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Determination of bacteriocin activity with bioassays carried out on solid and liquid substrates: assessing the factor "indicator microorganism".
Open accessAbstract
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.
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The agar diffusion assay primarily produces qualitative results by indicating the presence or absence of antimicrobial activity through inhibition zones rather than providing precise quantitative measurements or minimum inhibitory concentrations (MICs).
“The assay primarily produces qualitative results by indicating the presence or absence of antimicrobial activity through the formation of inhibition zones that serve as a relative measure of antimicrobial activity rather than providing precise quantitative measurements or minimum inhibitory concentrations (MICs).”
From Methods for screening and evaluation of antimicrobial activity: A review of protocols, advantages, and limitations (2024) · cites this paper as [33]
The Results and discussion (Agar diffusion assay; page unavailable) use inhibition-zone diameters to quantify responses across specified nisin concentrations, contrary to the claim that the assay primarily yields qualitative presence-or-absence results. The Background acknowledges limitations in precision, but the paper does not address MIC measurement. (LLM, conf=0.86)
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Experimental — accuracy not yet measuredThe selection of specific microorganisms and the choice of growth media in the agar diffusion assay may not represent all relevant clinical or environmental conditions.
“Researchers should also consider that the selection of specific microorganisms and the choice of growth media in the assay may not represent all relevant clinical or environmental conditions [33].”
From Methods for screening and evaluation of antimicrobial activity: A review of protocols, advantages, and limitations (2024) · cites this paper as [33]
The Background says assay success depends on the chosen indicator microorganism’s sensitivity, and the Agar diffusion assay methods describe the media used. These passages support caution about organism selection and assay conditions, but they do not address whether those choices represent clinical or environmental conditions. No page numbers are provided in our copy. (LLM, conf=0.82)
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Research works printed but not linked: 18
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Hossain, T. J. (2024) · European Journal of Microbiology and Immunology · doi:10.1556/1886.2024.00035 · Cited by 392
Metadata
- Authors
- Papagianni, M., Avramidis, N., Filioussis, G., Dasiou, D., & Ambrosiadis, I.
- Affiliations
- Aristotle University of Thessaloniki (GR)
- Corresponding author
- Maria Papagianni
- Venue
- Microbial Cell Factories
- Published
- 2006
- Volume
- 5
- Issue
- 1
- Pages
- 30
- ISSN
- 1475-2859 (ISSN-L)
- PMC
- PMC1622751
- OpenAlex
- W1822201252
- Cited by
- 50
- References
- 0
- Citations by year
- 2026: 2 · 2025: 2 · 2024: 3 · 2023: 2 · 2022: 3 · 2021: 1 · 2020: 3 · 2019: 2 · 2017: 3 · 2016: 2 · 2015: 3 · 2014: 3 · 2013: 5 · 2012: 4
- FWCI
- 4.84
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- cc-by
- Locations
- Microbial Cell Factories (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Topics
- Life Sciences › Agricultural and Biological Sciences › Food Science › Probiotics and Fermented Foods
- Life Sciences › Agricultural and Biological Sciences › Food Science › Essential Oils and Antimicrobial Activity
- Life Sciences › Immunology and Microbiology › Microbiology › Antimicrobial Peptides and Activities
- Keywords
- nisin, indicator microorganisms, agar diffusion, Lactobacillus curvatus, Pediococcus acidilactici, Micrococcus, Leuconostoc, fermented foods
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