The research team started from a simple but consequential observation: aging is accompanied by progressive functional decline and morphological defects in cholinergic neurons, the nerve cells that use the neurotransmitter acetylcholine and whose degeneration is a hallmark of disorders such as Alzheimer’s disease. Because the human gut harbors between 10^13 and 10^14 bacteria comprising at least a thousand different species, and because bacterial metabolites can enter the circulation and cross the blood-brain barrier, the so-called microbiota-gut-brain axis has become a major focus of research into both neuroprotection and neurological disease. Lowered biodiversity and dysbiosis of the gut microbiota have been documented in elderly people and in patients with Alzheimer’s and Parkinson’s disease, raising the question of whether specific probiotic bacteria might counteract these changes.

Biofilms were central to the investigation. These are complex three-dimensional structures of adherent, cooperative bacterial communities embedded in a secreted extracellular matrix, and growth within them helps bacteria survive hostile environments. Previous work in rodent and C. elegans models had identified biofilm proficiency as a crucial determinant of both pro- and anti-neurodegenerative actions of gut bacteria. In particular, neuroprotective effects of probiotic B. subtilis strains have been linked to biofilm formation in the gut and the release of biofilm-derived metabolic and signaling factors. Some B. subtilis strains also secrete proteolytic enzymes, including the fibrin-degrading enzyme nattokinase first isolated from the Japanese food natto, which can degrade amyloid fibrils characteristic of Alzheimer’s disease.

To find the most promising candidate, the researchers screened several B. subtilis strains in vitro for biofilm production and fibrinolytic activity. The proprietary strain B4U 50, deposited at the German Collection of Microorganisms and Cell Cultures as DSM 34350, was grown for 24 hours at 37 degrees Celsius in a simulated-colonic-environment medium in 96-well plates, after which attached biofilms were stained with crystal violet and quantified spectrophotometrically. B4U 50 developed extensive biofilms under these sessile growth conditions, clearly outperforming the widely used laboratory strain NCIB3610, which showed only modest biofilm formation, while the strains 168 and JH642 produced none. On fibrin agar plates, B4U 50 also displayed the highest fibrinolytic activity of the four strains tested, generating the largest transparent hydrolysis halos, whereas strain 168 showed no detectable fibrinolytic activity at all. The biofilm- and protease-deficient strain 168 therefore served as the control in all subsequent feeding experiments.

The experimental animal was the transgenic nematode strain LX929, in which cholinergic neurons are labeled with green fluorescent protein, allowing researchers to watch neuronal aging unfold in living animals. C. elegans offers distinct advantages for this kind of work: the worms nourish on bacteria, some ingested bacteria survive to become part of their gut flora, key longevity and stress-response pathways are evolutionarily conserved between worm and human, the nervous system contains only 302 well-characterized neurons, and the animals’ transparency and short lifespan permit rapid, longitudinal evaluation of dietary interventions. In aging nematodes, cholinergic neurons undergo progressive damage without cell loss, a process that mirrors the progressive dendritic, axonal and synaptic degeneration of cholinergic neurons seen in aging humans.

Synchronized worms were fed from their first larval stage for up to 15 days on either B4U 50 or the control strain 168, and their neurons were imaged by fluorescence microscopy on days 3, 7, 11 and 15. Three blinded researchers classified the neurons into three categories: normal neurons showed a continuous, uniform GFP signal along the ventral nerve cord; mildly damaged neurons displayed scattered minor swellings or knots without breaks in fluorescence; and severely damaged neurons showed multiple large swellings or visible ruptures in the nerve cord. From these categories the team calculated a neurodegeneration index, a composite score obtained by weighting normal, mildly damaged and severely damaged neurons with values of 0, 1 and 2 respectively.

