Related Experiment Video
Updated: Mar 22, 2026
Evaluation of Microbial Safety of Dairies using Bacterial Proteomic Profiling via MALDI Approach
Published on: October 7, 2025
Use of propidium monoazide for selective profiling of viable microbial cells during Gouda cheese ripening
Oylum Erkus1, Victor C L de Jager2, Renske T C M Geene3
1NIZO food research BV, P.O. Box 20, 6710 BA Ede, The Netherlands; Top Institute Food and Nutrition (TIFN), Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 557, 6700 AN, Wageningen, The Netherlands; Wageningen University, Laboratory of Microbiology, Dreijenplein 10, 6703 HB Wageningen, The Netherlands.
Abstract:
DNA based microbial community profiling of food samples is confounded by the presence of DNA derived from membrane compromised (dead or injured) cells. Selective amplification of DNA from viable (intact) fraction of the community by propidium monoazide (PMA) treatment could circumvent this problem. Gouda cheese manufacturing is a proper model to evaluate the use of PMA for selective detection of intact cells since large fraction of membrane compromised cells emerges as a background in the cheese matrix during ripening. In this study, the effect of PMA on cheese community profiles was evaluated throughout manufacturing and ripening using quantitative PCR (qPCR). PMA effectively inhibited the amplification of DNA derived from membrane compromised cells and enhanced the analysis of the intact fraction residing in the cheese samples. Furthermore, a two-step protocol, which involves whole genome amplification (WGA) to enrich the DNA not modified with PMA and subsequent sequencing, was developed for the selective metagenome sequencing of viable fraction in the Gouda cheese microbial community. The metagenome profile of PMA treated cheese sample reflected the viable community profile at that time point in the cheese manufacturing.
Insights
Propidium monoazide (PMA) treatment selectively amplifies DNA from viable microbial cells in food, improving community profiling accuracy. This method enhances the analysis of intact microbial communities during cheese production.
Area of Science:
- Microbiology
- Food Science
- Molecular Biology
Background:
- DNA-based microbial profiling in food is hindered by DNA from dead or injured cells.
- Propidium monoazide (PMA) can selectively amplify DNA from viable cells with intact membranes.
- Gouda cheese ripening generates a significant background of membrane-compromised cells, making it a suitable model.
Purpose of the Study:
- To evaluate the effectiveness of PMA treatment for selective microbial DNA detection in Gouda cheese.
- To assess PMA's impact on microbial community profiles during cheese manufacturing and ripening.
- To develop a protocol for selective metagenome sequencing of viable microbial communities in cheese.
Main Methods:
- Quantitative PCR (qPCR) was used to assess the effect of PMA on DNA amplification.
- A two-step protocol involving whole genome amplification (WGA) and sequencing was developed.
- PMA treatment was applied to cheese samples throughout manufacturing and ripening.
Main Results:
- PMA effectively inhibited the amplification of DNA from membrane-compromised cells.
- PMA enhanced the analysis of the intact microbial fraction in cheese samples.
- The developed protocol enabled selective metagenome sequencing of the viable microbial community.
Conclusions:
- PMA treatment is a valuable tool for accurate DNA-based microbial community profiling in food matrices like cheese.
- The developed WGA and sequencing protocol allows for selective analysis of viable microbial communities.
- This approach provides a more representative understanding of the functional microbial populations during food production.
