Construction of a Single-batch Fermentation System to Simulate Human Intestinal Microbiota

Recently, various functional food components called prebiotics, probiotics, and biogenics have attracted attention as potential ways to manipulate the gut microbiota and to improve human health. Functionality of such food components usually has been evaluated by human intervention trials or animal feeding trials. However, human trials often are constrained by ethical considerations, while the animal feeding trials often yield results that are not reproducible in humans. Therefore, it was necessary to develop a relatively simple and short-term in vitro evaluation system. The aim of the present study thus was to construct a single-batch fermentation system that was as simple as possible, to prove a human intestinal model.

The anaerobic conditions in our system were carefully constructed and maintained by using a previously defined medium for the growth of anaerobes, Gifu anaerobic medium (GAM), and constantly flushing the medium with N₂/CO₂ gas before and during the fermentation. Operation of the batch fermentation system was initiated by inoculating with small amounts of human volunteer fecal sample. The total number of the eubacteria was calculated to range from 60.9 × 10¹¹ to 128 × 10¹¹ copies/wet-g of feces. After 24 h of fermentation, eubacterial copy numbers reached 4.86 × 10¹¹ to 6.93 × 10¹¹ copies/mL. 16S rRNA gene sequence analysis of bacteria grown in the system revealed that occupancies of the major phyla, including Bacteroidetes, Firmicutes, and Actinobacteria, as well as overall species diversity, were consistent with those of the original feces. At the earlier stages of fermentation (up to 9h), trace mixtures of acetate, lactate, and succinate were detectable; at the later stages (after 24 h), larger amounts of acetate accumulated along with low levels of propionate and butyrate. These patterns were similar to those observed in the original feces. Thus, this system could serve as a simple model to simulate the diversity as well as the metabolism of human intestinal microbiota.

Finally, in order to substantiate the practicality using the mode, we evaluated the functionality of prebiotics, in this case using oligosaccharides. Supplementation of the system with several prebiotic oligosaccharides (including fructo-, galacto-, isomalto-, and xylo-oligosaccharides; lactulose; and lactosucrose) yielded elevation of the population of bacteria belonging to the genus Bifidobacterium, consistent with significant increases in acetate production. Thus, the system was able to detect the known bifidogenic effects of prebiotic oligosaccharides. Therefore, our single-batch fermentation system is expected to serve as a simple but versatile tool for evaluating the functionality of various food components.

Keywords: single batch fermentation system, human intestinal model, microbiota, prebiotics