Related Experiment Video
Updated: Mar 26, 2026
Bioreactor Assembly for Continuous Culture of Complex Fecal Communities
Published on: April 25, 2025
Assessing the impact of long term frozen storage of faecal samples on protein concentration and protease activity
Laura S Morris1, Julian R Marchesi2
1School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, UK; College of Medicine, Swansea University, Swansea SA2 8PP, UK.
Background:
The proteome is the second axis of the microbiome:host interactome and proteases are a significant aspect in this interaction. They interact with a large variety of host proteins and structures and in many situations are implicated in pathogenesis. Furthermore faecal samples are commonly collected and stored frozen so they can be analysed at a later date. So we were interested to know whether long term storage affected the integrity of proteases and total protein and whether historical native faecal samples were still a viable option for answering research questions around the functional proteome.
Methods:
Faecal samples were collected from 3 healthy volunteers (3 biological replicates) and processed in order to be stored at both -20 °C and -80 °C and in a variety of storage buffers. Protein extraction, protein content and protease activity were assessed at the time of collection, after 24h, 1 week, 1 month, 3 months 6 months and finally 1 year.
Results:
Beadbeating impacted the quantity of protein extracted, while sodium azide did not impact protease assays. Long term storage of extracted proteins showed that both total protein and protease activity were affected when they were stored as extracted protein. Intact faecal samples were shown to maintain both protein levels and protease activity regardless of time and temperature.
Conclusions:
Beadbeating increases the protein and protease activity when extracting from a faecal sample, however, the extracted protein is not stable and activity is lost, even with a suitable storage buffer. The most robust solution is to store the proteins in an intact frozen native faecal matrix and extract at the time of assay or analysis, this approach was shown to be suitable for samples in which, there are low levels of protease activity and which had been frozen for a year.
Insights
Storing intact fecal samples preserves protein and protease activity over long periods, unlike extracted proteins which degrade. This makes historical fecal samples viable for microbiome-host interactome research.
Area of Science:
- Microbiome research
- Host-microbiome interactions
- Proteomics
Background:
- The microbiome's proteome is crucial for host interactions, with proteases playing a significant role.
- Proteases can influence host proteins and structures, often contributing to pathogenesis.
- Faecal samples are frequently stored long-term, raising questions about sample integrity for proteomic analysis.
Purpose of the Study:
- To investigate the impact of long-term storage on protease and total protein integrity in faecal samples.
- To determine if historical faecal samples remain viable for functional proteome research.
Main Methods:
- Faecal samples from 3 healthy volunteers were stored at -20°C and -80°C in various buffers.
- Protein extraction, content, and protease activity were assessed at multiple time points up to 1 year.
- The effect of beadbeating and sodium azide on protein and protease activity was evaluated.
Main Results:
- Beadbeating increased protein yield but did not stabilize extracted proteins.
- Long-term storage of extracted proteins led to degradation of total protein and loss of protease activity.
- Intact faecal samples maintained protein levels and protease activity irrespective of storage temperature and duration.
Conclusions:
- While beadbeating aids extraction, the resulting proteins lack stability for long-term storage.
- Storing faecal samples intact as a frozen matrix is the most robust method for preserving protein and protease integrity.
- This approach ensures the viability of historical samples for proteomic analysis, even with low protease activity after a year of storage.
