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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: determination of nisin in L. lactis fermentation broths using L. curvatus, P. acidilactici, and M. varians.
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Figure 8: Turbidometric assay: determination of nisin in L. lactis fermentation broths using L. curvatus, P. acidilactici, and M. varians.

Mentions: Following treatment, samples were analyzed for nisin with both methods and all test-microorganisms. L. curvatus and P. acidilactici were the test-microorganisms performing better- in terms of produced zones of inhibition and percentages of growth inhibition- in both assays than the other examined microorganisms. Figures 7 and 8 show the results produced with the agar diffusion and the turbidometric assays, for L. lactis fermentation samples, using L. curvatus, P. acidilactici and M. varians. Nisin concentrations in L. lactis fermentation broths can be quantified successfully using L. curvatus as an indicator microorganism using the standard curves produced with standard nisin solutions, made with nisin from Sigma, in the agar diffusion and the turbidometric assays.


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: determination of nisin in L. lactis fermentation broths using L. curvatus, P. acidilactici, and M. varians.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Turbidometric assay: determination of nisin in L. lactis fermentation broths using L. curvatus, P. acidilactici, and M. varians.
Mentions: Following treatment, samples were analyzed for nisin with both methods and all test-microorganisms. L. curvatus and P. acidilactici were the test-microorganisms performing better- in terms of produced zones of inhibition and percentages of growth inhibition- in both assays than the other examined microorganisms. Figures 7 and 8 show the results produced with the agar diffusion and the turbidometric assays, for L. lactis fermentation samples, using L. curvatus, P. acidilactici and M. varians. Nisin concentrations in L. lactis fermentation broths can be quantified successfully using L. curvatus as an indicator microorganism using the standard curves produced with standard nisin solutions, made with nisin from Sigma, in the agar diffusion and the turbidometric assays.

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.