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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.


Turbidometric assay: plot of log10 nisin concentrations vs percentage inhibition of growth with L. plantarum, L. curvatus and L. sakei.
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Figure 4: Turbidometric assay: plot of log10 nisin concentrations vs percentage inhibition of growth with L. plantarum, L. curvatus and L. sakei.

Mentions: Percentages of growth inhibition of test-microorganisms were plotted against log nisin concentrations in Figs 4, 5, and 6. Total inhibition was observed with L. curvatus at 75 IU/ml nisin, with L. sakei at 100 IU/ml, while with L. plantarum only at 800 IU/ml nisin. The lower response limit for all three Lactobacilli was 1 IU/ml nisin, at which growth inhibition was 33% for L. curvatus, 41% for L. sakei, and 5.20% for L. plantarum. This method is obviously more sensitive than the agar diffusion method since lower detected sensitivity levels in the agar were 40 IU/ml for L. sakei and 75 IU/ml for L. plantarum. L. plantarum appeared to be less sensitive to nisin compared to the other two Lactobacillus strains, but it produced a linear response to nisin doses ranging from 1 to 100 IU/ml. L. curvatus responded linearly within 1 to 75 IU/ml nisin (R > 0.98) -the response producing a plateau at higher nisin concentrations- suffering a much stronger inhibition effect. The results produced with L. curvatus in the turbidometric assay are in agreement with those produced in the agar diffusion assay. L. sakei response, according to Fig. 5, cannot be regarded as linear.


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)

Turbidometric assay: plot of log10 nisin concentrations vs percentage inhibition of growth with L. plantarum, L. curvatus and L. sakei.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Turbidometric assay: plot of log10 nisin concentrations vs percentage inhibition of growth with L. plantarum, L. curvatus and L. sakei.
Mentions: Percentages of growth inhibition of test-microorganisms were plotted against log nisin concentrations in Figs 4, 5, and 6. Total inhibition was observed with L. curvatus at 75 IU/ml nisin, with L. sakei at 100 IU/ml, while with L. plantarum only at 800 IU/ml nisin. The lower response limit for all three Lactobacilli was 1 IU/ml nisin, at which growth inhibition was 33% for L. curvatus, 41% for L. sakei, and 5.20% for L. plantarum. This method is obviously more sensitive than the agar diffusion method since lower detected sensitivity levels in the agar were 40 IU/ml for L. sakei and 75 IU/ml for L. plantarum. L. plantarum appeared to be less sensitive to nisin compared to the other two Lactobacillus strains, but it produced a linear response to nisin doses ranging from 1 to 100 IU/ml. L. curvatus responded linearly within 1 to 75 IU/ml nisin (R > 0.98) -the response producing a plateau at higher nisin concentrations- suffering a much stronger inhibition effect. The results produced with L. curvatus in the turbidometric assay are in agreement with those produced in the agar diffusion assay. L. sakei response, according to Fig. 5, cannot be regarded as linear.

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.