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Related Experiment Video

Updated: Oct 31, 2025

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota
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Effect of Freezing on Gut Microbiota Composition and Functionality for In Vitro Fermentation Experiments.

Sergio Pérez-Burillo1,2, Daniel Hinojosa-Nogueira1, Beatriz Navajas-Porras1

  • 1Centro de Investigación Biomédica, Departamento de Nutrición y Bromatología, Instituto de Nutrición y Tecnología de los Alimentos, Universidad de Granada, 18071 Granada, Spain.

Nutrients
|July 2, 2021
PubMed
Summary

Freezing fecal samples for gut microbiota studies is feasible. While freezing alters microbial community structure, its impact is less significant than individual variations, and microbial function is preserved for in vitro fermentation after stabilization.

Keywords:
bioactive compoundsfoodsfreezinggut microbiotastorage

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Area of Science:

  • Microbiology
  • Human Health
  • Gastroenterology

Background:

  • The gut microbiota significantly influences human health.
  • In vitro batch fermentation using fecal material is a common method to study gut microbiota interactions.
  • Experimental designs may necessitate the use of previously frozen fecal samples.

Purpose of the Study:

  • To investigate the viability of using frozen fecal material for in vitro fermentation.
  • To assess the impact of freezing and subsequent stabilization on gut microbial community structure and function.

Main Methods:

  • Pilot study involving in vitro batch fermentations with fresh and frozen fecal samples (preserved with glycerol).
  • Fecal samples were stabilized in a nutrient medium for 6 days prior to fermentation.
  • Microbial community structure was analyzed, and potential functionality was predicted using in silico genome-scaled metabolic reconstruction.

Main Results:

  • Microbial community structure differed significantly between fresh and frozen samples, but variation from freezing was less than inter-individual variation.
  • Potential microbial functionality remained largely unchanged, likely due to functional redundancy.
  • The genus *Bacteroides* was most affected by freezing.

Conclusions:

  • Freezing fecal material for in vitro fermentation is a viable option.
  • Stabilization can help preserve the functionality of the microbial community in frozen samples.
  • The impact of freezing on microbial structure is less significant than inherent individual differences.