RESUMO
Faecal microbiota transplantation is an emerging medical concept for the treatment of gastrointestinal diseases. This concept, however, has disadvantages as low storability of stool and intensive donor screening. A solution to overcome these problems would be the preservation of an in vitro microbiota through freeze-drying. However, the influence of the entire preservation process, including cultivation and lyophilization, has not been assessed so far. In this study, the influences of the process steps cultivation, drying and re-cultivation were determined with cell count, production of metabolites, microbial composition and diversity in the system as evaluation criteria. All pH conditions resulted in stable, culturable communities after re-cultivation. Cell count, richness, diversity and microbial composition were affected by freeze-drying, but these effects were reversible and vanished during re-cultivation. Hence, the re-cultivated system did not differ from the system before drying. The metabolism, measured by short-chain fatty acids as indicators, showed slight changes due to natural dynamics. Consequently, the cultivation prior to drying was identified to have more influence than the drying itself on the preservation process and therefore the biggest potential for optimization. Hence, the highest similarity with the initial stool sample was obtained with pH 6.0 - 6.5 during cultivation.
Assuntos
Microbioma Gastrointestinal , Dessecação , Liofilização/métodos , Humanos , Concentração de Íons de HidrogênioRESUMO
Fecal microbiota transplantation (FMT) is an alternative method for the treatment of gastrointestinal diseases with a high recovery rate. Disadvantages are ethical concerns, high donor requirements and the low storability of stool samples. The cultivation of an in vitro microbiota in a continuous bioreactor was established as an alternative to FMT to overcome these problems. In this study, the influence of the system parameters and donor stool characteristics was investigated. Each continuous colonic fermentation system was inoculated with feces from three different donors until a stable state was established. The influence of the fermentation conditions on the system's behavior regarding cell count, metabolic activity, short-chain fatty acid profile and microbiota composition as well as richness and diversity was assessed. Cultivation conditions were found to affect the microbial system: the number of cells and the production of short-chain fatty acids increased. The abundance of Actinobacteria and Firmicutes decreased, Bacteroidetes increased, while Proteobacteria and Verrucomicrobia remained largely unaffected. Diversity in the in vitro system decreased, but richness was unaffected. The cultivation of stool from different donors revealed that the performance of the created in vitro system was similar and comparable, but unique characteristics of the composition of the original stool remained.
RESUMO
Bifidobacterium longum, one of the main microorganisms in the human gut, is used as an adjunct to lactic acid starter cultures or sold as a probiotic product. Therefore, Bifidobacterium longum cell suspensions get freeze-dried with protective additives to prevent activity losses. To date, investigations covering growth and inactivation kinetics of Bifidobacterium longum during the whole process (cultivation, drying, and storage) have been lacking. In this study, the effect of cultivation conditions and shelf temperature as well as the influence of protectants (maltodextrin, glucitol, trehalose) at various concentrations on cell survival during freeze-drying was assessed. Drying was followed by a storage at + 4 °C and + 20 °C for 70 days to evaluate inactivation kinetics. The impact of the different factors was assessed by measuring surival rate and residual moisture content at various points of time over the whole process. In parallel cell membrane integrity and glass transition were determined to reveal inactivation effects. Cultivation strategy had a strong influence on survival with a huge potential for process improvement. A pH of 6.0 at the growth optimum of the strain provides better conditions regarding cell survival after drying than free acidification (non-regulated pH conditions). During the drying step, membrane leakage due to the removal of water is the main reason for the inactivation in this process step. In this study, the highest survival of 49% was obtained with cells dried at + 35 °C shelf temperature with an addition of maltodextrin (75% bacterial dry matter, w/w). The results show that Bifidobacterium longum cells are mostly inactivated during drying, whereas storage conditions at + 4 °C with an addition of 75% BDM maltodextrin relative to bacterial dry mass prevent cell loss completely.
