and linearity of responses to bacteriocin varied significantly among different test-microorganisms in both applied methods, the lower sensitivity limits depending on both the test-microorganism and the applied method. In both methods, however, only two of the nine tested microorganisms (Lactobacillus curvatus ATCC 51436 and Pediococcus acidilactici ATCC 25740) were sensitive to very low concentrations of the bacteriocin and produced a linear-type of response in all kinds of samples used in this work. In all cases, very low bacteriocin concentrations, e.g. 1 IU/ml nisin, were more accurately determined in the turbidometric assay.

Conclusion: The present work shows that in growth inhibition techniques used in bacteriocin quantification, the choice of the indicator microorganism is critical. Evaluation of sensitivity levels and type of produced responses showed that they can vary widely among different test-microorganisms and different applied methods, indicating that not all microorganisms can be used successfully as indicators and that measurements of growth inhibition in liquid media produce more reliable results.

No MeSH data available.


P. acidilactici, P. pentosaceus and L. mesenteroides in agar diffusion assay: plots of log10 nisin concentrations vs average zone diameters of growth inhibition.
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Figure 3: P. acidilactici, P. pentosaceus and L. mesenteroides in agar diffusion assay: plots of log10 nisin concentrations vs average zone diameters of growth inhibition.

Mentions: Log nisin concentrations in the range of 1 to 1.000 IU/ml were plotted against average diameters of inhibition zones of inhibition as shown in Figs 1, 2, and 3. According to Fig. 1, from the three Lactobacilli tested, L. curvatus produced a linear response having an R value greater than 0.98. L. curvatus, appeared to be the most sensitive to nisin, producing the largest zones, with 12 mm corresponding to the dose of 1.000 IU/ml nisin, and a minimal detectable level of 1 IU/ml. A linear response was also observed with L. sakei to nisin levels between 40 and 1.000 IU/ml. However, L. sakei sensitivity was by far lower compared to the exhibited by L. curvatus, since no response was observed to doses up to 40 IU/ml nisin. L. plantarum produced inhibition zones at nisin concentrations above 75 IU/ml, while its response appeared sigmoidal. L. sakei and L. plantarum produced significantly smaller inhibition zones compared to those produced by L. curvatus, ranging from 0.25 to 7 mm and 0.5 to 5.3 mm, respectively, for nisin concentrations ranging from 40 to 1.000 IU/ml.


Determination of bacteriocin activity with bioassays carried out on solid and liquid substrates: assessing the factor "indicator microorganism".

Papagianni M, Avramidis N, Filioussis G, Dasiou D, Ambrosiadis I - Microb. Cell Fact. (2006)

P. acidilactici, P. pentosaceus and L. mesenteroides in agar diffusion assay: plots of log10 nisin concentrations vs average zone diameters of growth inhibition.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1622751&req=5

Figure 3: P. acidilactici, P. pentosaceus and L. mesenteroides in agar diffusion assay: plots of log10 nisin concentrations vs average zone diameters of growth inhibition.
Mentions: Log nisin concentrations in the range of 1 to 1.000 IU/ml were plotted against average diameters of inhibition zones of inhibition as shown in Figs 1, 2, and 3. According to Fig. 1, from the three Lactobacilli tested, L. curvatus produced a linear response having an R value greater than 0.98. L. curvatus, appeared to be the most sensitive to nisin, producing the largest zones, with 12 mm corresponding to the dose of 1.000 IU/ml nisin, and a minimal detectable level of 1 IU/ml. A linear response was also observed with L. sakei to nisin levels between 40 and 1.000 IU/ml. However, L. sakei sensitivity was by far lower compared to the exhibited by L. curvatus, since no response was observed to doses up to 40 IU/ml nisin. L. plantarum produced inhibition zones at nisin concentrations above 75 IU/ml, while its response appeared sigmoidal. L. sakei and L. plantarum produced significantly smaller inhibition zones compared to those produced by L. curvatus, ranging from 0.25 to 7 mm and 0.5 to 5.3 mm, respectively, for nisin concentrations ranging from 40 to 1.000 IU/ml.

Bottom Line: Sensitivity limits and linearity of responses to bacteriocin varied significantly among different test-microorganisms in both applied methods, the lower sensitivity limits depending on both the test-microorganism and the applied method.The present work shows that in growth inhibition techniques used in bacteriocin quantification, the choice of the indicator microorganism is critical.Evaluation of sensitivity levels and type of produced responses showed that they can vary widely among different test-microorganisms and different applied methods, indicating that not all microorganisms can be used successfully as indicators and that measurements of growth inhibition in liquid media produce more reliable results.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Hygiene and Technology of Food of Animal Origin, Laboratory of Food Technology, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece. mp2000@vet.auth.gr

ABSTRACT

Background: Successful application of growth inhibition techniques for quantitative determination of bacteriocins relies on the sensitivity of the applied indicator microorganism to the bacteriocin to which is exposed. However, information on indicator microorganisms' performance and comparisons in bacteriocin determination with bioassays is almost non-existing in the literature. The aim of the present work was to evaluate the parameter "indicator microorganism" in bioassays carried out on solid -agar diffusion assay- and liquid -turbidometric assay- substrates, applied in the quantification of the most studied bacteriocin nisin.

Results: The performance of characterized microorganisms of known sources, belonging to the genera of Lactobacillus, Pediococcus, Micrococcus and Leuconostoc, has been assessed in this work in the assays of plate agar diffusion and turbidometry. Dose responses and sensitivities were examined and compared over a range of assay variables in standard bacteriocin solutions, fermentation broth filtrates and processed food samples. Measurements on inhibition zones produced on agar plates were made by means of digital image analysis. The data produced were analyzed statistically using the ANOVA technique and pairwise comparisons tests. Sensitivity limits and linearity of responses to bacteriocin varied significantly among different test-microorganisms in both applied methods, the lower sensitivity limits depending on both the test-microorganism and the applied method. In both methods, however, only two of the nine tested microorganisms (Lactobacillus curvatus ATCC 51436 and Pediococcus acidilactici ATCC 25740) were sensitive to very low concentrations of the bacteriocin and produced a linear-type of response in all kinds of samples used in this work. In all cases, very low bacteriocin concentrations, e.g. 1 IU/ml nisin, were more accurately determined in the turbidometric assay.

Conclusion: The present work shows that in growth inhibition techniques used in bacteriocin quantification, the choice of the indicator microorganism is critical. Evaluation of sensitivity levels and type of produced responses showed that they can vary widely among different test-microorganisms and different applied methods, indicating that not all microorganisms can be used successfully as indicators and that measurements of growth inhibition in liquid media produce more reliable results.

No MeSH data available.