OBJECTIVES:
Recent research has recognized humans as metaorganisms— multispecies consortia consisting of the human host and its associated microbial inhabitants commonly referred to as the microbiome. The subsequent flood of research in this field has led to a dramatic improvement of our understanding into how the host, its microbiome, and the environment interact to determine human health and disease. On Earth, probiotics have been shown to interact with the host and its GI microbiome to improve the immune response, protect against pathogens, and improve gut barrier function.
Long-duration missions in space expose astronauts to chronic stresses imposed by microgravity, ionizing radiation, and confinement. These aspects of space flight have been shown to alter astronaut physiology, affecting nearly every system including musculoskeletal, neurological, endocrine, cardiovascular, respiratory, excretory, cognitive, and immune systems. Development of effective countermeasures to maintain astronaut health and performance under the extreme conditions of deep space exploration is an actively ongoing NASA endeavor.
Research into changes to astronaut microbiomes has revealed evidence of both compositional and functional changes during long-duration space flight. Dysregulation of the astronaut immune system in space has been identified as a major contributor to numerous spaceflight syndromes. Further, The Design Reference Mission (DRM) for a Mars mission states that all nutrients sufficient for three plus years will be preserved and stored onboard, and no resupply activity or cultivation of fresh food is planned. This necessitates research into the stability and shelf-life of food items, supplements, and pharmaceuticals in space over the course of a Mars DRM. Probiotics stored as freeze-dried powders in capsules are a promising option for long-term stability. However, probiotics consisting of bacterial spores may serve as a better, albeit unexplored, option for long-duration missions due to their extreme longevity and documented resistance to ionizing radiation.
The hypothesis of this experiment was that exposure of probiotic bacteria to simulated space radiation would result in a decrease in survival and potency at a rate which can be empirically measured. Probiotics containing
Bacillus spores were hypothesized to demonstrate enhanced long-term stability and potency compared to traditional
Lactobacillus- or
Bifidobacterium-containing probiotics.
This experiment had three specific aims.
(1) Select probiotic candidates to test.
(2) Expose probiotic samples to simulated Galactic Cosmic Radiation (GCRSim) and Solar Particle Events (SPESim) at Mars DRM-relevant doses.
(3) Characterize probiotic viability of pre- and post-irradiation probiotic samples and potency of these samples in simulated gastric and intestinal juices in vitro.
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APPROACH:
For Aim 1, the researchers applied a set of criteria to select three candidate probiotic formulations. These criteria included (1) the probiotics contain only a single species, (2) the selected species should be readily cultured on standard laboratory media, (3) the candidate probiotics are commercially available in a form amenable to long-duration space flight, and (4) the candidate probiotics should have a proven record of safety and efficacy documented in the peer-reviewed literature. The selected probiotics included Lactobacillus acidophilus DDS-1, Bifidobacterium longum BB536, and Bacillus subtilis HU58.
Following the selection of the probiotic strains, 16S taxonomy was used to ensure the species identity of selected commercial probiotic cultures. The universal bacterial primers B27F (5'-GAGTTTGATCMTGGCTCAG-3') and B1512R (5'-AAGGAGGTGATCCANCCRCA-3') were used to amplify the selected 16S sequence, which were subsequently be identified to the species level using the Ribosomal Database Project (RDP) and National Center for Biotechnology Information (NCBI) BLASTN servers.
In Aim 2, the samples were exposed to Mars DM-relevant doses of GCRSim or SPESim beams at the NASA Space Radiation Laboratory (NSRL). GCRSim consists of a mixture of atomic nuclei (H, He, C, O, Si, Ti, and Fe) delivered in series at a variety of energies and dosages to simulate the flux of GCR expected to be encountered during a three-year Mars DRM. SPESim consists of protons delivered at a variety of different energies simulating a typical SPE. Each probiotic will be loaded into capsules and arranged on 28-place blister cards according to a random number generator.
In Aim 3, the treated samples and control samples of each strain were compared across three assays: (1) viability assessment by plating, (2) survival in simulated gastric juices, and (3) survival in simulated intestinal juices in vitro. Working suspensions of each sample were prepared from probiotic lyophilized powder within the capsules for all four assays. Aliquots of serial tenfold dilutions were plated in duplicate on either LB (Bacillus subtilis) or MRSC (Lactobacillus acidophilus, Bifidobacterium longum) agar plates and incubated at 37 °C for 24 hours under aerobic conditions or 48 under anaerobic conditions. Plates yielding at least 100 well-separated colonies were counted and the number of Colony Forming Units (CFUs) were extrapolated. For in vitro experiments, aliquots of the working suspension were transferred to commercial preparations of simulated gastric and intestinal juices and incubated in a 37 °C shaking water bath at intervals between 0 to 3 hours (gastric) or between 0 to 24 hours (intestinal). The potency of cells exposed to simulated gastric or intestinal fluids was evaluated using plating.
RESULTS:
This study sought to assess the viability and potency of three common probiotics, Lactobacillus acidophilus, Bifidobacterium longum, and Bacillus subtilis, after exposure to ionizing radiation simulating a three-year Mars DRM. The researchers hypothesized that all three bacteria would be inactivated to some extent by radiation exposure, but that Bacillus subtilis spores would retain a higher degree of viability and potency than the other two strains due to the well-documented enhanced radiation resistance of spores. The investigators found that freeze-dried samples of B. subtilis spores maintained high viability compared to the two probiotics tested. This does not completely rule out the utility of Lactobacillus acidophilus and Bifidobacterium longum in spaceflight contexts as postbiotics, however. The results of this research demonstrate B. subtilis spores as a strong candidate probiotic with a long and robust shelf-life in the spaceflight environment.
Crew health and performance is critical to successful human exploration beyond low Earth orbit.
The Human Research Program (HRP) investigates and mitigates the highest risks to human health
and performance, providing essential countermeasures and technologies for human space exploration.
Risks include physiological and performance effects from hazards such as radiation, altered gravity,
and hostile environments, as well as unique challenges in medical support, human factors,
and behavioral health support. The HRP utilizes an Integrated Research Plan (IRP) to identify
the approach and research activities planned to address these risks, which are assigned to specific
Elements within the program. The Human Research Roadmap is the web-based tool for communicating the IRP content.
The Human Research Roadmap is located at:
https://humanresearchroadmap.nasa.gov/
+ Click here for information of how this experiment is contributing to the HRP's path for risk reduction.