The results were striking. Neuronal damage increased progressively with age in all groups, but the decline was markedly more pronounced in worms kept on the control strain: by days 11 and 15 these animals showed a steep drop in the proportion of normal neurons and a higher incidence of both mild and severe defects. Worms continuously fed B4U 50 retained a greater proportion of morphologically intact neurons throughout the observation period, and their neurodegeneration index remained significantly lower. The team also ran dietary switch experiments, transferring worms initially raised on strain 168 to B4U 50 on day 4, day 8 or day 12. Switching early, on day 4, produced a neuroprotective effect comparable to continuous feeding with B4U 50; switching on day 8 still conferred a moderate benefit; but switching on day 12 no longer protected the neurons. This defines a window of early and middle adulthood for optimal dietary intervention.

The researchers also tested whether the bacterial diet could be combined with a known neuroprotective compound. They used TEBONIN intens, a standardized extract prepared from Ginkgo biloba leaves, which has been reported to enhance cognitive performance in patients with mild and moderate dementia and to extend lifespan and alleviate amyloid-beta-induced pathology in C. elegans models. Supplementation with the Ginkgo biloba preparation was neuroprotective in worms fed on either B. subtilis strain, reducing both the occurrence and the timing of mild and severe neuronal defects. Notably, the combination of B4U 50 and the Ginkgo biloba extract yielded the highest proportion of morphologically intact neurons by day 15 and the lowest neurodegeneration index values of any group, while worms on the control strain without the extract fared worst. Representative fluorescence images of day-15 animals made the difference visible: worms receiving the combined intervention displayed largely intact GFP-labeled ventral nerve cords, whereas those fed only the control strain exhibited pronounced swellings and discontinuities.

Why might a biofilm-forming bacterium protect nerve cells? The authors point to a proposed model in which B. subtilis spores ingested with food survive gastric transit, germinate in the intestine, and grow in biofilms that produce and secrete substances spreading from the gut throughout the body, exerting anti-aging and neuroprotective effects. Previous studies have identified the quorum-sensing pentapeptide known as competence sporulation-stimulating factor and the gasotransmitter nitric oxide as key mediators of the benefits of B. subtilis biofilms, and probiotic biofilm-producing strains have been shown to counteract neurodegeneration in wild-type worms as well as in transgenic models of Alzheimer’s and Parkinson’s disease expressing human amyloid-beta and alpha-synuclein. Metabolic remodeling during biofilm development is complex, affecting cellular respiration, lipid metabolism and the production of secondary metabolites and signaling molecules.

The authors are careful about the limits of their work. They did not examine whether B4U 50 actually forms a biofilm inside the worm gut, nor did they identify the specific signaling or metabolic factors mediating the neuroprotective effect, and they note this as a limitation of the study. It is also worth noting that the research was funded by Evonik Operations GmbH, the company that maintains the proprietary B4U 50 strain, and that three of the co-authors are Evonik employees, though the other authors report no competing interests. Nevertheless, the study extends a promising line of research suggesting that B. subtilis strains with strong biofilm proficiency, whether laboratory strains like NCIB3610 or probiotic isolates from East Asian fermented foods, can extend lifespan and protect the nervous system in C. elegans. If the findings translate beyond the nematode, combining biofilm-proficient probiotic bacteria with established natural products such as standardized Ginkgo biloba extract could yield dietary supplements with enhanced neuroprotective capacity for the prevention and amelioration of neurodegenerative disorders.

Subject of Research: Neuroprotective effects of a biofilm-forming Bacillus subtilis probiotic strain on aging cholinergic neurons in C. elegans

Article Title: A Bacillus subtilis strain with high proficiency for biofilm formation delays the onset of aging-associated morphological defects in cholinergic neurons of C. elegans

Article References: Gong, W., Priebs, J., Ohse, V. A., Bremges, A., Dieck, H. T., Klotz, L.-O., Dargatz, M., & Steinbrenner, H. (2026). A Bacillus subtilis strain with high proficiency for biofilm formation delays the onset of aging-associated morphological defects in cholinergic neurons of C. elegans. GeroScience. https://doi.org/10.1007/s11357-026-02594-0

Image Credits: AI Generated

DOI: 10.1007/s11357-026-02594-0

Keywords: Bacillus subtilis, C. elegans, biofilm, cholinergic neurons, neurodegeneration, probiotics, Ginkgo biloba, microbiota-gut-brain axis, aging, nattokinase, neuroprotection, GeroScience